CN109342711B - ELISA kit for simultaneous determination of multi-species IL-1Ra and IL-1β and their ratio - Google Patents

Abstract

The invention relates to an ELISA kit for synchronous determination of multi-species IL-1Ra and IL-1β and their ratios, belonging to the field of biotechnology detection. Including anti-IL-1β and IL-1Ra rabbit-derived polyclonal capture antibody pre-coated microtiter plate, IL-1β and IL-1Ra recombinant protein standard, diluent, 20×PBS-T concentrated washing solution, blue fluorescence Guinea pig-derived anti-IL-1β polyclonal detection antibody labeled with microspheres, and guinea pig-derived anti-IL-1R polyclonal detection antibody labeled with green fluorescent microspheres. The present invention has the advantages of high sensitivity, strong specificity, and a wide range of detection species, and can be used for the determination of IL-1β and IL-1Ra content in sheep, human, cow, horse, pig, mouse, rabbit serum, plasma and other tissue samples. And the ratio can be calculated, the simultaneous detection of the two targets reduces the error in ratio calculation, and the sensitivity reaches pg level. The kit can be used to determine the ratio of IL-1Ra and IL-1β for the differential diagnosis of brucellosis natural infection and vaccine immunity. And other studies of disease diagnosis markers related to the change of this ratio.

Description

ELISA kit for simultaneous determination of multi-species IL-1Ra and IL-1β and their ratio

technical field

The invention belongs to the field of biotechnology detection, and analyzes the amino acid sequences of IL-1β and IL-1Ra proteins of sheep, humans, cattle, horses, pigs, mice and rabbits through homologous comparison, and determines the homologous and conserved IL-1β of multiple species and IL-1Ra domain gene and protein fragments, using prokaryotic expression system to prepare IL-1β and IL-1Ra homologous conserved domain genetically engineered recombinant protein, and use it as an immunogen to prepare separately and simultaneously bind multi-species IL- Rabbit-derived polyclonal capture antibodies for 1β and IL-1Ra, guinea pig-derived polyclonal detection antibodies that specifically bind to multi-species IL-1β, and guinea pig-derived polyclonal detection antibodies that specifically bind to multi-species IL-1Ra. Two kinds of detection antibodies were coupled with blue and green fluorescent microspheres respectively to establish a double-fluorescence ELISA detection method for simultaneous determination of multi-species IL-1β and IL-1Ra double antibody sandwich, and assembled into an easy-to-apply detection kit. It can be used to determine the content of IL-1β and IL-1Ra in serum, plasma and other tissue samples of sheep, humans, cattle, horses, pigs, mice, rabbits, etc., and can calculate the ratio of IL-1Ra/IL-1β. Synchronous detection of targets reduces errors in ratio calculations.

Background technique

The interleukin 1 (IL-1) cytokine family includes 11 members, mainly interleukin 1α (IL-1α), interleukin 1β (IL-1β), interleukin 1 receptor antagonist (IL-1Ra), etc. . IL-1α and IL-1β are two members of the IL-1 family that mainly activate downstream signaling pathways to exert biological functions. It exerts biological functions mainly by binding to specific receptors, recruiting co-receptors, activating intracellular signals, and inducing inflammatory responses. Studies have found that IL-1β itself does not have a signal peptide sequence and is secreted through unconventional pathways. In addition, the IL-1β precursor is biologically inactive and only becomes active after its N-terminal region is cleaved by caspase-1. The activity of IL-1β is tightly controlled by inhibitors of protein translation, processing, maturation, receptor binding and receptor spontaneous signaling, of which IL-1Ra is the main IL-1 family antagonist. Studies have found that the imbalance between IL-1 and IL-1Ra can lead to excessive inflammatory response, IL-1β and IL-1Ra have different differential expression patterns in some diseases, and their ratios are also different in different diseases. Therefore, the ratio of the two expression levels has certain indicative significance in measuring the occurrence, development, and outcome of certain related diseases. At present, the balance relationship between IL-1 and IL-1Ra has been widely studied in various diseases such as arthritis and inflammatory bowel disease (IBD). Local overproduction of IL-1 and/or underproduction of IL-1Ra predisposes to disease, and therapeutic administration of IL-1Ra effectively prevents tissue damage. The detection methods of IL-1β and IL-1Ra content mainly include intracellular cytokine detection method, immunological detection method, biological activity detection method and molecular biology method and so on. The first three methods are too single to detect species, with poor specificity and low reliability of detection values. Molecular biology methods mainly focus on the detection of nucleic acids, and the detection operations are relatively cumbersome, requiring the steps of sample pretreatment and nucleic acid extraction, and the extraction efficiency directly affects the detection results.

Brucellosis (Brucellosis, referred to as "brucellosis") is one of the most serious zoonotic diseases in the world. In the brucellosis purification work, there is no effective detection method to distinguish between animals vaccinated against brucellosis and diseased animals, which brings resistance to the culling of diseased animals and seriously affects the effective prevention and control of brucellosis.

Contents of the invention

The invention provides an ELISA kit for synchronous determination of multi-species IL-1Ra and IL-1β and their ratios, which can simultaneously detect the concentrations and ratios of IL-1Ra and IL-1β in a sample through one experiment, solving the problem of measuring IL-1 in a single experiment. - The problem of inaccurate concentration ratio of 1β and IL-1Ra, while using fluorescence detection can more sensitively detect the concentration of IL-1β and IL-1Ra in serum, plasma, and various tissue samples. Antibodies prepared from the homologous conserved domains of IL-1β and IL-1Ra of sheep, humans, cattle, horses, pigs, mice, and rabbits and their tandem fusion genes designed by homologous comparison, and their genetically engineered expression proteins It can be combined with natural IL-1β and IL-1Ra of sheep, human, cow, horse, pig, mouse, rabbit, dog, chicken and duck, and can be used to detect serum, plasma and tissues and organs of multiple species The content of IL-1β and IL-1Ra in the sample, and the ratio of IL-1Ra/IL-1β.

The technical scheme adopted by the present invention is: including anti-IL-1β and IL-1Ra rabbit-derived polyclonal capture antibody pre-coated microtiter plate, IL-1β and IL-1Ra recombinant protein standard, diluent, 20×PBS- T concentrated washing solution, guinea pig-derived anti-IL-1β polyclonal detection antibody labeled with blue fluorescent microspheres, and guinea pig-derived anti-IL-1R polyclonal detection antibody labeled with green fluorescent microspheres.

this invention:

(1) The preparation steps of the anti-IL-1β and IL-1Ra rabbit-derived polyclonal capture antibody pre-coated microtiter plate are as follows:

(1) Coating with anti-IL-1β and IL-1Ra rabbit-derived polyclonal capture antibody: Dilute the anti-IL-1β and IL-1Ra rabbit-derived polyclonal capture antibody with diluent to a final concentration of 0.078 μg/mL, Add to the microtiter plate, 100 μL/well, and coat overnight at 4°C;

(2) Use PBS containing 0.2% Tween-20 as the PBS-T washing solution, wash the plate 3-4 times, 200 μL/well, 1 min each time, then block with 0.1M ammonium chloride solution, 200 μL/well, 37 ℃1h, drain the liquid;

(2) Guinea pig-derived anti-IL-1β polyclonal detection antibody labeled with blue fluorescent microspheres, with an excitation wavelength of 360 nm and an emission wavelength of 450 nm; the coupling steps are as follows: add 10 μL of blue fluorescent microspheres to MES buffer at pH 4.5-7.5 Mix 90 mL of the solution, centrifuge at 15000 rpm for 15 minutes, discard the supernatant, collect the precipitated microspheres, and wash twice; add 4.8 μL of EDC (1 mg/mL ), activate for 20 minutes; then add 5.6 μL NHS, (1 mg/mL), and mix for 20 minutes; centrifuge at 15,000 rpm at 4°C for 15 minutes to collect the pellet; resuspend with 90 μL MES buffer solution with pH 4.5-7.5 Add 10 μL guinea pig-derived anti-IL-1β polyclonal detection antibody (1 mg/mL) to the microspheres, and react with vortex shaking at room temperature for 4 hours; add 10 μL blocking solution (10% BSA), and react at room temperature for 30 min; 4°C, 15,000 rpm Centrifuge for 15 minutes, discard the supernatant, add 100 μL of MES buffer solution with pH 4.5-7.5, and store in the dark at 4°C;

(3) Guinea pig-derived anti-IL-1R polyclonal detection antibody labeled with green fluorescent microspheres, the excitation wavelength is 480nm, and the emission wavelength is 520nm; 10 μL of green fluorescent microspheres is added to 90 mL of MES buffer solution with pH 4.5-7.5, mixed well, and 15000 Centrifuge at rpm for 15 minutes, discard the supernatant, collect the pellet, and wash twice; add 4.8 μL of EDC (1 mg/mL) to 90 μL of 0.2M MES buffer solution with pH 4.5-7.5, and activate for 20 minutes; then Add 5.6 μL of NHS (1 mg/mL), mix for 20 minutes; centrifuge at 15,000 rpm at 4°C for 15 minutes to collect the pellet; resuspend the microspheres with 90 μL of MES buffer solution with pH 4.5-7.5, and then add 10 μL of guinea pig source Anti-IL-1R polyclonal detection antibody (1mg/mL), vortex reaction at room temperature for 4 hours; add 10 μL of blocking solution (10% BSA), react at room temperature for 30 minutes; centrifuge at 15,000 rpm at 4°C for 15 minutes, discard the supernatant , add 100 μL of MES buffer solution with pH 4.5-7.5, and store in the dark at 4°C.

The preparation steps of the anti-IL-1β and IL-1Ra rabbit-derived polyclonal capture antibody, guinea pig-derived anti-IL-1β polyclonal detection antibody, and guinea pig-derived anti-IL-1R polyclonal detection antibody of the present invention are as follows:

(1) Download the full-length IL-1β and IL-1Ra gene and protein sequences of sheep, humans, cattle, horses, pigs, mice, and rabbits from the GenBank database of NCBI, and use DNAMAN version 6 for sequence identity comparison analysis, Determine the sequences of homologous conserved regions of IL-1β and IL-1Ra proteins and nucleic acids in sheep, humans, cattle, horses, pigs, mice, and rabbits, and analyze the differences between the homologous conserved regions of IL-1β and IL-1Ra sex;

(2) Select two sequences with large differences and low sequence identity between the homologous conserved regions of IL-1β and IL-1Ra, and name them respectively IL-1β-1 and IL-1Ra-1, and prokaryotic expression of recombinant The protein was used as the immunogen for preparing anti-IL-1β detection antibody and anti-IL-1Ra detection antibody respectively. In addition, two sequences with partial sequence consistency between IL-1β and IL-1Ra homologous conserved regions were selected and named respectively For IL-1β-2 and IL-1Ra-2, connect IL-1β-2 and IL-1Ra-2 through a DNA linker whose amino acid sequence is FFMSH, and construct a tandem gene of the homologous conserved region of IL-1β and IL-1Ra IL-1β-1Ra-2, its prokaryotic expression recombinant protein is used as an immunogen to prepare simultaneous anti-IL-1β and IL-1Ra capture antibodies;

(3) Requirements for the selection of multi-species homologous sequences for the preparation of immunogens: IL-1β-2 and IL-1Ra-2 have partial sequence identity, and IL-1β-2 and IL-1Ra-2 are tandemly fused to obtain tandem Gene IL-1β-1Ra-2, using its prokaryotic expression genetically engineered recombinant protein to immunize rabbits to prepare rabbit-derived anti-IL-1β and IL-1Ra capture antibodies, which can simultaneously bind IL-1β and IL-1Ra, IL-1β- 1 has low sequence identity with IL-1Ra, IL-1Ra-1 and IL-1β, so that the guinea pig-derived anti-IL prepared by IL-1β-1 and IL-1Ra-1 genetic engineering recombinant protein expression products respectively -1β detection antibody and guinea pig-derived anti-IL-1Ra detection antibody can specifically bind IL-1β and IL-1Ra respectively;

(4) Design a pair of upstream and downstream primers IL-1β-1-S and IL-1β-1-A for amplifying IL-1β-1, and use the cDNA recombinant plasmid containing full-length IL-1β as a PCR template to use The DNA of IL-1β-1 was amplified under conventional PCR reaction conditions, and the DNA of IL-1β-1 was purified using a DNA gel recovery kit;

(5) Design a pair of upstream and downstream primers IL-1Ra-1-S and IL-1Ra-1-A for amplifying IL-1Ra-1, and use the cDNA recombinant plasmid containing full-length IL-1Ra as a PCR template to use The DNA of IL-1Ra-1 was amplified under conventional PCR reaction conditions, and the DNA of IL-1Ra-1 was purified using a DNA gel recovery kit;

(6) Design a pair of upstream and downstream primers IL-1β-2-S and IL-1β-2-A for amplifying IL-1β-2, and use the cDNA recombinant plasmid containing full-length IL-1β as a PCR template to use The DNA of IL-1β-2 was amplified under conventional PCR reaction conditions, and the DNA of IL-1β-2 was purified using a DNA gel recovery kit;

(7) Design a pair of IL-1Ra-2-S and IL-1Ra-2-A of upstream and downstream primers for amplifying IL-1Ra-2, and use the cDNA recombinant plasmid containing full-length IL-1Ra as a PCR template to utilize The DNA of IL-1Ra-2 was amplified under conventional PCR reaction conditions, and the DNA of IL-1Ra-2 was purified using a DNA gel recovery kit;

(8) Utilize the linker sequence designed in the downstream primer IL-1β-2-A used to amplify IL-1β-2 and the upstream primer IL-1Ra-2-S used to amplify IL-1Ra-2, using conventional Overlap extension PCR method, using IL-1β-2 DNA and IL-1Ra-2 DNA as templates, IL-1β-2-S and IL-1Ra-2-A as upstream primers, IL-1β-2 Concatenated with IL-1Ra-2 gene through Linker, amplified IL-1β-1Ra-2 fusion DNA, and purified IL-1β-1Ra-2 DNA using DNA gel recovery kit;

(9) The DNA fragments of IL-1β-1 and IL-1Ra-1 obtained were respectively cloned into the pMCSG9 vector by a ligase-independent T4 DNA polymerase method, and the DNA fragments of IL-1β-1Ra-2 were Connect with pET-30a vector to construct recombinant expression plasmids pMCSG9-IL-1β-1, pMCSG9-IL-1Ra-1 and pET30a-IL-1β-1Ra-2;

(10) Transform the recombinant expression plasmid into Escherichia coli BL21-Codonplus cells to induce expression of the recombinant protein, pick the correct single colony identified by sequencing of the recombinant plasmid, and inoculate it in liquid LB medium containing 50 mg/L ampicillin resistance, at 37°C Incubate for 2 hours, then inoculate 200 mL of fresh liquid LB medium containing 50 mg/L ampicillin resistance at a ratio of 1:500, culture with shaking at 37°C for about 1 hour, add 300 μL of 1mol/mL IPTG, and continue shaking for 5.5 hours After centrifugation at 4°C and 10,000 rpm for 5 minutes, the genetically engineered E. coli pellets of pMCSG9-IL-1β-1, pMCSG9-IL-1Ra-1, and pET30a-IL-1β-1Ra-2 were harvested respectively, and resuspended in PBS , lyse with an ultrasonic breaker, the power is 40W, ultrasonic 10s, intermittent 10s, a total of 30min, at 12000 rpm, centrifuged for 30min, to obtain supernatant and inclusion body precipitation;

(11) The recombinant protein expressed in the Escherichia coli system is fused with the His tag, and the supernatant of the broken bacterial body of the genetically engineered expression bacteria is purified using a nickel column according to the natural sedimentation method of the conventional nickel ion affinity chromatography to purify the target antigen protein IL-1β-1 , IL-1β-2 and IL-1β-1Ra-2, use SDS-PAGE and Western-blot analysis to verify the purified recombinant protein, dialyze with a dialysis bag with a molecular weight cut-off of 8000-14000Da at 4°C, and the dialysate is 0.01M pH 8.0 PBS, change the dialysate every 2 hours, a total of 5 times, use PEG20000 for reverse dialysis concentration, SDS-PAGE to identify the purity, and put it in a -80°C refrigerator for later use;

(12) Animal immunity

Take 1 mg of IL-1β-1, IL-1Ra-1 and IL-1β-1Ra-2 protein for the first immunization, mix and emulsify with equal volume of complete adjuvant, and emulsify IL-1β-1 and IL-1β-1 respectively -1Ra-1 protein immunized guinea pigs to prepare guinea pig-derived anti-IL-1β detection antibody and guinea pig-derived anti-IL-1Ra detection antibody, and immunized New Zealand white rabbits with emulsified IL-1β-1Ra-2 protein to prepare rabbit-derived anti-IL-1 1β and IL-1Ra capture antibodies were used for subcutaneous multi-point immunization on the back, 15 days as an immunization cycle, the second immunization began to use incomplete adjuvant to prepare emulsified immunogen for immunization, the method was the same as above, after the third immunization, Serum titer was measured every 7 days, and the titer reached more than 50,000, and heart blood was collected to collect serum to be tested for titer and purified antibody;

(13) Antibody titer detection and purification

Take a small amount of serum before immunization as negative blood, collect blood from rabbits and guinea pigs after immunization, incubate at 37°C for 0.5h, clot at 4°C for 2h, centrifuge at 3000 rpm for 5min, draw serum, and use conventional ELISA method to detect titer , using the AKATA protein purification system and conventional protein G affinity chromatography to purify the antibody;

(14) Removal of interfering antibodies

By interfering with the antibody removal experiment, the non-specific binding of the two kinds of guinea pig-derived antibodies for detection can be reduced. The specific steps are as follows: routinely induce the E. coli expression bacteria transformed into the pMCSG9 empty vector, take 1mL-5mL bacterial culture fermentation broth, 1300 rpm , Centrifuge for 1 min, discard the supernatant to collect the bacteria, take two kinds of guinea pig-derived antibodies for detection of 100 μL suspension bacteria, incubate at 37 ° C for 45-90 min, centrifuge at 3000 rpm for 5 min, and take the supernatant to remove the detection of interfering antibodies Antibody.

In the present invention:

The nucleotide sequence of IL-1β-1 is as described in SEQ ID No.1;

The amino acid sequence of IL-1β-1 is as described in SEQ ID No.2;

The nucleotide sequence of IL-1β-2 is as described in SEQ ID No.3;

The amino acid sequence of IL-1β-2 is as described in SEQ ID No.4;

The nucleotide sequence of IL-1Ra-1 is as described in SEQ ID No.5;

The amino acid sequence of IL-1Ra-1 is as described in SEQ ID No.6;

The nucleotide sequence of IL-1Ra-2 is as described in SEQ ID No.7;

The amino acid sequence of IL-1Ra-2 is as described in SEQ ID No.8;

The nucleotide sequence of IL-1β-1Ra-2 is as described in SEQ ID No.9;

The amino acid sequence of IL-1β-1Ra-2 is described in SEQ ID No.10.

In the present invention:

(1) IL-1β-1-S:5 ^/ -tacttccaatccaatgcCTATACCTGTCTTGTGTGACCCA-3 ^/ ;

IL-1β-1-A:5 ^/ -ttatccacttccaatgtcAGAAGACGAATCGCTTTTCC-3 ^/ ;

(2) IL-1Ra-1-S:5 ^/ -tacttccaatccaatgcCAAGCCTTCAGGATCTGGG-3 ^/ ;

IL-1Ra-1-A:5 ^/ -ttatccacttccaatgtcAACCTTGCAAGTATCCAGC-3 ^/ ;

(3) IL-1β-2-S:5 ^/ -ccgcatatgTACAAGACAGAAATCAAGAACACAGT-3 ^/ ;

IL-1β-2-A:5 ^/ -atgcatgctgaagaaTATATCCTGGCCACCTCTAAAAC-3 ^/ ;

(4) IL-1Ra-2-S:5 ^/ -ttcttcagcatgcatGACAAGCGCTTCGCCTTCATC-3 ^/ ;

IL-1Ra-2-A:5 ^/ -ccgctcgagATTGGTGAGGCCCACGGG-3 ^/ ;

(5) The linker sequence ttcttcagcatgcat of the tandem gene IL-1β-1Ra-2 in the homologous conserved region of IL-1β and IL-1Ra.

The IL-1β recombinant protein standard product and IL-1Ra recombinant protein standard product described in the present invention are respectively IL-1β recombinant protein and IL-1Ra recombinant protein expressed by Escherichia coli expression system genetic engineering, and the specific preparation method is conventional Prokaryotic expression technology, the open reading frames of IL-1β and IL-1Ra were inserted into the prokaryotic expression vector pET-30a, respectively, and the recombinant expression vectors pET-30a-IL-1β and pET-30a-IL-1Ra fused with the His tag were constructed. Conventional induced expression purification technology to obtain high-purity genetically engineered IL-1β recombinant protein and IL-1Ra recombinant protein, and prepare standard protein.

The diluent in the present invention is a 0.1M PBS solution with a pH of 7.2-7.6.

An operation method of an ELISA kit for simultaneous determination of multi-species IL-1β and IL-1Ra and their ratios, the steps are as follows:

(1) According to the number of samples to be tested, take out the black flat-bottomed microtiter plate coated with anti-IL-1β and IL-1Ra capture antibodies, set standard wells, positive wells, negative wells, blank wells and sample wells, each well Add 100 μL of liquid each time. The standard wells were filled with two standard mixed solutions diluted with diluent, the final concentrations of the two standard protein IL-1β and IL-1Ra were 78.125ng/ml, 39.625ng/ml, 19.531ng/ml, 9.766 ng/ml, 4.883ng/ml, 2.441ng/ml, 1.221ng/ml, 0.610ng/ml, 0.305ng/ml, 0.153ng/ml, 0.076ng/ml and 0.038ng/ml 12 concentration gradients, sample well Add 2 times diluted serum to be tested, add PBS diluent to negative wells, add no liquid to positive wells and blank wells, incubate at 37°C for 1 hour, add 0.1% PBST to wash the plate 3 times, 1 minute each time;

(2) Add 0.125 μg/mL detection antibody to the sample well, standard well and negative well, 100 μL per well, incubate at 37°C for 1 hour, add 0.1% PBST to wash the plate 3 times, 1 minute each time;

(3) Do not add any liquid to the blank well, add 100 μL of the fluorescent microsphere mixture of the two kinds of fluorescent microspheres with a concentration of 0.125 μg/mL to the positive well, add 100 μL of diluent to the standard well, negative well, and sample well,

(4) Use a fluorescence microplate detector to perform dual fluorescence detection readings, detect the IL-1β concentration at an excitation wavelength of 360nm and an emission wavelength of 450nm, and detect the concentration of IL-1Ra at an excitation wavelength of 480nm and an emission wavelength of 520nm;

(5) Take the fluorescence value B of the blank well as the instrument blank zeroing value, deduct the signal noise interference of the instrument, set the fluorescence value of the standard well as ST, the fluorescence value of the sample well as SA, the fluorescence value of the negative well as N, and the fluorescence value of the positive well is P, corrected with the fluorescence value P of the positive well as the denominator, and calculated the corrected fluorescence value ST _a of the standard well and the corrected fluorescence value SA _a of the sample well, that is, ST _a =ST/P or SA _a =SA/P, and plotted IL- 1β and IL-1Ra standard curve, take the logarithm of the standard IL-β and IL-1Ra protein concentration as the abscissa, take the calibration fluorescence value ST _a of the standard well as the ordinate, draw the standard curve, and calculate the standard curve equation and correlation coefficient R ² , substituted the calibration fluorescence value SA _a of the sample hole into the standard curve equation, calculated the concentration logarithm X, 10 ^X was the concentration of IL-1β and IL-1Ra in the sample, and calculated the concentration ratio of IL-1Ra/IL-1β;

(6) Positive wells and negative wells are set up to control the effectiveness of fluorescence detection and make multiple test results comparable.

The antibody of the present invention can specifically bind and react with IL-1β and IL-1Ra of sheep, humans, cattle, horses, pigs, mice, rabbits, dogs, chickens, and ducks, and is used to detect sheep, humans, The content of IL-1β and IL-1Ra in the serum, plasma and tissue and organ samples of cattle, horses, pigs, mice, rabbits, dogs, chickens and ducks, and the ratio of IL-1Ra/IL-1β.

The ratio IL-1Ra/IL-1β of IL-1Ra and IL-1β is measured synchronously by the double-antibody sandwich double-fluorescent ELISA of the present invention, and it is confirmed by the detection and analysis of clinical sheep blood samples that the blood serum of sheep with brucellosis The difference of IL-1Ra/IL-1β and serum IL-1Ra/IL-1β between brucellosis vaccine immunized sheep and healthy sheep was extremely significant. Moreover, it was confirmed by statistical ROC analysis that IL-1Ra/IL-1β in sheep serum has differential diagnosis significance in distinguishing brucellosis infection and immune clinical testing. The invention can be used for the differential diagnosis of Brucellosis infection and immunity, and can also be used for the diagnosis of other IL-1Ra/IL-1β-related diseases and the research on the marking function of occurrence, development and outcome.

The beneficial effects of the present invention are: the present invention provides a double-antibody sandwich fluorescence ELISA detection method for synchronous determination of multi-species IL-1β and IL-1Ra and their ratios and its application in the differential diagnosis of brucellosis infection and immunity Method, the established fluorescent ELISA detection method can accurately obtain IL-1β and IL-1Ra and its IL-1Ra/IL-1β ratio through one experiment at the same time. It has the advantages of small size, accurate measurement results, and a wide range of detection species. It can be used to determine the content of IL-1β and IL-1Ra in serum, plasma and other tissue samples of sheep, human, cow, horse, pig, mouse, rabbit and other species. Synchronously detect and calculate the ratio of the two targets, reduce the error in the calculation of the IL-1Ra/IL-1β ratio, and the sensitivity reaches the picogram (pg) level, which solves the problem that there is currently no accurate method for calculating the IL-1Ra/IL-1β ratio. At the same time, the detection method of the present invention has wide species versatility, expands the application range of the method, and can more accurately compare and study the participation of IL-1β and IL-1Ra in different species in physiological and pathological functions of the body. The method for determining the ratio of IL-1Ra/IL-1β can be used for the differential diagnosis of brucellosis natural infection and vaccine immunity, as well as other diseases related to the change of the ratio. Solve the problem that there is currently no reliable differential diagnosis method for distinguishing brucellosis infection and immunity, resulting in a large number of brucellosis immune animals being culled as brucellosis sick animals due to positive serum brucellosis antibody reaction, bringing huge economic benefits loss. The invention can effectively assist in the grouping of sick and healthy brucellosis, reduce unnecessary economic losses in animal husbandry, provide valuable detection and diagnosis tools for animal brucellosis prevention and control, and provide scientific purification of brucellosis Animals provide applicable methods.

Description of drawings

Figure 1 is a sequence comparison analysis of the homologous conserved domains of IL-1β and IL-1Ra in multiple species, in which:

A: Sequence alignment analysis of IL-1β-1 homologous conserved protein domain; B: Sequence alignment analysis of IL-1Ra-1 homologous conserved protein domain; C: Structure of IL-1β-2 homologous conserved protein Domain sequence alignment analysis diagram; D: IL-1Ra-2 homologous conserved protein domain sequence alignment analysis diagram;

Fig. 2 is the electrophoresis diagram of the tandem PCR amplification products of IL-1β-2 and IL-1Ra-2 genes;

Fig. 3 is the SDS-PAGE electrophoresis analysis diagram of IL-1β-1, IL-1Ra-1 and IL-1β-1Ra-2 recombinant protein expression and purification, wherein:

M: Marker; 1: IL-1β-1 induced expression; 2: IL-1Ra-1 induced expression; 3: IL-1β-1 uninduced control; 4: IL-1Ra-1 uninduced control; 5: IL- 1β-1Ra-2 uninduced control; 6: IL-1β-1Ra-2 induced expression; 7: pMCSG9 empty vector uninduced control; 8: pMCSG9 empty vector induced control;

Figure 4 is an SDS-PAGE analysis chart of the purification of antibodies for anti-IL-1β detection, anti-IL-1Ra detection antibodies, and simultaneous anti-IL-1β and IL-1Ra capture antibodies, wherein:

M, Marker; 1. Anti-IL-1β and IL-1Ra capture antibody; 2. Anti-IL-1β detection antibody; 3. Anti-IL-1Ra detection antibody;

Figure 5 is a Western blot analysis diagram of the specific binding of the capture antibody and the detection antibody to the genetically engineered expression proteins IL-1β and IL-1Ra, wherein:

A: Anti-IL-1β and IL-1Ra capture antibody analysis diagram; B: Anti-IL-1β detection antibody analysis diagram; C: Anti-IL-1Ra detection antibody analysis diagram; 1: Genetic engineering expression protein IL-1β; 2: Genetic engineering expression protein IL-1Ra;

Figure 6 is a Western blot analysis diagram of the specific binding of the capture antibody and the detection antibody to the multi-species natural IL-1β and IL-1Ra proteins, wherein:

A: Anti-IL-1β detection antibody and multi-species natural IL-1β protein binding diagram; B: Anti-IL-1Ra detection antibody and multi-species natural IL-1Ra protein specific binding diagram; C: Anti-IL-1β -1 and IL-1Ra capture antibody specific binding to multi-species natural IL-1β and IL-1Ra proteins;

Figure 7 is an ultraviolet scanning analysis diagram of antibody-coupled fluorescent microspheres for detection, wherein:

A: Anti-IL-1β antibody-coupled blue fluorescent microspheres; B: Anti-IL-1Ra antibody-coupled green fluorescent microspheres;

Fig. 8 is to determine the optimal antibody pair working concentration analysis diagram;

A: The relationship between the concentration of the capture antibody and the detection fluorescence value; B: The relationship between the concentration of the detection antibody and the fluorescence value;

Fig. 9 is a standard curve diagram of IL-1β and IL-1Ra;

Fig. 10 is to detect the fluorescence density attenuation analysis figure;

Fig. 11 is the clinical actual serum sample detection analysis diagram of the brucellosis vaccine immunization sheep group, the brucellosis vaccine non-immunization sheep negative group and the infection brucellosis sheep positive group, wherein:

A: Determination of IL-1β concentration in goat serum samples; B: Determination of IL-1Ra concentration in goat serum samples; C: Determination of IL-1Ra/IL-1β ratio in goat serum samples;

Fig. 12 is the statistical ROC curve analysis chart of the differential diagnostic significance of the IL-1Ra/IL-1β ratio in the brucellosis infection group, the vaccine immunization group, and the negative control group in the sheep serum sample, wherein:

A: Non-immunized brucellosis vaccine negative group and brucellosis vaccine immunized group; B: Non-immunized brucellosis vaccine negative group and brucellosis positive group; C: brucellosis vaccine immunized group and brucellosis positive group.

Detailed ways

Example 1 Selection of IL-1β and IL-1Ra Homologous Conserved Sequence

Search and download the IL-1β and IL-1Ra gene and protein sequences of sheep, human, cattle, horse, pig, mouse, and rabbit in the GenBank database of NCBI, and use DNAMAN version 6 for sequence identity comparison analysis to obtain sheep, human IL-1β and IL-1Ra proteins and nucleic acid multi-species homologous conserved region sequences of cattle, horses, pigs, mice and rabbits, and analyze the differences between IL-1β and IL-1Ra conserved regions;

Select homologous conserved sequences with large differences between IL-1β and IL-1Ra, named as IL-1β-1 and IL-1Ra-1, respectively, for the preparation of immunogens for detection antibodies; select the other two paragraphs with partial The similar IL-1β and IL-1Ra are named as IL-1β-2 and IL-1Ra-2 respectively, and the linker (amino acid sequence is FFSMH) with the sequence ttcttcagcatgcat is used to connect the two to construct a fusion tandem gene, Named as IL-1β-1Ra-2, it was used to prepare the immunogen for the capture antibody.

Requirements for the selection of multi-species homologous conserved sequences for the preparation of immunogens: except IL-1β-2 and IL-1Ra-2 have partial sequence identity, other IL-1β-1, IL-1β-2, IL-1Ra -1. The sequences of IL-1Ra-2 are not similar between each other. See Figure 1 for the selection results of conservative sequence analysis by homologous comparison.

Preparation of embodiment 2 recombinant protein

(1) Design and synthesis of primers for amplifying IL-1β-1, IL-1β-2, IL-1Ra-1, IL-1Ra-2 DNA sequences are shown in Table 1:

Table 1: Primers designed and synthesized by the present invention

Primer name Primer sequence IL-1β-1-S 5^/-tacttccaatccaatgcCTATACCTGTCTTGTGTGACCCA-3^/ IL-1β-1-A 5^/-ttatccacttccaatgtcAGAAGACGAATCGCTTTTCC-3^/ IL-1Ra-1-S 5^/-tacttccaatccaatgcCAAGCCTTCAGGATCTGGG-3^/ IL-1Ra-1-A 5^/-ttatccacttccaatgtcAACCTTGCAAGTATCCAGC-3^/ IL-1β-2-S 5^/-ccgcatatgTACAAGACAGAAATCAAGAACACAGT-3^/ IL-1β-2-A 5^/-atgcatgctgaagaaTATATCCTGGCCACCTCTAAAAC-3^/ IL-1Ra-2-S 5^/-ttcttcagcatgcatGACAAGCGCTTCGCCTTCATC-3^/ IL-1Ra-2-A 5^/-ccgctcgagATTGGTGAGGCCCACGGG-3^/

(2) Amplify the target DNA sequences of IL-1β-1, IL-1Ra-1, IL-1β-2, and IL-1Ra-2 using conventional PCR methods. The PCR system is as follows: Es Taq: 25 μl; upstream primer: 1 μl ; Downstream primer: 1 μl; IL-1β or IL-1Ra full-length plasmid template: 2 μl; ddH ₂ O: 21 μl; Total: 50 μl; ℃30s, 62℃30s, 72℃90s, a total of 32 cycles; 72℃10min; IL-1β-2 and IL-1Ra-2 reaction conditions are as follows: 94℃3min; 94℃30s, 60℃30s, 72℃90s , a total of 32 cycles; 72 ℃ 10min;

(3) IL-1β-2 and IL-1Ra-2 gene in tandem: IL-1β-2 downstream primer IL-1β-2-A and IL-1Ra-2 upstream primer IL-1Ra-2-S contain a linker The sequence ttcttcagcatgcat, therefore, was amplified by PCR using IL-1β-2-S as the upstream primer, IL-1Ra-2-A as the downstream primer, and IL-1β-2 and IL-1Ra-2 gel recovery products as templates The amplification conditions are as follows: 94°C for 3min; 94°C for 30s, 62°C for 30s, 72°C for 90s, a total of 32 cycles; 72°C for 10min; the PCR system is as follows: EsTaq: 25μl; IL-1β-2-S: 1μl; IL -1Ra-2-A: 1 μl; PCR product of IL-1β-2: 1 μl; PCR product of IL-1Ra-2: 1 μl; ddH ₂ O: 21 μl; Total: 50 μl. figure 2;

(4) Treat the pMCSG9 vector with SSPⅠ enzyme digestion, the system is 4 μl of SSPⅠ enzyme, 32 μl of pMCSG9 plasmid, 4 μl of 10×Buffer, overnight at 37°C, without gel recovery;

(5) Ligate the SSPⅠdigested product of pMCSG9 with the recovered product of IL-1β-1 and IL-1Ra-1 respectively, construct the reaction system according to Table 2, mix well, keep it at 70°C for 5min, and transfer it to a constant temperature bath at 37°C , add 1 μl T4 DNA pol polymerase, the final volume is 10 μl, mix gently and incubate at 37°C for 5 minutes, mix 2 μl of IL-1β-1 or IL-1Ra-1 reaction system with 2 μl of pMCSG9 reaction system, and then incubate at 22 After reacting at ℃ for 15 minutes, add 2 μl of 25mM EDTA, and react at 22℃ for 10 minutes, successfully constructing recombinant expression plasmids pMCSG9-IL-1β-1 and pMCSG9-IL-1Ra-1;

Table 2; Reaction system

(6) The pET-30a plasmid and the recovered product of IL-1β-1Ra-2 gel were subjected to double enzyme digestion with restriction endonucleases NdeI and XhoI respectively, and the products after digestion were ligated to successfully construct recombinant expression Plasmid pET30a-IL-1β-1Ra-2;

(7) Transform the recombinant expression plasmid into E.coli BL21(DE3) expression bacteria, induce expression with IPTG at a final concentration of 0.4mM according to conventional methods, purify by His tag-nickel column affinity chromatography, and obtain prokaryotic expression gene engineering Recombinant proteins IL-1β-1, IL-1Ra-1 and IL-1β-1Ra-2, see Figure 3; specific steps are as follows:

Transform the recombinant expression plasmid into Escherichia coli BL21-Codonplus cells to induce the expression of the recombinant protein, pick the correct single colony identified by sequencing of the recombinant plasmid, inoculate it in liquid LB medium containing 50 mg/L ampicillin resistance, and incubate at 37°C for 2 hours , and then inoculated in 200 mL of fresh liquid LB medium containing 50 mg/L ampicillin resistance at a ratio of 1:500, cultured with shaking at 37°C for about 1 hour, added 300 μL of 1 mol/mL IPTG, continued shaking culture for 5.5 hours, and then cultured for 4 hours. Centrifuge at 10000rpm for 5min, harvest pMCSG9-IL-1β-1, pMCSG9-IL-1Ra-1, and pET30a-IL-1β-1Ra-2 genetically engineered Escherichia coli pellets, resuspend in PBS, and Crack with a breaker with a power of 40W, ultrasonic for 10s, intermittent for 10s, for a total of 30min, centrifuge at 12000 rpm for 30min to obtain supernatant and inclusion body precipitation;

The recombinant protein expressed in the Escherichia coli system was fused with a His tag, and the supernatant of the broken bacterial body of the genetically engineered expression bacteria was purified using a nickel column according to the natural sedimentation method of conventional nickel ion affinity chromatography to purify the target antigen protein IL-1β-1, IL- 1β-2 and IL-1β-1Ra-2, use SDS-PAGE and Western-blot analysis to verify the purified recombinant protein, dialyze with a dialysis bag with a molecular weight cut-off of 8000-14000Da at 4°C, and the dialysate is 0.01M PBS with pH 8.0 , the dialysate was changed every 2 hours, a total of 5 times, reverse dialysis concentration was performed with PEG20000, the purity was identified by SDS-PAGE, and the aliquots were placed in a -80°C refrigerator for later use.

Example 3 Preparation of anti-IL-1β detection antibody, anti-IL-1Ra detection antibody, and simultaneous anti-IL-1β and IL-1Ra capture antibody

(1) For the first immunization, take 1 mg of IL-1β-1, IL-1Ra-1 and IL-1β-1Ra-2 proteins and mix them with an equal volume of Freund’s complete adjuvant and fully emulsify them. Spot immunization method, immunize guinea pigs with IL-1β-1 and IL-1Ra-1, and immunize New Zealand white rabbits with IL-1β-1Ra-2. One immunization cycle is 15 days, and Freund’s incomplete adjuvant is used for the second immunization Mix with the immunogen to prepare emulsified immunogen, immunize according to the above method, after the third immunization, measure the serum titer every 7 days, after the titer reaches 50,000, collect blood from the heart, and collect serum;

(2) Antibody titer test: take a small amount of serum before immunization as negative blood, take a small amount of blood from rabbits and guinea pigs after immunization, incubate at 37°C for 0.5h, coagulate at 4°C for 2h, and then centrifuge at 3000 rpm for 5min. Aspirate the serum and use the conventional ELISA method to detect the titer. When the ELISA detects that the antibody titer reaches 50,000, use the AKATA protein purification system and conventional protein G affinity chromatography to purify the antibody, as shown in Figure 4;

(3) Removal of interfering antibodies: Induce E.coli BL21(DE3) expressing bacteria transformed into the pMCSG9 empty vector, take 1mL-5mL bacterial culture fermentation broth, centrifuge at 1300 rpm for 1min, discard the supernatant to obtain the bacteria; Mix 100 μL of the two guinea pig antibodies with the above-mentioned different amounts of bacteria, and incubate at 37°C for 45min-90min respectively; after the incubation is completed, centrifuge at 3000 rpm for 5min, and take the supernatant as the detection antibody for removing interfering antibodies.

Example 4 Identification and Analysis of Capture Antibody and Detection Antibody

(1) Using the conventional Western Blot method to hybridize and analyze the specificity and cross-reactivity of rabbit-derived capture antibodies and guinea-pig-derived detection antibodies to their target genetically engineered recombinant proteins IL-1β and IL-1Ra, see Figure 5;

(2) Using the conventional Western Blot method to hybridize and analyze the specificity and cross-reactivity of rabbit-derived capture antibodies and guinea pig-derived detection antibodies to their target multi-species natural proteins IL-1β and IL-1Ra, natural protein extraction: using conventional RIPA The tissue protein was extracted from the lysate, and the 0.1M PMSF isopropanol stock solution was diluted 100 times with a conventional RIPA lysate to prepare a complete RIPA lysate. The specific extraction steps are as follows: Cut an appropriate amount of tissue pieces into a 2mL flat-bottomed grinding tube, add 8mL of complete RIPA lysate per gram of tissue, add an appropriate volume of complete RIPA lysate, fully grind and homogenate for 10min, 4°C, 13000 rpm Centrifuge for 20 minutes, transfer the supernatant to a 1.5mL centrifuge tube, measure the total protein content, and analyze the binding characteristics of the capture antibody and detection antibody to the multi-species natural proteins IL-1β and IL-1Ra by conventional Western blot hybridization method, see Figure 6 .

The preparation of embodiment 5IL-1β standard substance and IL-1Ra standard substance

The genetically engineered IL-1β recombinant protein and IL-1Ra recombinant protein were respectively prepared by the E. coli expression system according to the conventional prokaryotic expression technology as the standard protein of the present invention. The specific preparation method is to insert the open reading frames of IL-1β and IL-1Ra into the prokaryotic expression vector pET-30a respectively, and construct the recombinant expression vectors pET-30a-IL-1β and pET-30a-IL-1Ra fused with the His tag. Conventional induced expression and purification technology to obtain high-purity genetically engineered IL-1β recombinant protein and IL-1Ra recombinant protein to make standard protein.

Example 6 Detection of antibody-coupled fluorescent microspheres

The two guinea pig-derived polyclonal detection antibodies were coupled to fluorescent microspheres of different colors, among which the anti-IL-1β detection antibody was coupled to blue fluorescent microspheres (excitation wavelength 360nm, emission wavelength 450nm), anti-IL-1Ra The detection antibody is coupled to green fluorescent microspheres (excitation wavelength 480nm, emission wavelength 520nm), and the detection antibody with fluorescent microspheres is prepared, which can be used for direct detection of IL-1β and IL-1Ra; the specific coupling steps are as follows: Add 90 mL of MES buffer solution with pH 4.5-7.5 to 10 μL of microspheres, mix well, centrifuge at 15,000 rpm for 15 minutes, discard the supernatant, collect the precipitate of microspheres, and repeat washing twice; Add 4.8μL EDC (1mg/mL) to the solution and activate for 20 minutes; then add 5.6μL NHS (1mg/mL) and mix for 20 minutes; Resuspend the microspheres in 4.5-7.5 MES buffer solution, then add 10 μL antibody (1 mg/mL), vortex and shake at room temperature for 4 hours; add 10 μL blocking solution (10% BSA), and react at room temperature for 30 minutes; 4 °C 15000 rpm Centrifuge for 15 minutes, discard the supernatant, add 100 μL of MES buffer solution with pH 4.5-7.5, store in the dark at 4°C, and analyze the conjugated fluorescent microsphere antibody with full-wavelength ultraviolet scanning, as shown in Figure 7.

Embodiment 7 checkerboard method determines optimal antibody to working concentration

Coat the simultaneous anti-IL-1β and IL-1Ra rabbit-derived antibody on a 96-well black microwell plate, add IL-1β and IL-1Ra mixed protein standards, and then add the fluorescence of IL-1β and IL-1Ra The detection antibody is used to detect the content of IL-1β and IL-1Ra in the mixed protein, and finally use a fluorescence microplate analyzer to detect its fluorescence value. The specific operation steps are as follows:

(1) Coating capture antibody: use 0.1M PBS with pH 7.2-7.6 as the diluent to coat the capture antibody, and dilute the capture antibody to a final concentration of 1.248 μg/mL, 0.624 μg/mL mL, 0.312μg/mL, 0.156μg/mL, 0.078μg/mL, 0.039μg/mL, added to the microtiter plate, 100μL/well, each column is the same concentration of capture antibody, coated at 4°C overnight;

(2) Use PBS containing 0.2% Tween-20 as the PBS-T washing solution, wash the plate 3-4 times, 200 μL/well, 1 min each time, and then block with 0.1M ammonium chloride solution, 200 μL/well, 1h at 37°C, drain the liquid;

(3) Dilute IL-1β and IL-1Ra mixed standard protein with diluent until the final concentration of IL-1β and IL-1Ra is 1 μg/mL, add it to the microtiter plate, react for 1 hour, replace the standard with PBS diluent The product was used as a negative control, and a negative control was set up for each antibody pair, and a blank well without any reagent was used as a blank control;

(4) Wash the plate 3-4 times with PBS-T, and make up the mixed fluorescent detection antibody by IL-1β-1 detection antibody and IL-1Ra-1 detection antibody, and dilute the mixed fluorescent detection antibody with diluent Until the final concentrations of the two detection antibodies are 2 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.25 μg/mL, 0.125 μg/mL, 0.0625 μg/mL, react at 37 ° C for 1 h;

(5) Wash the plate 3-4 times with PBS-T, dilute the naked fluorescent microspheres without conjugated antibody to the same multiple, set up a group of positive control groups separately, and use a fluorescent microplate analyzer to separate the fluorescent microspheres at excitation wavelengths of 360nm, The emission wavelength is 450nm, the excitation wavelength is 480nm, and the emission wavelength is 520nm to measure the fluorescence density value.

(6) Set the fluorescence value of the standard well as S, the fluorescence value of the blank well as B, and the fluorescence value of the negative well as N, and determine the optimal antibody pair concentration used in this method according to the strength and change trend of the fluorescence value corresponding to each antibody concentration , the optimal working concentration of the capture antibody for coating is 0.078 μg/mL, and the optimal working concentration of the detection antibody is 0.125 μg/mL, see Figure 8.

Example 8 Determine the blocking time, antigen action time, and detection antibody action time

The action time of 0.1M ammonium chloride blocking solution is set to four time gradients of 0.5h, 1h, 1.5h, and 2h; the action time of antigen is set to five time gradients of 45min, 60min, 75min, 90min, and 120min; Five time gradients of 45min, 60min, 75min and 90min, the ELISA detection reaction was completed with the optimal antibody pair concentration and final concentration of 1 μg/mL IL-1β and IL-1R mixed standard protein, and the fluorescence values were measured respectively to determine the optimal operating hours. The results show that the blocking effect is not complete before the blocking solution is applied for 1 hour, and the fluorescence value is stable after 1 hour, and the blocking effect is basically the same. ; The fluorescence value of the detection antibody is relatively stable before 1h, and the fluorescence value begins to decrease after 1h. The best reaction time of the detection antibody is 1h.

Example 9 Construction of Standard Curve for Synchronous Determination of IL-1β and IL-1Ra by Double Antibody Sandwich Double Fluorescent ELISA Detection Method

Dilute the IL-β and IL-1Ra standard mixture with 0.1M PBS diluent of pH 7.2-7.6 to the final concentrations of the two standards are 1250ng/mL, 625ng/mL, 312.5ng/mL, 156.25ng/mL , 78.125ng/mL, 39.625ng/mL, 19.531ng/mL, 9.766ng/mL, 4.883ng/mL, 2.441ng/mL, 1.221ng/mL, 0.610ng/mL, 0.305ng/mL, 0.153ng/mL . For the control, the blank well without any reagent was used as the blank control, and only 100 μL of the fluorescent microsphere mixture with the concentration of the two fluorescent microspheres at 0.125 μg/mL was added as the positive control; the fluorescence value B of the blank well was used as the instrument blank Adjust the zero value to deduct the signal and noise interference of the instrument. Set the fluorescence value of the standard well as ST, the fluorescence value of the negative well as N, and the fluorescence value of the positive well as P; use the fluorescence value of the positive well P as the denominator to correct, and calculate the corrected fluorescence value ST _a of the standard well, that is, ST _a = ST/ p. Take the logarithm of the protein concentration of the standard IL-β and IL-1Ra as the abscissa, and take the standard well-corrected fluorescence value ST _a as the ordinate, draw the standard curves of IL-1β and IL-1Ra respectively, and calculate the standard curve equation and correlation coefficient ^R2 . Finally, it was determined that the linear range of IL-1β and IL-1Ra was 76pg/mL-625ng/mL, the standard curve equation of IL-1β was y=0.0076x-0.0058, and R2 was ^0.9937 ; the standard curve equation of IL-1Ra was y=0.0027x+0.0251, R ² is 0.9904. Substitute the average fluorescence value of 8 negative wells into the standard curve equation, and calculate the corresponding concentration value as the minimum detection limit of this method. The minimum detection limit of IL-1β and IL-1Ra is both 11pg/mL, as shown in Figure 9.

Embodiment 10 recovery rate experiment

Mix IL-1β standard substance and IL-1Ra standard substance according to the ratio of 1:3, 1:2, 1:1, 2:1, 3:1 respectively, detect the fluorescence value, and calculate the corresponding protein concentration according to the standard curve equation , divided by the known standard protein concentration to calculate the recovery rate, the recovery rate of IL-1β ranged from 80.89% to 101.13.52%, and the average recovery rate was 87.842%; the recovery rate of IL-1Ra ranged from 80.07% to 114.18%, and the average recovery rate The rate is 93.93%, see Table 3;

Table 3: Recovery analysis results

Embodiment 11 Fluorescent signal decay period analysis

Dilute IL-1β and IL-1Ra standard protein with PBS diluent to the final concentration of the two proteins are 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 10ng/mL, 5ng/mL, to establish The double-antibody sandwich double-fluorescence ELISA method was used for detection, and the fluorescence value was measured after adding the fluorescence detection antibody for 0h, 0.5h, 1h, and 2h respectively. The results show that within 0.5 hours of adding the fluorescent antibody, the fluorescence density value has no significant change; within 2 hours of adding the fluorescent antibody, the target can be effectively detected; it is recommended to complete the fluorescence detection within 0.5 hours of adding the fluorescent antibody, see Figure 10.

Example 12 Operation steps of a double-antibody sandwich double-fluorescent ELISA detection method for simultaneous determination of multi-species IL-1β and IL-1Ra and their ratios

(1) According to the number of samples to be tested, take out the black flat-bottomed microtiter plate coated with anti-IL-1β and IL-1Ra capture antibodies, set standard wells, positive wells, negative wells, blank wells and sample wells, each well Add 100 μL of liquid each time. The standard wells were filled with two standard mixed solutions diluted with diluent, the final concentrations of the two standard protein IL-1β and IL-1Ra were 78.125ng/ml, 39.625ng/ml, 19.531ng/ml, 9.766 ng/ml, 4.883ng/ml, 2.441ng/ml, 1.221ng/ml, 0.610ng/ml, 0.305ng/ml, 0.153ng/ml, 0.076ng/ml and 0.038ng/ml 12 concentration gradients, sample well Add 2 times diluted serum to be tested, add PBS diluent to negative wells, add no liquid to positive wells and blank wells, incubate at 37°C for 1 hour, add 0.1% PBS-T to wash the plate 3 times, 1 minute each time;

(2) Add the two detection antibodies at a concentration of 0.125 μg/mL to the sample well, standard well and negative well, 100 μL per well, incubate at 37°C for 1 hour, add 0.1% PBS-T to wash the plate 3 times, each time 1 minute each time;

(3) Do not add any liquid to the blank well, add 100 μL of the fluorescent microsphere mixture with the concentration of the two fluorescent microspheres at 0.125 μg/mL to the positive well, and add 100 μL of PBS diluent to the standard well, negative well, and sample well;

(4) Use a fluorescence microplate analyzer to perform dual fluorescence detection readings. The concentration of IL-1β was detected at the excitation wavelength of 360nm and the emission wavelength of 450nm, and the concentration of IL-1Ra was detected at the excitation wavelength of 480nm and the emission wavelength of 520nm;

(5) The fluorescence value B of the blank well was used as the zero value of the instrument blank, and the signal-to-noise interference of the instrument was deducted. Set the fluorescence value of the standard well as ST, the fluorescence value of the sample well as SA, the fluorescence value of the negative well as N, and the fluorescence value of the positive well as P, and use the fluorescence value of the positive well as P as the denominator to correct, and calculate the corrected fluorescence value of the standard well as ST _a Correct the fluorescence value SA _a with the sample well, that is, ST _a =ST/P or SA _a =SA/P, draw the IL-1β and IL-1Ra standard curves respectively, and use the logarithm of the standard IL-β and IL-1Ra protein concentration As the abscissa, use the calibration fluorescence value ST _a of the standard well as the ordinate, draw the standard curve, calculate the standard curve equation and correlation coefficient R ² , substitute the calibration fluorescence value SA _a of the sample hole into the standard curve equation, and calculate the concentration logarithm X, 10 ^X is the concentration of IL-1β and IL-1Ra in the sample, and calculate the concentration ratio of IL-1Ra/IL-1β;

(6) Positive wells and negative wells are set up to control the effectiveness of fluorescence detection and make multiple test results comparable.

Example 13 Application of a double-antibody sandwich double-fluorescent ELISA detection method for simultaneous determination of multi-species IL-1β and IL-1Ra and their ratio in the differential diagnosis of brucellosis infection and immunity

(1) 904 goat serum and plasma samples were collected and analyzed by tiger red plate agglutination test for brucellosis. 196 sheep serum samples from the brucellosis-negative healthy group that were not immunized with brucellosis vaccine showed negative tiger red plate agglutination test results; 592 sheep serum samples from the brucellosis immune group immunized with Brucella S2 vaccine showed positive tiger red plate agglutination test results ; 115 sheep serum samples from the brucellosis-positive group infected with brucellosis were positive in the agglutination test of tiger bengal plate. Current immunological detection methods based on antigen-antibody reactions, such as tiger red plate agglutination test, cannot distinguish natural infection of brucellosis from vaccine immunity;

(2) Use the double-antibody sandwich double fluorescence ELISA detection method of IL-1β and IL-1Ra and its ratio to measure the concentration of IL-1β and IL-1Ra in serum, and calculate IL-1Ra/IL- 1 beta ratio. The concentration of IL-1β in the brucellosis infection group was significantly higher than that in the brucellosis immune group and the brucellosis negative healthy group; the IL-1Ra concentration in the brucellosis infection group was lower than that in the brucellosis immune group and the brucellosis negative healthy group, and the IL-1Ra/IL The -1β ratio was significantly lower than that of the negative healthy group and the immune group, and the statistical analysis showed a significant difference, P﹤0.01, see Figure 11;

(3) Statistical ROC curve analysis IL-1Ra/IL-1β ratio has different diagnostic significance in the brucellosis infection group, vaccine immunization group and negative control group. The results showed that the area under the curve (AUC) of the brucellosis-negative group and the brucellosis-immune group was 0.155﹤0.5, and the IL-1Ra/IL-1β levels in the brucellosis-negative group and the brucellosis-immune group had no identification significance; The AUC and AUC of the brucellosis immunization group and the brucellosis infection group were both 1.000﹥0.5, and the ratio of IL-1Ra/IL-1β could be used for the differential diagnosis of brucellosis natural infection and vaccine immunity, as shown in Figure 12. The distribution ranges of IL-1β, IL-1Ra, and IL-1Ra/IL-1β ratios of brucellosis-infected sheep, brucellosis-immune sheep, and brucellosis-negative healthy sheep were calculated using the state distribution method, as shown in Table 4;

Table 4: Statistical analysis of IL-1β, IL-1Ra, IL-1Ra/IL-1β ratio in sheep serum

(4) The recommended operating procedures for differential diagnosis of sheep brucellosis between natural infection and vaccine immunity are as follows:

① Tiger red plate agglutination test

Take 30 μL goat serum to be tested and 30 μL tiger bengal agglutinated antigen on a glass plate and mix well. Judgment of results: If there is no agglutination in the tested serum within 5 minutes, and the reaction system is evenly pink, then the tiger bengal test is negative, and it is judged as no brucellosis-infected sheep; if agglutination is visible to the naked eye, it is positive for the tiger bengal test , judged as brucellosis infected or immune sheep, the next step is to measure the ratio of IL-1Ra/IL-1β.

②Determination of IL-1Ra/IL-1β ratio

Utilize IL-1β and IL-1Ra and its ratio of the present invention to simultaneously measure the double-antibody sandwich double-fluorescent ELISA detection method, according to the operation steps of Example 12, measure the IL-1Ra/IL-1β ratio in the tiger bengal test-positive serum samples. Judgment of results: If IL-1Ra/IL-1β is in the range of 44.42-53.93, then it is judged as Brucellosis-affected sheep; if IL-1Ra/IL-1β is in the range of 74.12-139.45, it is judged as not suffering from Brucella Bacterial disease sheep; IL-1Ra/IL-1β is not in the above two ranges, it needs to be further tested and analyzed in combination with clinical symptoms or other brucellosis detection methods.

Example 14 Application of a double-antibody sandwich double-fluorescence ELISA detection method for simultaneous determination of multi-species IL-1β and IL-1Ra and their ratios in serum and plasma samples

Collect the serum and plasma samples of 100 sheep simultaneously according to conventional methods, utilize IL-1β of the present invention and IL-1Ra and its ratio synchronously to measure the double-antibody sandwich double fluorescent ELISA detection method, according to embodiment 12 operation steps, measure serum and plasma The ratio of IL-1β, IL-1Ra, and IL-1Ra/IL-1β in serum showed no significant difference between the measured IL-1β, IL-1Ra, IL-1Ra/IL-1β ratio in serum and plasma, The method of the invention can effectively measure IL-1β, IL-1Ra and the ratio of IL-1Ra/IL-1β in serum and plasma.

Example 15 Application of a double-antibody sandwich double-fluorescence ELISA detection method for simultaneous determination of multi-species IL-1β and IL-1Ra and their ratios in healthy mice and human serum samples

Collect 50 healthy mouse serum samples and 57 healthy human serum samples according to conventional methods, use the IL-1β and IL-1Ra and their ratios of the present invention to simultaneously measure the double-antibody sandwich double fluorescent ELISA detection method, according to the operation steps of Example 12, Determine the ratio of IL-1β, IL-1Ra, IL-1Ra/IL-1β in mouse serum and human serum, and calculate IL-1β, IL-1Ra, IL-1Ra, IL-1Ra, The distribution range of the ratio of IL-1Ra/IL-1β is shown in Table 5; the double-antibody sandwich double fluorescence ELISA detection method for simultaneous determination of IL-1β and IL-1Ra and their ratios of the present invention can be used for IL-1β and IL-1Ra of multiple species and its ratio determination.

Table 5: Statistical analysis of IL-1β, IL-1Ra, IL-1Ra/IL-1β ratio in mouse and human serum

The ratio IL-1Ra/IL-1β of IL-1Ra and IL-1β is measured synchronously by the double-antibody sandwich double-fluorescent ELISA of the present invention, and it is confirmed by the detection and analysis of clinical sheep blood samples that the blood serum of sheep with brucellosis The difference of IL-1Ra/IL-1β and serum IL-1Ra/IL-1β between brucellosis vaccine immunized sheep and healthy sheep was extremely significant. Moreover, it was confirmed by statistical ROC analysis that IL-1Ra/IL-1β in sheep serum has differential diagnosis significance in distinguishing brucellosis infection and immune clinical testing. The invention can be used for the differential diagnosis of Brucellosis infection and immunity, and can also be used for the diagnosis of other IL-1Ra/IL-1β-related diseases and the research on the marking function of occurrence, development and outcome.

sequence listing

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<213> sheep (sheep)

<400> 9

tacaagacag aaatcaagaa cacagttgaa tttgagtctg tcctgtaccc taactggtac 60

atcagcactt ctcaaatcga agaaaagccc gtcttcctgg gacgttttag aggtggccag 120

gatatattct tcagcatgca tgacaagcgc ttcgccttca tccgctctga caacggcccc 180

accaccagct tcgagtcggc tgcctgcccc ggctggttcc tctgcacatc actggaggcc 240

gaccagcccg tgggcctcac caat 264

<210> 10

<211> 88

<212> PRT

<213> sheep (sheep)

<400> 10

Tyr Lys Thr Glu Ile Lys Asn Thr Val Glu Phe Glu Ser Val Leu Tyr

1 5 10 15

Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ile Glu Glu Lys Pro Val Phe

20 25 30

Leu Gly Arg Phe Arg Gly Gly Gln Asp Ile Phe Phe Ser Met His Asp

35 40 45

Lys Arg Phe Ala Phe Ile Arg Ser Asp Asn Gly Pro Thr Thr Ser Phe

50 55 60

Glu Ser Ala Ala Cys Pro Gly Trp Phe Leu Cys Thr Ser Leu Glu Ala

65 70 75 80

Asp Gln Pro Val Gly Leu Thr Asn

85

Claims (6)

1. the ELISA kit of a kind of several species IL-1Ra and IL-1 β and its ratio Simultaneous Determination, it is characterised in that: including anti- The polyclonal capture antibody pre-coated elisa plate in IL-1 β and IL-1Ra rabbit source, IL-1 β recombinant protein standard items and IL-1Ra recombination The polyclonal inspection of cavy source anti-il-i-beta of protein standard substance, dilution, 20 × PBS-T concentrated cleaning solution, blue-fluorescence microballoon label The polyclonal detection antibody of the anti-IL-1Ra in cavy source of survey antibody, green fluorescent microspheres label；Wherein:

The polyclonal capture antibody of the anti-il-i-beta and IL-1Ra rabbit source, cavy source anti-il-i-beta polyclonal detection antibody, globefish The preparation step of the polyclonal detection antibody of the anti-IL-1Ra of source of mouse is as follows:

(1) IL-1 β and the IL-1Ra base of overall length sheep, people, ox, horse, pig, mouse, rabbit is downloaded in the GenBank database of NCBI Cause and protein sequence, using DNAMAN version 6 carry out sequence identity compare analysis, determine sheep, people, ox, horse, pig, Mouse, IL-1 β and the IL-1Ra albumen of rabbit and the homologous conserved region sequence of nucleic acid several species, and analyze the homologous guarantor of IL-1 β and IL-1Ra Otherness between defending zone；

(2) select the homologous conserved region IL-1 β and IL-1Ra between have differ greatly, two sections of sequences that sequence identity is low, point It is not named as IL-1 β -1 and IL-1Ra-1, IL-1 β -1 nucleotide sequence is as described in SEQ ID No.1, IL-1Ra-1 nucleotide Sequence is as described in SEQ ID No.5；Its prokaryotic expression recombinant protein is used separately as preparing anti-il-i-beta detection antibody and anti-IL- The 1Ra detection immunogene of antibody, wherein IL-1 β -1 amino acid sequence is as described in SEQ ID No.2, IL-1Ra-1 amino acid sequence Column are as described in SEQ ID No.6；In addition select that there are consistent two sections of partial sequence between the homologous conserved region IL-1 β and IL-1Ra Sequence is respectively designated as IL-1 β -2 and IL-1Ra-2, and IL-1 β -2 nucleotide sequence is as described in SEQ ID No.3, IL-1 β -2 Amino acid sequence is as described in SEQ ID No.4, and IL-1Ra-2 nucleotide sequence is as described in SEQ ID No.7, IL-1Ra-2 amino Acid sequence is as described in SEQ ID No.8；And IL-1 β -2 and IL-1Ra-2 is passed through into the DNA that amino acid sequence is FFMSH Linker connection, building IL-1 β and the homologous conserved region tandem gene IL-1 β -1Ra-2 of IL-1Ra, the IL-1 β -1Ra-2 nucleotide Sequence as described in SEQ ID No.9, IL-1 β -1Ra-2 amino acid sequence as described in SEQ ID No.10, prokaryotic expression recombination Albumen is used as the immunogene that antibody is used in preparation anti-il-i-beta and IL-1Ra capture simultaneously；

(3) the several species homologous sequence selection for being used to prepare immunogene requires: IL-1 β -2 and IL-1Ra-2 has partial sequence one IL-1 β -2 and IL-1Ra-2 fused in tandem are obtained tandem gene IL-1 β -1Ra-2, with its prokaryotic expression genetic engineering by cause property Recombinant protein immunizing rabbit prepares rabbit source anti-il-i-beta and IL-1Ra capture antibody, can be in combination with IL-1 β and IL-1Ra, IL- Sequence identity is not low homologous between 1 β -1 and IL-1Ra, IL-1Ra-1 and IL-1 β, so that respectively with IL-1 β -1 and IL-1Ra- The anti-IL-1Ra detection of the cavy source anti-il-i-beta detection antibody of 1 genetic engineering recombinant protein expression product preparation and cavy source is used Antibody can specifically bind IL-1 β and IL-1Ra respectively；

(4) upstream and downstream primer a pair of IL-1 β -1-S and IL-1 β -1-A of design amplification IL-1 β -1, to include overall length IL-1 β CDNA recombinant plasmid be pcr template, using PCR react amplification IL-1 β -1 DNA, purified using DNA gel QIAquick Gel Extraction Kit The DNA of IL-1 β -1；

(5) upstream and downstream primer a pair of the IL-1Ra-1-S and IL-1Ra-1-A of design amplification IL-1Ra-1, to include overall length IL- The cDNA recombinant plasmid of 1Ra is pcr template, and the DNA of amplification IL-1Ra-1 is reacted using PCR, uses DNA gel QIAquick Gel Extraction Kit Purify the DNA of IL-1Ra-1；

(6) upstream and downstream primer a pair of IL-1 β -2-S and IL-1 β -2-A of design amplification IL-1 β -2, to include overall length IL-1 β CDNA recombinant plasmid be pcr template, using PCR condition amplification IL-1 β -2 DNA, purified using DNA gel QIAquick Gel Extraction Kit The DNA of IL-1 β -2；

(7) upstream and downstream primer a pair of the IL-1Ra-2-S and IL-1Ra-2-A of design amplification IL-1Ra-2, to include overall length IL- The cDNA recombinant plasmid of 1Ra is pcr template, and the DNA of amplification IL-1Ra-2 is reacted using PCR, uses DNA gel QIAquick Gel Extraction Kit Purify the DNA of IL-1Ra-2；

(8) drawn using upstream used in design the downstream primer IL-1 β -2-A used in amplification IL-1 β -2 and amplification IL-1Ra-2 Linker sequence in object IL-1Ra-2-S, using Overlap extension PCR method, with the DNA of the DNA of IL-1 β -2 and IL-1Ra-2 IL-1 β -2 and IL-1Ra-2 gene are gone here and there by Linker using IL-1 β -2-S and IL-1Ra-2-A as upstream primer for template Connection expands IL-1 β -1Ra-2 fusion dna, uses the DNA of DNA gel QIAquick Gel Extraction Kit purifying IL-1 β -1Ra-2；

(9) respectively by the DNA fragmentation of the IL-1 β -1 of acquisition and IL-1Ra-1, with the T4 archaeal dna polymerase of disconnected enzyme dependence Method is cloned on pMCSG9 carrier, and DNA and the pET-30a carrier of IL-1 β -1Ra-2 is connect, and constructs recombinant expression plasmid PMCSG9-IL-1 β -1, pMCSG9-IL-1Ra-1 and pET30a-IL-1 β -1Ra-2；

(10) recombinant expression plasmid is converted into the inducing expression recombinant protein into e. coli bl21-Codonplus cell, picking Correct single colonie is identified in recombinant plasmid sequencing, is inoculated in the LB liquid medium of the benzyl resistance of ammonia containing 50mg/L, 37 DEG C of cultures 2 Hour, it is then inoculated in the fresh liquid LB culture medium of the benzyl resistance of ammonia containing 50mg/L of 200mL in 1:500 ratio, 37 DEG C of vibrations Culture 1 hour is swung, the IPTG of 300 μ L1mol/mL is added, is centrifuged 5min through 4 DEG C, 10000rpm after continuing shaken cultivation 5.5 hours, The gene engineering colibacillus bacterium of pMCSG9-IL-1 β -1, pMCSG9-IL-1Ra-1, pET30a-IL-1 β -1Ra-2 are harvested respectively Body precipitating, after being resuspended with PBS, is cracked, power 40W, ultrasonic 10s, interval 10s, total 30min with Ultrasonic Cell Disruptor, in 12000 revs/min, centrifugation 30min, obtain supernatant and inclusion body precipitates；

(11) recombinant protein of E. coli system expression has merged His label, will be on the broken thallus of gene engineering expression bacterium Clear liquid presses nickel ion affinity chromatograph natural subsidence method purification of target antigen protein IL-1 β -1, IL-1 β -2 and IL-1 using nickel column β -1Ra-2, using the recombinant protein of SDS-PAGE and Western-blot analysis verifying after purification, with molecular cut off 8000- 4 DEG C of the bag filter dialysis of 14000Da, dialyzate are the PBS of 0.01M pH 8.0, and every 2h replaces a dialyzate, replaces 5 altogether It is secondary, concentration of instead dialysing is carried out with PEG20000, SDS-PAGE identifies purity, and it is spare that packing is put into -80 DEG C of refrigerators；

(12) animal immune

Immune IL-1 β -1, IL-1Ra-1 the and IL-1 β -1Ra-2 albumen for taking 1mg respectively for the first time, it is mixed with isometric Freund's complete adjuvant Merge emulsification, IL-1 β -1 after emulsification and IL-1Ra-1 protein immunization cavy are prepared into cavy source anti-il-i-beta detection antibody respectively With the anti-IL-1Ra detection antibody in cavy source, the IL-1 β -1Ra-2 protein immunization new zealand white rabbit after emulsification is prepared into rabbit source Anti-il-i-beta and IL-1Ra capture antibody, carry out dorsal sc multiple spot and are immunized, and 15 days are an immune period, and second immune Freund's incomplete adjuvant is begun to use to prepare emulsified immunogen for being immunized, method is same as above, after third time is immune, measurement one in every 7 days Secondary serum titer, potency carry out Culling heart blood up to 50000 or more, collect serum potency to be measured and antibody purification；

(13) antibody titer detection and purifying

It leaves and takes a small amount of serum before immune to ask as negative blood, the rabbit and cavy after being immunized acquire blood, are incubated in 37 DEG C 0.5h, 4 DEG C of blood coagulations 2h, 3000 revs/min of centrifugation 5min draw serum, detect potency using ELISA method, use AKATA egg White purification system and protein g affinity chromatography method antibody purification；

(14) interference antibody removal

By interference antibody removal experiment, the non-specific binding of two kinds of cavy source detection antibody is reduced, the specific steps of which are as follows: Induction is transferred to the Bacillus coli expression bacterium of pMCSG9 empty carrier, takes 1mL-5mL Bacteria Culture fermentation liquid, 1300 revs/min, centrifugation 1min abandons supernatant and collects thallus, takes two kinds of cavy source detection antibody, 100 μ L suspension thallus respectively, 37 DEG C of incubation 45-90min, 3000 revs/min of centrifugation 5min, taking supernatant is the detection antibody for removing interference antibody.

2. the ELISA reagent of a kind of several species IL-1Ra according to claim 1 and IL-1 β and its ratio Simultaneous Determination Box, it is characterised in that:

(1) preparation step of the polyclonal capture antibody pre-coated elisa plate of anti-il-i-beta and IL-1Ra rabbit source is as follows:

(1) be coated with anti-il-i-beta and the polyclonal capture antibody in IL-1Ra rabbit source: by anti-il-i-beta and IL-1Ra rabbit source is polyclonal catches It obtains with antibody diluted to final concentration of 0.078 μ g/mL, is added in ELISA Plate, 100 holes μ L/, 4 DEG C of coatings are overnight；

(2) to contain the PBS of 0.2% Tween-20 as PBS-T cleaning solution, board-washing 3-4 times, 200 holes μ L/, each 1min, so It is closed afterwards with 0.1M ammonium chloride solution, 200 holes μ L/, 37 DEG C of 1h, draining liq；

(2) the polyclonal detection antibody of the cavy source anti-il-i-beta of blue-fluorescence microballoon label, excitation wavelength 360nm, transmitting Wavelength 450nm；Coupling step is as follows: the MES buffer solution 90mL of pH 4.5-7.5 is added in 10 μ L blue-fluorescence microballoons, mixes, 15000 revs/min are centrifuged 15 minutes, abandon supernatant, collect microballoon precipitating, and repetition is washed twice；In 90 μ L 0.2M pH 4.5-7.5 MES buffer solution in, be added 4.8 μ L 1mg/mL EDC, activate 20 minutes；Then 5.6 μ L 1mg/mL NHS are added, mix 20 minutes；4 DEG C 15000 revs/min are centrifuged 15 minutes, collect microballoon precipitating；With the MES buffer solution of 90 μ L pH 4.5-7.5 Microballoon is resuspended, adds the 10 polyclonal detection antibody of μ L 1mg/mL cavy source anti-il-i-beta, room temperature vortex concussion reaction 4 is small When；10 μ L 10%BSA confining liquids are added, react at room temperature 30min；4 DEG C 15000 revs/min are centrifuged 15 minutes, abandon supernatant, are added The MES buffer solution of 100 μ L pH 4.5-7.5,4 DEG C are kept in dark place；

(3) the polyclonal detection antibody of the anti-IL-1Ra in cavy source of green fluorescent microspheres label, excitation wavelength 480nm, transmitting Wavelength 520nm；10 μ L green fluorescent microspheres be added pH 4.5-7.5 MES buffer solution 90mL, mix, 15000 revs/min from The heart 15 minutes, supernatant is abandoned, collects microballoon precipitating, repetition is washed twice；In the MES buffer solution of 90 μ L 0.2M pH 4.5-7.5 In, 4.8 μ L 1mg/mL EDC are added, activate 20 minutes；Then 5.6 μ L 1mg/mL NHS are added, mix 20 minutes；4℃ 15000 revs/min are centrifuged 15 minutes, collect microballoon precipitating；Microballoon is resuspended with the MES buffer solution of 90 μ L pH 4.5-7.5, then The 10 polyclonal detection antibody of the μ L 1mg/mL cavy anti-IL-1Ra in source, room temperature vortex concussion reaction 4 hours is added；10 μ L are added 10%BSA confining liquid reacts at room temperature 30min；4 DEG C 15000 revs/min are centrifuged 15 minutes, abandon supernatant, and 100 μ L pH are added The MES buffer solution of 4.5-7.5,4 DEG C are kept in dark place.

3. the ELISA reagent of a kind of several species IL-1Ra according to claim 1 and IL-1 β and its ratio Simultaneous Determination Box, it is characterised in that:

(1) IL-1 β -1-S:5^/-tacttccaatccaatgcCTATACCTGTCTTGTGTGACCCA-3^/；

IL-1 β -1-A:5^/-ttatccacttccaatgtcAGAAGACGAATCGCTTTTCC-3^/；

(2) IL-1Ra-1-S:5^/-tacttccaatccaatgcCAAGCCTTCAGGATCTGGG-3^/；

IL-1Ra-1-A:5^/-ttatccacttccaatgtcAACCTTGCAAGTATCCAGC-3^/；

(3) IL-1 β -2-S:5^/-ccgcatatgTACAAGACAGAAATCAAGAACACAGT-3^/；

IL-1 β -2-A:5^/-atgcatgctgaagaaTATATCCTGGCCACCTCTAAAAC-3^/；

(4) IL-1Ra-2-S:5^/-ttcttcagcatgcatGACAAGCGCTTCGCCTTCATC-3^/；

IL-1Ra-2-A:5^/-ccgctcgagATTGGTGAGGCCCACGGG-3^/；

(5) the linker sequence ttcttcagcatgcat of IL-1 β and the homologous conserved region tandem gene IL-1 β -1Ra-2 of IL-1Ra.

4. the ELISA reagent of a kind of several species IL-1Ra according to claim 1 and IL-1 β and its ratio Simultaneous Determination Box, it is characterised in that: the IL-1 β recombinant protein standard items and IL-1Ra recombinant protein standard items, are with large intestine bar respectively The IL-1 β recombinant protein and IL-1Ra recombinant protein of bacterium expression system gene engineering expression, specific preparation method press prokaryotic expression The open reading frame of IL-1 β and IL-1Ra are inserted into prokaryotic expression carrier pET-30a respectively by technology, building fusion His label Recombinant expression carrier pET-30a-IL-1 β and pET-30a-IL-1Ra obtain the gene of high-purity with inducing expression purification technique Engineering IL-1 β recombinant protein and IL-1Ra recombinant protein, prepare standard items albumen.

5. the ELISA reagent of a kind of several species IL-1Ra according to claim 1 and IL-1 β and its ratio Simultaneous Determination Box, it is characterised in that: the dilution is the 0.1M PBS solution of pH7.2-7.6.

6. the ELISA reagent of a kind of several species IL-1Ra according to claim 1 and IL-1 β and its ratio Simultaneous Determination Box, it is characterised in that: the antibody can with sheep, people, ox, horse, pig, mouse, rabbit, dog, chicken, the multiple species of duck IL-1 β and IL- 1Ra occur specific binding reaction, for detect sheep, people, ox, horse, pig, mouse, rabbit, dog, chicken, the multiple species of duck serum, blood The ratio of IL-1 β and IL-1Ra content and IL-1Ra/IL-1 β in slurry and histoorgan sample.