CN101363856B - Test paper strip for rapidly detecting morbilli and rubella virus IgG antibody colloidal gold - Google Patents

Abstract

The invention provides a rapid detection test strip for simultaneous detection of measles and rubella virus-specific IgG antibodies, including a reaction membrane and a conjugate release pad. Antigen detection zone and quality control zone coated with secondary antibody IgG; the conjugate release pad is coated with colloidal gold-labeled anti-human IgG, and the test strip of the present invention is used for detection, which is simple, convenient, fast and simple to operate, No special equipment or professional training is required, the results are clear and easy to distinguish, the operation is simple, and it is easy to promote. It is suitable for the grassroots, suitable for on-site detection and epidemiological investigation, and plays an auxiliary and differential diagnosis role for measles and rubella virus infections. It can be used to observe the immune effect after vaccination.

Description

A kind of measles, rubella virus IgG antibody colloidal gold fast detecting test paper strip

Technical field

The invention belongs to field of biological detection, be specifically related to a kind of measles, rubella virus specific IgG antibodies test strip and application thereof.

Background technology

(Measles virus MV) infects decapacitation and causes outside the acute infection symptoms such as heating, respiratory tract catarrh symptom and whole body maculopapule measles virus, also with the cell-mediated immunization of inhibition, thereby causes scabies secondary infections such as pneumonia, diarrhoea.In the minority case even can also cause severe complications such as encephalitis and continuation central nervous system infection.MV H albumen (hemagglutinin) belongs to II type transmembrane glycoprotein, and molecular weight is 73-78kD, can stimulate body to produce effective humoral immunity and cellular immunity.MV.H albumen is the main site of MV two receptoroid CD46 and SLAM effect, the infection of initial virus; Simultaneously can also with the acting in conjunction of F albumen, induce the fusion of viral after birth and cell membrane.

The clinical symptoms of current measles case is typical case inadequately often, therefore, the laboratory diagnosis of doubtful measles case is seemed important unusually.At present, measles IgM antibody is confirmed in clinical EUSA commonly used (ELISA) the method detection patient serum.But, specific IgM antibodies at the initial stage of a disease during 1～3d and convalescent positive rate lower, be merely 50%～73%, the positive rate that especially detects in the morbidity same day and next day is lower.Therefore, cause easily and fail to pinpoint a disease in diagnosis. cause various severe complications.It is reported that to clinical meeting of prescription on individual diagnosis at eruption initial stage, but the patient of measles IgM negative antibody adopts the method for RT-PCR to detect the MV-H gene, can improve the positive rate of detection.But be subject to cost and round pcr itself, be not suitable for being applied to large-scale Clinical detection.

(Rubella virus RV) is unique member of Togaviridae rubella virus genus to rubella virus.Usually the RV infection symptoms is lighter, show as acute, light-duty, the course of disease short, self limiting eruption property disease.RV is its teratogenesis characteristic to the biggest threat of public health.Woman gestationperiod, infection RV can cause the neonate to produce a series of organ deformity in especially preceding 3 months, was called congenital rubella syndrome (CRS) (CRS).RV is the sub-thread positive chain RNA virus, three kinds of albumen of structural gene coding of its 3 ' end, i.e. capsid protein C and two kinds of envelope glycoprotein E1 and E2.In 3 kinds of primary structure albumen E1, E2 and C albumen of RV, epitope mainly is present on the E1 memebrane protein.E1 is an envelope glycoprotein, constitutes the furcella of peplos jointly with E2.The E1 relative molecular mass is 57000, is made up of 412-418 amino acid residue.Using monoclonal antibody confirmed to exist on the E1 blood clotting and in and antigenic determinant.The full-length gene of external existing E1 memebrane protein is expressed, but complete E1 memebrane protein poorly water-soluble is difficult for purifying, is difficult to be used for as antigen the serology detection of RV virus infections.According to bibliographical information, in the E1 memebrane protein most of antigenic determinant accumulation area between the 202nd～305 amino acids, and this antigenic determinant accumulation area sequence high conservative.

Since HIA (blood clotting inhibition) set up, the laboratory diagnostic method that RV infects had obtained continuous renewal and improvement.Though ELISA is the main conventional method that Diagnosis of Congenital infects, cross reaction takes place in RV specific antibody and piconavirus B19, Epstein-Barr virus etc., and positive reaction also can appear in type i diabetes, chronic hepatitis patients, and its specificity is restricted.And PCR exists the gesture that additive method hardly matches in the diagnosis of virus infections.Detect RV diagnosis of infection standard but still lack PCR at present, remain further to be studied to illustrate.So. it is insecure infecting from an aspect diagnosis RV separately, and two or more method such as ELISA and PCR Combined application are only the RV infection and have more convictive diagnostic method.Yet this method for combined use program is complicated, needs the long time of cost, and needs certain experiments supporting instrument, and it is high to detect cost.

Summary of the invention

The purpose of invention provides a kind of easy to use, quick; Be used to detect the test strips of measles virus, rubella virus specific IgG antibodies; Detect measles in the human sample, rubella virus specific IgG antibodies, be used for the auxiliary diagnosis and the discriminating of measles, rubella virus infection.

Another object of the present invention provides a kind of method for preparing measles, rubella virus IgG antibody colloidal gold colloidal gold detection test paper strip.

Test strip of the present invention comprises reaction film and bond releasing cushion, and said reaction film has detection band that encapsulates measles virus H antigen and rubella virus E 1 specific antigen respectively and the quality control band that encapsulates two anti-IgG; Said bond releasing cushion is coated with the anti-human IgG of colloid gold label.

Wherein, the package amount of measles virus H antigen, rubella virus E 1 antigen is respectively 2-3mg/ml

Wherein, reaction film can be nitrocellulose membrane, and the bond releasing cushion can be glass fibre membrane.

Wherein, measles virus H antigen, rubella virus E 1 antigen can prepare through prokaryotic expression respectively.

Wherein the IgG antiantibody is a rabbit anti-mouse igg antibody.

The present invention also provides a kind of method for preparing above-mentioned test strip, and it comprises the steps:

1) prepares measles virus H antigen, rubella virus E 1 specific antigen respectively;

2) antigen of step 1) preparation and IgG antiantibody are encapsulated form detection line and nature controlling line on the reaction film respectively, subsequent use;

3) the anti-human IgG with colloid gold label encapsulates on the bond pad;

4) with 2) and 3) bond releasing cushion, reaction film and the sample pad, adsorptive pads and the reaction holder that prepare be assembled into test card.

The present invention obtains measles virus H antigen and rubella virus E 1 specific antigen through genetic engineering means, can significantly improve the sensitivity and the specificity of diagnosis.

Preferably the invention also discloses the application of said test strips in detecting measles virus, rubella virus specific IgG antibodies.

Technical scheme of the present invention is: the measles virus H antigen, rubella virus E 1 specific gene engineering antigen and the anti-mouse IgG difference solid phase (NC film) on nitrocellulose membrane that adopt purifying; The anti-human IgG of association colloid gold mark is used rete and is analysed measles virus, the rubella virus specific IgG antibodies of catching in the ratio juris detection sample.

Test strips of the present invention utilizes colloidal gold-labeled method and rete to analyse technology, is used for measles, rubella virus specific antibody that fast qualitative half-quantitative detection sample possibly exist, reaches quick screening patient, in time controls the purpose of epidemic situation.Can save a large amount of manpower and materials, easily and fast, simple and direct, not need special instruments and equipment; Do not need professional training, clear being prone to of result distinguished, and be simple to operate; Be easy to promote; Be fit to basic unit, be suitable for on-the-spot the detection and epidemiology survey, auxiliary and antidiastole effect are played in the infection of measles, rubella virus.

Description of drawings

Fig. 1: the front schematic view of A test strips of the present invention; The side schematic view of B test strips of the present invention.Wherein, 1: adsorptive pads; 2: nitrocellulose membrane (T1 and T2: two test strips of measles virus H, rubella virus E 1 specific antigen; C: the Quality Control band that encapsulates anti-mouse IgG); 3: the glass fibre membrane that contains the anti-human IgG of colloid gold label; 4: golden labeling antibody diaphragm; 5: the reaction holder.

Fig. 2: testing result synoptic diagram.Wherein, be followed successively by from left to right: T1, C or T2, two line positives of C; Line feminine gender of C; T, two line feminine genders of C are invalid.

Embodiment

Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.

If do not specialize the conventional means that used technological means is well known to those skilled in the art among the embodiment.

The clonal expression of embodiment 1 measles virus H, rubella virus E 1 specific antigen

(1) amplification of MV.H GFP

According to Trizol method extracting MV geneome RNA, then according to synthetic cDNA first chain of reverse transcription kit instructions.(the GenBank number of landing of MV.H GFP is AB045300)

Use primer 5 ' CGCA AGCTTCTATCTGCGATTGGTTCCA 3 ' and 5 ' TTAGGATCCATGTCACCACA ACGAGACCG 3 ' again under the effect of high-fidelity DNA polymerase, with 95 ℃ of 3min; 94 ℃ of 30S, 54 ℃ of 35s, 72105S, 30 circulations; Again in 72 ℃ of condition amplification MV.H genes that extend 10min.Product is through PCR product (in a small amount) purification kit purifying.

The reorganization of virus

Respectively MV.H gene and carrier pGEMEX-1 (Promega) are placed the double digestion system of BamHI and Hind-III; 37 ℃ of water-bath 1h, product reclaims kit (TAKARA Agarose Gel DNA Purification Kit Ver.2.0 article No.: DV805A) purifying with rubber tapping.The enzyme of MV-H and pGEMEX-1 is cut product, under the effect of T4 dna ligase, and 24 ℃ of water-bath 1h.Immediately 10 μ L are connected product and 100 μ L large intestine Erichsen bacterium DH5 α competent cell mixings, behind the ice bath 30min, 42 ℃ of water-bath heat shock 90S, ice bath 5min adds 450 μ L LB nutrient culture media, then in 37 ℃ of shaking table joltings of 180r/min 1h again.Evenly coat the solid LB culture medium flat plate surface that contains 100 μ g/mL ampicillins with transforming back bacterium liquid 200 μ L, 37 ℃ of incubators spend the night, and screening positive clone also carries out the enzyme evaluation of cutting and check order to it, and the result has obtained 10 strain positive colonies.

Expression of Fusion Protein and evaluation are with the measles virus hemagglutinin

Gene recombination plasmid MV-H gene and carrier pGEMEX-1 transform EHEC JM109 (DE3) and BL21 (DE3), and method is the same.Positive strain increases in the LB nutrient culture media behind 3～4h, adds IPTG (final concentration is 1mmol/L) abduction delivering 4h and 6h respectively.With the bacterium liquid centrifugation that obtains, wash with PBS.After the PBS dissolving, boil 5 minutes cracking bacterium liquid.At last, with 8%SDS-PAGE proteins gel electrophoresis checking expression product.With GST-fusion purification kit (B-PER bacterium GST tag fusion protein pillar purifying Kit article No.: the silent generation that science and technology that flies of 78200 matches); Carry out purifying by kit recommendation step and system; Purified product carries out the SDS-PAGE electrophoresis to judge purification effect, records productive rate more than 95% through the ultraviolet thin layer.

The Western-Blot of fusion identifies

Cutting half glue changes on nitrocellulose filter with the half-dried electroporation that BioRad company produces, and carries out the trace test, and one anti-ly is MV monoclonal antibody (Beijing Bo Aosen Bioisystech Co., Ltd), and after the TMB colour developing, the result has the specific proteins band to occur.

(2)RV-E1

Choose and at first select a fragment (nt 9345～9365) in the E1 gene (number of landing EU622506), design reverse transcription primer P3 in genes of interest to be amplified downstream.Introduce BamHI restriction enzyme site and protection base and EcoR I restriction enzyme site and protection base respectively simultaneously according to target gene sequences design PCR primer P1 and P2, and at 5 ends and 3 ends of primer.Primer is given birth to worker company by Shanghai and is synthesized.Primer sequence is following:

P15?ATGAATGGATCCGGCAATCAGCAGTCCCGGT?3

P25?GCCGGAATTCTG?TCAGGGGAATGGCGT?3

P35?GGTGAGATGGCAATTGCCT?3

The extraction of virus total RNA and RT-PCR

The total RNA extraction reagent box of Using P romega company, and by providing method extraction intracellular virus geneome RNA.With the RNA that extracts as template; With ReverseTranscription System Kit (TAKARA RNA PCR Kit (AMV) Ver.3.0 article No.: DRR019A) carry out reverse transcription; Reverse transcription product is with performing PCR again after 5 times of the deionized water dilutions, and the PCR response parameter is: 94 ℃ of preparatory sex change 5min; 94 ℃ of 30S, 52 ℃ of 30S, 72 ℃ of 30S, totally 35 circulations; 72 ℃ are extended 10min.The PCR product is in the up electrophoresis detection of Ago-Gel, and with Wizard PCR preps DNA purificaton system kit (TaKaRa DNA Fragment Purification Kit Ver.2.0 article No.: DV807A) purifying and recovering.

The structure of recombination, amalgamation and expression plasmid

The PCR product is cloned into carrier pGEX4T-2 (Pharmacia) with conventional method after order-checking is identified, transform DH-5 α bacterial strain, goes out positive recombinant clone, called after pGEX4T-2/E1 with BamH I and EcoR I double digestion evaluation and screening.

The expression of recombinant plasmid in DH-5 α bacterial strain chosen the single bacterium colony that contains former plasmid of pGEX4T-2 and recombinant plasmid and is inoculated in respectively in the 5ml LB nutrient solution, and 37 ℃ of thermal agitations are cultured to A ₆₀₀Be about 0.5.With 1: 100 ratio activation bacterium to A ₆₀₀Be about 0.2, add IPTG and induce 3～5h to final concentration 1mmol/L.Every separated 1h gets the 1ml culture and detects the situation of inducing.With before inducing and different induction times collected respectively manage bacterium liquid respectively under 4 ℃ with 12, the centrifugal 2min of 000r/min abandons supernatant, collects bacterium.It is resuspended that each pipe adds 1 * SDS protein gel sample loading buffer of 50 μ L, boils 5min behind the mixing, and with the centrifugal 5min of 12000r/min, it is subsequent use to suct clear liquid-20 ℃ preservation.

The SDS-PAGE of expression product and purifying

Get each the 20 μ L of supernatant and the capable simultaneously SDS PAGE of middle relative molecular mass standard protein Marker of above-mentioned preparation, after electrophoresis finished, with Coomassie brilliant blue R-250 dyeing, destainer decolouring back observation had or not the purpose band.With GST-fusion purification kit (B-PER bacterium GST tag fusion protein pillar purifying Kit article No.: the silent generation that science and technology that flies of 78200 matches); Carry out purifying by kit recommendation step and system, purified product carries out the SDS-PAGE electrophoresis to judge that purification effect records productive rate more than 95% through the ultraviolet thin layer.

The Western-Blot of fusion identifies

Cutting half glue changes on nitrocellulose filter with the half-dried electroporation that BioRad company produces, and carries out the trace test, and one anti-ly is RV monoclonal antibody (Beijing Bo Aosen Bioisystech Co., Ltd), and after the TMB colour developing, the result has the specific proteins band to occur.

Embodiment 2: measles, rubella virus IgG antibody colloidal gold fast detecting test paper strip (referring to Fig. 1)

(1) preparation of colloidal gold antibody bond:

Confirm that through experiment its best combination pH value of anti-human IgG monoclonal antibody colloid mark is 8.0, the proportioning of collaurum and antibody is respectively 20 μ g/ml collaurums.The mark collaurum by the amount of every square centimeter 65 μ l, is got collaurum-antibody conjugates solution after stabilizing agent (0.5%BSA, pH8.0,0.01M Tris damping fluid) is handled, evenly is adsorbed on the spun glass, and freeze drying, and in dry environment, preserve.

(2) envelope antigen is in nitrocellulose membrane:

Measles virus H antigen, the rubella virus E 1 antigen of embodiment 1 preparation are diluted to 3.5mg/ml with 0.01MPBS.To resist mouse IgG polyclonal antibody to be diluted to 2mg/ml with 0.01MPBS.With Membrane jetter the two speed with 1 μ l/cm is sprayed on the nitrocellulose membrane, forms detection line and control line respectively.Article three, be spaced apart 0.5cm between the line.

Put the cellulose nitrate that is fixed with antigen, antibody in 37 ℃ of baking boxs dry 2 hours.Preserve subsequent use in the dry environment.

(3) measles, rubella virus IgG antibody detectable bar are formed

The reaction holder is 6.5cm * 0.4cm PCV plate; Adsorptive pads is the filter paper for oil of 2cm * 0.4cm; 1.8cm the nitrocellulose membrane of * 0.4cm encapsulates anti-mouse IgG successively, measles virus H antigen, rubella virus E 1 antigen, the spun glass of the anti-human IgG monoclonal antibody that contains colloid gold label of 0.4cm * 0.4cm; Gold labeling antibody diaphragm (sample pad) is the filter paper fibre of 2.7cm * 0.4cm; Measles virus, rubella virus IgG antibody test strip (colloidal gold method) have promptly been formed.

(4) measles, rubella virus IgG antibody detectable bar specificity and susceptibility detect:

Specificity experiment: the negative quality-control product that this product design is following: influenza virus serum, Respiratory Syncytial Virus(RSV) serum, parainfluenza virus serum, mumps virus serum, mycoplasma pneumoniae serum, normal human serum, measles serum, rubella serum.All serum all screen with corresponding ELISA kit, are the IgG positive serum.Testing result shows, two detection lines of kit only with corresponding IgG seroreaction, and with other no cross reaction.

The susceptibility experiment: measles G positive serum, rubella IgG positive serum is all demarcated through the ELISA screening.Detection show with, this product is 1: 20 (ELISA tires) to the limit of identification of measles IgG positive serum, is 1: 20 (ELISA tires) to the limit of identification of rubella IgG positive serum.

Embodiment 3: method of application (referring to Fig. 2)

Sample to be checked (whole blood, blood plasma or serum) directly is added dropwise to test strips " 4 " locates, sample liquid is up along film, 10-15 minute sentence read result.

The result:

As containing measles virus, rubella virus IgG antibody in the sample; Then with test strips on the anti-human IgG monoclonal antibody of colloid gold label form corresponding compound; Up be coated on nitrocellulose membrane on measles virus H antigen, rubella virus E 1 specific antigen combine to form red lines, promptly in T1, T2 place formation red stripes.

No matter whether contain corresponding antibody, the monoclonal antibody of the anti-human IgG of colloid gold label continues upwards to creep and be coated on the anti-mouse IgG formation red precipitate line on the film, promptly locates to form red stripes at " C ".This line is a nature controlling line, loses efficacy like collaurum, and this line just can not occur, and explains that test strips lost efficacy.

Sequence table

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< 120>a kind of measles, rubella virus IgG antibody colloidal gold fast detecting test paper strip

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Claims (5)

1. a test strip comprises reaction film and bond releasing cushion, and said reaction film has detection band that encapsulates measles virus H antigen and rubella virus E 1 specific antigen respectively and the quality control band that encapsulates two anti-IgG; Said bond releasing cushion is coated with the anti-human IgG of colloid gold label, and the package amount of measles virus H antigen, rubella virus E 1 antigen is respectively 2-3mg/ml, and described two anti-IgG are rabbit anti-mouse igg; Measles virus H antigen is that measles virus H gene cloning and expression obtains, and measles virus H gene is through extracting measles virus geneome RNA, as template, obtains with the RT-PCR method, and used upstream and downstream primer sequence is:

5 '-CGCAAGCTTCTATCTGCGATTGGTTCCA-3 ' and

5’-TTAGGATCCATGTCACCACA?ACGAGACCG-3’；

Rubella virus E 1 antigen is that the rubella virus E 1 gene cloning and expression obtains, and the rubella virus E 1 gene is through extracting rubella virus genome group RNA, as template, obtains with the RT-PCR method, and used upstream and downstream primer sequence is:

P1：5-ATGAATG?GATCCGGCAATCAGCAGTCCCGGT-3，

P2：5-GCCGGAATTCTG?TCAGGGGAATGGCGT-3，

The reverse transcription primer is P3:5-GGTGAGATGGCAATTGCCT-3.

2. test strips as claimed in claim 1 is characterized in that, described measles virus H antigen, rubella virus E 1 specific antigen prepare through prokaryotic expression.

3. according to claim 1 or claim 2 test strips is characterized in that described reaction film is a nitrocellulose filter.

4. according to claim 1 or claim 2 test strips is characterized in that described bond releasing cushion is a glass fibre membrane.

5. a method for preparing each said test strips of claim 1～4 comprises the steps:

1) prepares measles virus H antigen, rubella virus E 1 specific antigen respectively;

2) antigen of step 1) preparation and IgG antiantibody are encapsulated form detection line and nature controlling line on the reaction film respectively, subsequent use;

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