CN106244560B - A kind of C-Ps monoclonal antibody and its preparation and application - Google Patents

Abstract

The invention relates to a hybridoma cell line 5B3B9 that can stably secrete a C-polysaccharide (C-Ps) monoclonal antibody of Pneumococcus pneumoniae. The C-Ps monoclonal antibody produced by the hybridoma cell has strong specificity and a simple preparation method. It can be used for the immunological detection and analysis of C-Ps. The C-Ps detection kit prepared from the C-Ps monoclonal antibody, especially the colloidal gold immunodetection system, is easy to use, has high detection accuracy and sensitivity, and has good stability. .

Description

A kind of C-Ps monoclonal antibody and its preparation and application

technical field

The invention relates to the technical field of biotechnology, in particular to a hybridoma cell line that can stably secrete a C-polysaccharide (C-Ps) monoclonal antibody of Pneumococcus pneumoniae, a C-Ps monoclonal antibody and a C-Ps detection kit.

Background technique

Streptococcus pneumoniae is a gram-positive bacterium and is a common pathogen that causes pneumonia, otitis media, meningitis and other diseases. In severe cases, it can cause a very high mortality rate. So far, more than 90 different types of pneumococcus have been isolated. Its serotype is closely related to the capsule, because the capsule can resist the phagocytosis of phagocytes in the human body and multiply and cause disease. The LD50 test of encapsulated and non-encapsulated pneumococci demonstrated that the capsule is the main virulence factor of pneumococci. Streptococcus pneumoniae C-polysaccharides (C-Ps) are antigens common to all pneumococcal serotypes and thus can be used as antigens for the detection of different subtypes of pneumococci.

Patent CN200510084709.5 discloses a rabbit anti-Streptococcus pneumoniae strain R6 antibody affinity purified using the C-polysaccharide antigen of Streptococcus pneumoniae strain R6, and the above antibody is coupled with gold particles, and the above antibody is used to prepare a sample For the capture line, goat anti-rabbit immunoglobulin (IgG) was used to prepare the control line, and an ICT device was prepared for the detection of Streptococcus pneumoniae antigens. Since the labeled antibody and the capture antibacterial were both polyclonal antibodies, the specificity was different. high, the detection sensitivity and accuracy are not high.

SUMMARY OF THE INVENTION

In order to overcome the deficiencies of the prior art, the present invention provides a hybridoma cell line 5B3B9, which can stably secrete C-polysaccharide monoclonal antibody, and the hybridoma cell line was deposited in the China Microorganism Culture Collection on July 21, 2016. General Microbiology Center, the deposit number is CGMCC No.12694.

Described hybridoma cell is prepared by the following method:

(1) Antigen preparation

Streptococcus pneumoniae was cultured on Columbia culture plate, washed with sterile saline to make bacterial suspension, and inactivated;

(2) Mice immunization

Select 4-8 week old Balb/C mice, take the inactivated Streptococcus pneumoniae suspension in step (1) as the immunization antigen, and the immunization dose is 50-200 μl/mouse, immunized 2-7 times;

(3) Cell fusion

Take the immunized mouse splenocytes in step (2), mix them with SP2/0 myeloma cells in a ratio of 10:1 to 1:1, centrifuge, add PEG solution, and then add RPMI-1640 medium to terminate the reaction , centrifuged, resuspended in RPMI-1640 medium, cultured, added HAT medium the next day to dilute to the working concentration, cultured until the cell mass grew, and assayed the cell supernatant;

(4) Screening of hybridoma cells

Determine the C-Ps antibody in the cell supernatant of step (3) by indirect ELISA, screen the positive parent cell line, and use the limiting dilution method to clone to determine the positive cell line 5B3B9;

(5) Identification of hybridoma cells

For the 5B3B9 cell line obtained in the culturing step (4), aspirate the cell supernatant, and identify the positive cells by indirect ELISA method;

Preferably, the above preparation method further comprises: (6) preservation of hybridoma cells.

Preferably, in step (1), the Columbia blood culture plate is Columbia culture medium containing 10% sheep blood; the culture time is 18-20h;

Preferably, the concentration of the bacterial suspension is 1×10 ⁷ -1×10 ⁹ cells/ml, more preferably 2×10 ⁸ cells/ml;

Preferably, in step (2), the selected mouse is a 4-6 week old Balb/C female mouse; the immunization method is intramuscular injection of immunized antigen in the mouse leg; the immunization dose is 100 μl per mouse; the number of immunizations is 3 times , more preferably 1 time every 14 days, 3 times at the same dose;

Preferably, in step (3), the mixing ratio of immunized mouse splenocytes and SP2/0 myeloma cells is 5:2; the PEG solution is a PEG1450 solution;

Preferably, in steps (3) and (4), the cell culture conditions after the fusion are cultured in a 37° C., 5% CO ₂ incubator;

Preferably, in steps (4) and (5), the process of measuring the antibody by the indirect ELISA method is as follows: using carbonate buffer as the coating liquid, the coated antigen standard C-polysaccharide is double-diluted Coated ELISA plates, 100 μl/well, coated overnight at 4 °C, patted dry the next day, washed twice, blocked at 37 °C for 1 h, and patted dry; 100 μl of the double-diluted cell supernatant was added to each well, and blocked at 37 °C Incubate for 1 h and wash 3 times with PBST; goat anti-rabbit IgG labeled with horseradish peroxidase was added to each well and incubated for 1 h and washed 4 times with PBST; finally, 100 μl of TMB chromogenic solution was added to each well, and the reaction was performed in the dark for 15 min. The absorbance value at the wavelength of 450nm detected by the instrument; more preferably, in the screening, the 100-fold dilution of the mouse serum before fusion is used as a positive control, and the blank medium is used as a negative control, and the OD ₄₅₀ value of the cell culture supernatant is screened out. The positive parent cell line with the highest OD ₄₅₀ value was 2.1 times higher than the negative control.

In one embodiment of the present invention, the step (3) includes: taking the spleen of the immunized mouse in step (2) under aseptic conditions and grinding to prepare a cell suspension, which is mixed with SP2/0 myeloma cells according to 5: After mixing in the ratio of 2, centrifuge at 600-1000rpm for 5-10min, add 1ml PEG 1450 solution dropwise, then add RPMI-1640 medium to 20ml, stop the reaction, centrifuge at 600-1000rpm for 5-10min, use 20% fetal bovine serum The RPMI-1640 medium was resuspended and cultured in a 96-well cell culture plate with 200 μl per well. The next day, the HAT medium was added to dilute to the working concentration, and cultured in a 37°C, 5% CO ₂ incubator until cell clusters grew. Cell supernatants were assayed.

In one embodiment of the present invention, the step (4) includes: using an indirect ELISA method, using 100-fold dilution of the mouse serum before fusion as a positive control, and using a blank medium as a negative control, screening out those with a high OD ₄₅₀ value. The positive parent cell line with the highest OD ₄₅₀ value 2.1 times of the negative control was expanded to a 24-well plate, cultured to 80% full, and diluted to contain 1 hybridoma cell per 200 μl by limiting dilution method, 37°C, 5% Cultivate in a CO ₂ incubator until the cell mass reaches 80%-90%, and then measure the cell supernatant; repeat the above dilution culture method three times to determine the final positive cell line 5B3B9 cell line.

Preferably, in step (6), 10% DMSO+90% fetal bovine serum is used as a freezing medium, and the hybridoma cells identified in step (5) are cryopreserved in liquid nitrogen.

The present invention also provides a C-Ps monoclonal antibody, which is produced by the above hybridoma cell line 5B3B9.

The C-Ps monoclonal antibody is prepared by the following method:

The suspension of the above hybridoma cells was taken and injected into Balb/C mice intraperitoneally. After the abdomen of the mice was enlarged, the mice were sacrificed, the ascites fluid was collected, centrifuged, and the supernatant was collected and purified to obtain the C-Ps monoclonal antibody.

Preferably, the dose of the hybridoma cell suspension injected into the mouse abdominal cavity is 0.1×10 ⁶ -5×10 ⁶ cells; more preferably 1×10 ⁶ cells;

In one embodiment of the present invention, the C-Ps monoclonal antibody is prepared by the following method:

(1) Take 6-8 week old Balb/C mice, and inject 0.5 ml of incomplete Freund's adjuvant into the mouse intraperitoneal cavity with a syringe;

(2) Hybridoma cells were cultured for about 7 days, the cultured cells were pipetted down, centrifuged (600-1000r/min, 8min), the supernatant was discarded, suspended with serum-free RPMI-1640 medium, and the number of cells was adjusted to 2×10 ⁶ , intraperitoneal injection of 0.5ml/piece;

(3) 8-15 days after the mice were inoculated with hybridoma cells, after the abdomen was enlarged, the mice were killed by pulling their necks immediately, and the abdominal skin was disinfected with alcohol cotton wool, and ascites was sucked with a syringe in a sterile environment;

(4) Mix the collected ascites, centrifuge (9500r/min, 30min) for 2 times, and collect the supernatant;

(5) Purify the antibody in the supernatant by Protein-A affinity chromatography.

The present invention also provides an application of the above-mentioned C-Ps monoclonal antibody in the detection and analysis of C-Ps.

The present invention also provides a C-Ps detection kit, the kit includes a C-Ps monoclonal antibody;

The C-Ps monoclonal antibody may be the monoclonal antibody against C-Ps disclosed in the prior art, or the above-mentioned C-Ps monoclonal antibody of the present invention; preferably, the above-mentioned C-Ps monoclonal antibody.

Preferably, the above-mentioned kit also includes a polyclonal antibody and/or a second antibody to C-Ps. The C-Ps polyclonal antibody can be purified from the serum of an animal after immunizing it with an antigen, and those skilled in the art can It is prepared according to the conventional production method of polyclonal antibody, which is not limited in the present invention; the second antibody is an antibody that can specifically bind to C-Ps monoclonal antibody or polyclonal antibody, that is, the anti-C-Ps antibody. Antibody;

Preferably, the tracer label in the above-mentioned kit is selected from: radioisotopes, enzymes, fluorescein, colloidal gold, chemical or bioluminescence systems, and the labeled substance can be antigen or antibody; more preferably, colloidal gold is the label thing.

In a preferred embodiment of the present invention, the kit includes a colloidal gold test strip, and the test strip includes a sample pad, a colloidal gold pad, a chromatography membrane, a water-absorbing pad, and a bottom plate, and the colloidal gold The pad is coated with a colloidal gold-labeled antibody, and the chromatographic membrane is coated with a detection antibody and a quality control antibody; the labeled antibody is a C-Ps polyclonal antibody, and the detection antibody is a C-Ps monoclonal antibody. The control antibody is a secondary antibody; or, the labeled antibody is a C-Ps monoclonal antibody, the detection antibody is a C-Ps polyclonal antibody, and the quality control antibody is a secondary antibody; preferably, the labeled antibody is C-Ps A polyclonal antibody, the detection antibody is a C-Ps monoclonal antibody;

Preferably, the quality control antibody is a goat anti-rabbit IgG antibody;

Preferably, the bottom plate is a polyvinyl chloride (PVC) bottom plate; and/or, the colloidal gold pad is a glass fiber membrane; and/or, the chromatography membrane is a nitrocellulose (NC) membrane, The NC film is coated with a detection line and a quality control line; and/or, the sample pad is a glass fiber membrane; and/or, the absorbent pad is absorbent paper;

Preferably, the distance between the detection line and the quality control line is 2-4mm;

Preferably, the sample pad, the colloidal gold pad, the chromatography film and the water-absorbing pad are attached to the bottom plate in sequence; the non-spotted surface of the chromatography membrane is attached to the bottom plate; and/or, the The overlap between the colloidal gold pad and the chromatography membrane is 1-3mm, preferably 1mm; and/or, the overlap between the water-absorbing pad and the chromatography membrane is 1-3mm, preferably 2mm; and/or, the sample The overlap between the pad and the colloidal gold pad is 1-3mm, preferably 2mm;

Preferably, the width of the colloidal gold test paper is 2-6mm, preferably 4mm.

In another embodiment of the present invention, the kit uses an enzyme as a marker, which can be used for quantitative detection of C-Ps, and the kit further includes: a solid phase carrier, a coating solution, a washing solution, a blocking solution liquid, substrate solution, etc., those skilled in the art can choose the form and material of the solid phase carrier according to actual needs (forms such as microplates, small test tubes, microbeads or membranes, etc., materials such as polystyrene, polyvinyl chloride, polystyrene Propylene, etc.), coating buffer (such as carbonate buffer, Tris-HCl buffer), washing solution (such as PBS, PBST), blocking solution (such as BSA, gelatin, nonfat dry milk, calf serum, Casein, Tween) -20, etc.) solute composition, pH, concentration, etc., as well as selection and addition of other reagents, etc., which are not limited in the present invention.

Those skilled in the art can also use other labeling methods (such as radiolabeling, fluorescent labeling, chemiluminescence labeling, etc.) and immunodetection analysis methods to prepare corresponding detection kits for the characterization of C-Ps by using the C-Ps monoclonal antibody of the present invention and quantitative detection, the kit is also within the protection scope of the present invention.

The present invention also provides an application of the above-mentioned C-Ps detection kit in C-Ps detection and analysis, wherein the detection and analysis includes qualitative detection and quantitative detection.

The hybridoma cell line 5B3B9 provided by the present invention can stably secrete C-Ps monoclonal antibody. The C-Ps monoclonal antibody provided by the present invention has strong specificity and simple preparation method, and can be used for the immunological detection and analysis of C-Ps. The C-Ps detection kit prepared by the monoclonal antibody, especially the colloidal gold immunodetection system, is convenient to use, has high detection accuracy and sensitivity, and has good stability.

Description of drawings

Fig. 1 shows the SDS-PAGE electrophoresis diagram of Example 2 of the present invention;

Figure 2 is a schematic diagram of the structure of the colloidal gold immunoassay strip in Example 4 of the present invention; wherein, 1- sample pad, 2- colloidal gold - Detection line T, 7- Quality control line C.

Biological deposit:

Hybridoma cell line 5B3B9,

On July 21, 2016, it was deposited in the General Microbiology Center of China Microorganism Culture Collection Management Committee, and the deposit number is CGMCC No.12694.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1 Preparation, screening and identification of hybridoma cells

1 Materials and methods

1.1 Materials

Cell culture medium and additives: RPMI-1640, newborn bovine serum, HAT screening medium, PEG1450 solution;

Reagents: mouse rapid immune adjuvant, standard C-Ps; SDS-PAGE drug;

Antibody: horseradish peroxidase (HRP)-labeled goat anti-mouse IgG;

Cell line: SP2/0AG14 mouse myeloma cells;

Equipment: CK41 inverted phase contrast microscope; carbon dioxide incubator; 96-well polystyrene microtiter plate; 24-well culture plate; electrophoresis and immunoblotting equipment.

1.2 Methods

1.2.1 Preparation of antigens

Streptococcus pneumoniae (ATCC39938, purchased from ATCC) was cultured on Columbia blood plate (Columbia medium containing 10% sheep blood) for 18-20 hours, washed with sterile saline to prepare bacterial suspension, and the bacterial concentration was adjusted to 2× by hemocytometer. 10 ⁸ /ml, heat inactivated.

1.2.2 Preparation of hybridoma cells

1.2.2.1Balb/C mice immunization

4-6 weeks old Balb/C female mice were selected and immunized with the antigen prepared in the previous step, and the immunization dose was 100 μl/mice. Immunize once every 12-16 days, and the same dose is immunized three times.

1.2.2.2 SP2/0 cell fusion

Under sterile conditions, the spleen of the mice after the previous immunization was ground to prepare a cell suspension, mixed with SP2/0 cells in a ratio (volume) of 10:1-10:3, centrifuged at 600-1000rpm for 5-10min, dropwise Add 1-2ml PEG1450 solution, then add RPMI-1640 medium to 20ml, stop the reaction, centrifuge at 600-1000rpm for 5-10min, resuspend in RPMI-1640 medium containing 20% fetal bovine serum, and culture in 96-well cell culture plate , 200 μl per well, the next day, add HAT medium to dilute to the working concentration, cultivate in a 37°C, 5% CO ₂ incubator, and measure the antibody in the cell supernatant after the cell mass is grown.

1.2.2.3 Screening of hybridoma cells

Antibodies were determined by indirect ELISA. Using carbonate buffer as coating solution, the coated antigen standard C-polysaccharide was double-diluted and coated on ELISA plate, 100 μl/well, coated overnight at 4°C, and shot the next day. Dry, wash the plate twice, block at 37°C for 1 h and pat dry; add 100 μl of 3-fold diluted cell supernatant to each well (1:100, 1:300, 1:900, 1:2700, 1:8100, 1:24300, 1:72900, 1% BSA as blank control), 100-fold dilution of mouse serum before fusion, blank medium, blocked and incubated at 37°C for 1 h, washed 3 times with PBST; horseradish peroxidase-labeled sheep Anti-rabbit IgG was added to each well and incubated for 1 h, washed 4 times with PBST; finally, 100 μl of TMB chromogenic solution was added to each well, reacted in the dark for 15 min, the reaction was terminated, and the absorbance value at a wavelength of 450 nm was detected by a microplate reader. The 100-fold dilution of the mouse serum before fusion was used as the positive control, and the blank medium was used as the negative control, and the positive mother cell line with the OD _{450 value 2.1 times higher than the negative control and the highest OD 450 value} was screened out, and expanded to 24-well plates. Cultivated to 80% confluent, diluted to contain 1 hybridoma cell per 200 μl by limiting dilution method, spread 5 96-well plates, cultivated in 37°C, 5% CO ₂ incubator, cultivated to 80% confluent, wait for growth Cell supernatant antibodies were measured after the cell pellet was released. Repeat the above dilution culture method three times to determine the final positive cell line 5B3B9 cell line that can stably secrete C-Ps monoclonal antibody.

1.2.2.4 Identification and cryopreservation of hybridoma cells

The 5B3B9 cell line after colonization was cultured to 80%-90% of the 10cm cell culture dish, the cell supernatant was aspirated, and by indirect ELISA method, 100-fold dilution of mouse serum before fusion was used as a positive control, and blank medium was used as a positive control. As a negative control, the identified cells are positive (refer to 1.2.2.3 Hybridoma cell screening process), and 10% DMSO+90% fetal bovine serum is used as a freezing medium, and the identified hybridoma cells are cryopreserved in liquid nitrogen.

Example 2 Preparation, purification and characteristic analysis of C-Ps monoclonal antibody

1 Materials and methods

1.1 Materials

Cell culture medium: RPMI-1640 and newborn bovine serum in a ratio of 9:1-7:1 to prepare a complete culture medium for hybridoma cells;

Reagent: Freund's incomplete adjuvant; SDS-PAGE drug;

Experimental animal: Balb/C mice of 6-8 weeks old strictly sterilely bred;

Antibody: horseradish peroxidase (HRP)-labeled goat anti-mouse IgG;

Hybridoma cell line: hybridoma cell line 5B3B9 prepared in Example 1;

Equipment: CK41 inverted phase contrast microscope; carbon dioxide incubator; 96-well polystyrene microtiter plate; 24-well culture plate; electrophoresis and immunoblotting equipment.

1.2 Methods

1.2.1 Preparation of monoclonal antibodies

1.2.1.1 Recovery of hybridoma cells

Take out the hybridoma cell line 5B3B9 stored in the -80°C ultra-low temperature refrigerator, put it in a 37°C water bath to thaw quickly, and centrifuge at 800-1000 r/min for 5 min, discard the supernatant, and use RPMI-1640 complete medium to prepare a cell suspension. The solution was transferred to a 24-well culture plate and cultured in a 37°C, 5% _CO2 incubator. Change the culture medium the next day and continue the culture. The cells to be recovered grow well, with plump shape, translucent and round grains, and subculture for 2-4 days.

1.2.1.2 Preparation of ascites

(1) Take 6-8 week old Balb/C mice, and inject 0.5ml of incomplete Freund's adjuvant into the mice intraperitoneally with a syringe;

(2) Hybridoma cells were cultured for about 7 days, the cultured cells were pipetted down, centrifuged (800r/min, 8min), the supernatant was discarded, suspended with serum-free RPMI-1640 medium, and the number of cells was adjusted to 2× 10 ⁶ /ml, intraperitoneal injection of 0.5ml/mice;

(3) 8-15 days after the mice were inoculated with hybridoma cells, after the abdomen was enlarged, the mice were killed by pulling their necks immediately, and the abdominal skin was sterilized with alcohol cotton, and the ascites was sucked with a syringe in a sterile environment;

(4) Mix the collected ascites, centrifuge (9500r/min, 30min) for 3 times, collect the supernatant, do not suck the insoluble material in the uppermost layer of the centrifuge tube, and place it in a -80°C ultra-low temperature freezer for future use.

1.2.2 Purification and concentration identification of ascites

1.2.2.1 Purification and identification of ascites

Antibodies were purified by Protein-A affinity chromatography. The purity of the purified monoclonal antibody was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method, as shown in Figure 1, the left lane is the Marker protein, and the right lane is the above monoclonal antibody , the band is clear at around 54KD, and the concentration of monoclonal antibody is higher.

1.2.2.2 Determination of monoclonal antibody concentration after purification

The purity of the polyclonal antibody was determined by the Bradford kit, and the operation was performed according to the kit instructions.

1.2.2.3 Determination of monoclonal antibody titer after purification

The titer of the purified monoclonal antibody was measured by indirect ELISA method. Using carbonate buffer as the coating liquid, the coated antigen standard C-polysaccharide was double-diluted and coated on an ELISA plate, 100 μl/well, 4°C. Coat overnight, pat dry the next day, wash the plate twice, block at 37°C for 1 h and pat dry; add 100 μl of monoclonal antibodies (1:100, 1:300, 1:900, 1:100, 1:900, 1:900, 1:900, 1:900) to each well in turn. : 2700, 1:8100, 1:24300, 1:72900, 1% BSA as blank control), blocked and incubated at 37°C for 1 h, washed 3 times with PBST; horseradish peroxidase-labeled goat anti-rabbit IgG was added to each well and incubated 1 h, washed 4 times with PBST; finally, 100 μl of TMB color developing solution was added to each well, reacted in the dark for 15 min, and the reaction was terminated. In 2.1, the maximum dilution of the antibody is the titer of the monoclonal antibody, and the detection is shown in Table 1. It can be seen from the result data in Table 1 that the titer of the above-mentioned monoclonal antibody is relatively high, greater than 1:24300.

Table 1 Determination results of monoclonal antibody titer after purification

Example 3 Preparation, purification and characteristic analysis of C-Ps polyclonal antibody

1 Materials and methods

1.1 Materials

Medium: Columbia medium, sheep blood;

Reagents: rapid immune adjuvant, enzyme-labeled goat anti-rabbit IgG;

Experimental animal: New Zealand white rabbit (female, 2-3kg);

Strain: Streptococcus pneumoniae (ATCC 39938, purchased from ATCC).

1.2 Methods

1.2.1 Preparation of antigens

Refer to the preparation process of the antigen in Example 1.

1.2.2 Preparation of polyclonal antibodies

1.2.2.1 Immunization of New Zealand rabbits

New Zealand white rabbits were subcutaneously immunized at 4-6 o'clock. Immunize once every 12-16 days, 2-4 times with the same dose.

1.2.2.2 Determination of the titer of unpurified polyclonal antibodies

The titer of rabbit polyclonal antibody was determined by indirect ELISA method. Using carbonate buffer as coating solution, the coated antigen standard C-polysaccharide was double-diluted and coated on ELISA plate, 100 μl/well, overnight at 4°C. Coated, patted dry the next day, washed twice, blocked at 37°C for 1 h and patted dry; 100 μl of rabbit antiserum (1:100, 1:300, 1:900, 1:900, 1:900, 1:900) was added to each well in turn. 2700, 1:8100, 1:24300, 1:72900, 1% BSA as blank control), blocked and incubated at 37°C for 1 h, washed 3 times with PBST; phosphatase-labeled goat anti-rabbit IgG diluted 10,000 times was added to each well and incubated 1 h, washed 4 times with PBST; finally, 100 μl of TMB color developing solution was added to each well, and the reaction was performed in the dark for 15 min. After the reaction was terminated, the absorbance value at a wavelength of 450 nm was detected by a microplate reader, and the P/N value of each well was calculated, P/N When ≥2.1, the maximum dilution of the antibody is the titer of the polyclonal antibody.

1.2.2.3 Collection of rabbit antiserum

After the serum titer determined by the indirect ELISA method meets the purification requirements, the rabbit serum is collected. The specific method is: anesthetize the New Zealand rabbit, inject anticoagulant into the ear vein, intubate the carotid artery for bloodletting, and store the test tubes at 4°C temporarily after packaging. 3h, then centrifuged at 9500r/min for 20min, collected the supernatant, aliquoted, and stored at -80°C.

1.2.3 Purification and concentration identification of rabbit antiserum

1.2.3.1 Purification and identification of rabbit antiserum

Antibodies were purified by Protein-A affinity chromatography. The purity of the purified polyclonal antibody was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

1.2.3.2 Determination of polyclonal antibody concentration after purification

The purity of the polyclonal antibody was determined by the Bradford kit, and the operation was performed according to the kit instructions.

1.2.3.3 Determination of polyclonal antibody titer after purification

The determination of the antibody titer of the polyclonal antibody after purification adopts the indirect ELISA method, using carbonate buffer as the coating liquid, and the coated antigen standard C-polysaccharide is double-diluted to coat the ELISA plate, 100 μl/well, 4 Coat overnight at ℃, pat dry the next day, wash the plate twice, block at 37 ℃ for 1 h and pat dry; add 100 μl of rabbit antiserum (1:100, 1:300, 1:900, 1: 2700, 1: 8100, 1: 24300, 1: 72900, 1% BSA as blank control), blocked and incubated at 37°C for 1 h, washed 3 times with PBST; diluted 10,000-fold horseradish peroxidase-labeled goat antibody Rabbit IgG was added to each well and incubated for 1 h, and washed 4 times with PBST; finally, 100 μl of TMB chromogenic solution was added to each well, and the reaction was performed in the dark for 15 min. After the reaction was terminated, the absorbance value at a wavelength of 450 nm was detected by a microplate reader, and the P/P of each well was calculated. N value, when P/N ≥ 2.1, the maximum dilution of the antibody is the titer of the polyclonal antibody.

Example 4 Establishment of colloidal gold immune system

1 nanometer gold preparation

Add 1% auric acid aqueous solution to purified water (the preparation time should not exceed half a year, and store at 4°C), stir and boil on a heating magnetic stirrer, add dropwise trisodium citrate aqueous solution (currently prepared), boil for 15 minutes, the solution The color gradually changed from light yellow to black and finally to wine red. After cooling completely, scan the maximum absorption peak on the visible spectrophotometer, and there is a maximum absorption peak at 525-535 nm.

2 colloidal gold markers

Use potassium carbonate solution to adjust the pH of the gold solution, add the polyclonal antibody prepared in Example 3 dropwise to the above gold solution, stir for 20-40 minutes, then add BSA dropwise to the above mixed solution, stir for 15 minutes, seal the colloidal gold, PEG (molecular weight 20000) was added dropwise to the mixed solution to a final concentration of 0.1%, and stirred for 15 minutes to increase the stability of colloidal gold. Centrifuge, speed 8000-9000rpm, 4°C, 0.5-1 hour, after centrifugation, suspend the precipitate and discard the supernatant, then add the diluent and mix well.

3 Colloidal Gold Pad Preparation

The preparation of colloidal gold pads is mainly carried out by immersion gold. The glass fiber is completely immersed in the above-marked gold solution, and the immersed colloidal gold strips are placed at 37 °C to dry, and the humidity is constant at 20%, then put away and sealed for storage. Note: When using, just cut the whole strip, pay attention to discard the edges of both ends of the gold strip, because there is edge effect.

4NC film preparation

The C-Ps monoclonal antibody and goat anti-rabbit IgG prepared in Example 2 were uniformly spread on the NC membrane at the appropriate concentration as the detection line T and the quality control line C, oven-dried, 37 ℃ for 2 hours to the humidity below 20% , and then sealed and packaged for later use.

5 Assembly of test strips

(1) Paste the non-spotted surface of the NC film on the PVC bottom plate;

(2) The colloidal gold pad is pasted on the top of the NC film, covering the NC film by 1mm;

(3) The absorbent pad is pasted on the top of the NC film, covering the NC film by 2mm;

(4) The sample pad is pasted above the colloidal gold pad, covering the colloidal gold pad by 2mm;

(5) Cut the pasted detection board into 4mm wide strips with a paper cutter;

The structure of the test strip is shown in Figure 1. When in use, drop the sample to be tested on the sample pad, and the sample flows in the direction of the absorbent pad, passing through the colloidal gold pad and the NC film in turn; when both the detection line and the quality control line develop color, the detection sample is Positive, when the quality control line develops color but the detection line does not develop color, the test sample is negative.

6Test results

The above test strips were used to test samples with antigen concentrations of 1000, 100, 50, 25, 12.5, 6.25, 3.25, 1.5, and 0.75 ng, respectively, and a negative control group was set. The test results are shown in Table 2, where "+" indicates color development, the number of which represents the color development effect, and "-" indicates no color development. As can be seen from the data in Table 2, the quality control lines in the test are all colored and have good color rendering effects, that is, all detections are effective tests, and the detection sensitivity of the above-mentioned test strips can reach the ng level, the colloidal gold test paper of the present invention. The strip can be used for the qualitative detection of C-Ps, and the detection accuracy and sensitivity are high, and the stability is good.

Table 2 Colloidal gold test strip test results

The above-mentioned test strips use the C-Ps monoclonal antibody as the coating antibody, the C-Ps polyclonal antibody as the labeling antibody, and the secondary antibody as the control to detect the antigen. Antigen is a coating antibody, the above-mentioned monoclonal antibody is used as a labeled antibody, and the secondary antibody is used as a control to detect the antigen.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention. within.

Claims (7)

1. the hybridoma cell strain that a kind of deposit number is CGMCC No.12694.

2. a kind of C-Ps monoclonal antibody, which is characterized in that the monoclonal antibody is by hybridoma described in claim 1 Strain generates.

3. a kind of detection kit of C-Ps, the kit includes C-Ps monoclonal antibody as claimed in claim 2.

4. kit as claimed in claim 3, which is characterized in that the kit further include C-Ps polyclonal antibody and Secondary antibody, the secondary antibody are that can specifically bind with the C-Ps monoclonal antibody and the C-Ps polyclonal antibody Antibody.

5. kit as claimed in claim 4, which is characterized in that the kit includes colloidal gold strip, described Test strips include sample pad, colloidal gold pad, chromatographic film, water absorption pad, bottom plate, and the colloidal gold pad is coated with colloid gold label Antibody, the chromatographic film are coated with detection antibody and Quality Control antibody；The antibody of the colloid gold label is that C-Ps is polyclonal Antibody, detection antibody are C-Ps monoclonal antibody as claimed in claim 2, and Quality Control antibody is secondary antibody；Or, the colloidal gold mark The antibody of note is C-Ps monoclonal antibody as claimed in claim 2, and detection antibody is C-Ps polyclonal antibody, and Quality Control antibody is two It is anti-；The secondary antibody is the antibody that can be specifically bound with the C-Ps monoclonal antibody and the C-Ps polyclonal antibody.

6. kit as claimed in claim 5, which is characterized in that the antibody of the colloid gold label is C-Ps Anti-TNF-α Body, the detection antibody are C-Ps monoclonal antibody as claimed in claim 2, and the Quality Control antibody is anti-for goat anti-rabbit igg Body.

7. kit as claimed in claim 5, which is characterized in that the sample pad, colloidal gold pad, chromatographic film, water absorption pad Successively it is attached on bottom plate；And/or the non-point sample face paste of the chromatographic film invests on bottom plate；And/or the colloidal gold It pads and 1-3mm be overlapped between chromatographic film；And/or be overlapped 1-3mm between the water absorption pad and chromatographic film；And/or it is described Be overlapped 1-3mm between sample pad and colloidal gold pad；And/or the width of the colloid gold test paper is 2-6mm.