CN115572716B

CN115572716B - Monoclonal antibody against group III-B streptococcus capsular polysaccharide and hybridoma cell strain

Info

Publication number: CN115572716B
Application number: CN202211214570.1A
Authority: CN
Inventors: 黄镇; 钱雯; 陈南萍; 陈玉秋; 吴凯; 苏贵超; 张胜祥
Original assignee: Yuxi Walvax Biotechnology Co ltd; Walvax Biotechnology Co ltd
Current assignee: Yuxi Walvax Biotechnology Co ltd; Walvax Biotechnology Co ltd
Priority date: 2022-09-30
Filing date: 2022-09-30
Publication date: 2023-06-16
Anticipated expiration: 2042-09-30
Also published as: CN115572716A

Abstract

The invention discloses a monoclonal antibody of group III-B streptococcus capsular polysaccharide and a hybridoma cell strain. The hybridoma cell line is named as WV-GBSIII-04, and the preservation number is as follows: CCTCCNO: c202028, said monoclonal antibody being secreted by said hybridoma cell line. The invention can be used for the diagnosis of the group B streptococcus capsular polysaccharide and the quantitative detection of the group III group B streptococcus capsular polysaccharide in the research and development process of the group B streptococcus vaccine and the combined vaccine, has strong specificity, can reach the Nake level of detection sensitivity, improves the detection sensitivity and is simple to operate.

Description

Monoclonal antibody against group III-B streptococcus capsular polysaccharide and hybridoma cell strain

Technical Field

The invention relates to the field of monoclonal antibodies, in particular to an anti-group III group B streptococcus capsular polysaccharide monoclonal antibody and a hybridoma cell strain.

Background

Group B streptococcus is a common conditional pathogen, a common pathogen that causes puerperal fever, and regardless of the mode of delivery by the pregnant woman, these bacteria can cause puerperal infections. Most of the puerperal infections caused by group B streptococcus are single bacterial infections, and symptoms of the puerperal infections are early, high fever (> 38.8 ℃), tachycardia and the like.

Group B streptococcus is also a main pathogenic bacteria causing bacteremia and wound infection of pregnant women, and can also generate streptococcal pneumonia, meningitis, liver abscess, septicemia and the like, and has high mortality rate which can reach 29% -52%.

Neonatal GBS infection has also attracted widespread attention by global health authorities, and neonatal and infant GBS infection is currently classified into two types based on the differential classification of the onset time and clinical manifestations of the infant. (1) early onset infection: the infant developed within 7d after birth and developed within 24h after birth, and the main clinical manifestations are pneumonia and septicemia, wherein GBS pneumonia can have severe symptoms such as cyanosis, apnea, respiratory distress, etc. (2) late onset infection: the infant takes place from 7d to 3 months after birth, usually in term infants, and usually has latent morbidity, and the main clinical manifestations are fever, somnolence, intracranial hypertension and the like.

With the demonstration of the safety of the vaccinated pertussis vaccine during gestation, the development of GBS vaccine is advanced. The currently widely used conjugate vaccine has polysaccharide covalently coupled with a protein carrier with high immunogenicity, so that the immunogenicity is greatly improved, and the protein carrier which is more commonly used recently is CRM197. This large random double blind trial by Madhi scholars et al, a GBS capsular polysaccharide trivalent vaccine conjugated to CRM197, comprising serotypes Ia, ib and III, could cover about 80% of GBS-infected neonatal invasive diseases worldwide.

However, as a plurality of serotypes of GBS have partially identical epitopes and the GBS capsular polysaccharide-protein conjugate vaccine has the possibility of space shielding effect in the preparation process, some epitopes of polysaccharide are shielded by protein; so that group III-B streptococcus capsular polysaccharide cannot be detected accurately, no monoclonal antibody against group III-B streptococcus capsular polysaccharide is available.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an anti-group III streptococcus group B capsular polysaccharide monoclonal antibody and a hybridoma cell strain, and the hybridoma cell strain provided by the invention is proved by a transfer stability experiment: can stably and specifically secrete monoclonal antibodies aiming at group III-B streptococcus capsular polysaccharide.

In order to solve the technical problems, the technical scheme of the invention is as follows: a hybridoma cell line secreting anti-group III streptococcus capsular polysaccharide monoclonal antibodies, designated WV-GBSIII-04, deposited with the chinese collection of typical cultures, accession number: CCTCCNO: c202028, 12 months and 11 days of the preservation time 2020, wherein the preservation address is China, the university of Wuhan and Wuhan.

In a second aspect, the invention also provides a monoclonal antibody against group III-B streptococcus capsular polysaccharide, which monoclonal antibody is secreted by the hybridoma cell strain.

As a further description of the above scheme: the monoclonal antibody can specifically identify group III-B streptococcus capsular polysaccharide, group III-B streptococcus capsular polysaccharide conjugate and conjugate vaccine containing group III-B streptococcus capsular polysaccharide.

As a further description of the above scheme: the monoclonal antibodies are obtained after mice are immunized with a combination of group III group B Streptococcus capsular polysaccharide and diphtheria non-toxic variant protein (CRM 197).

In a third aspect, the invention also provides application of the monoclonal antibody in detecting the content of the group III-B streptococcus capsular polysaccharide.

In a fourth aspect, the invention also provides a detection kit containing the monoclonal antibody, which is used for detecting the capsular polysaccharide content of group III-B streptococcus.

The working principle of the invention is as follows: the hybridoma cell strain capable of stably secreting the specific antibody of the group III streptococcus capsular polysaccharide is screened and preserved, and the hybridoma cell strain is proved by a pass-through stability experiment to stably and specifically secrete the monoclonal antibody aiming at the group III streptococcus capsular polysaccharide. The hybridoma cell strain is inoculated into the abdominal cavity of a mouse, and the prepared ascites monoclonal antibody is high in purity and high in antibody titer after being purified by a protein chromatography method, meets the requirements of subsequent experiments, and is strong in specificity, high in sensitivity and good in repeatability by using an indirect competition ELISA method established by the monoclonal antibody. The monoclonal antibodies specifically recognize group III group B Streptococcus capsular polysaccharides, group B Streptococcus capsular polysaccharide conjugates, or vaccines. The method can not cause the condition that the specific recognition cannot be realized due to the shielding effect of proteins in the combined vaccine, and does not generate cross reaction with Ia, ib, II, IV and V-type polysaccharide and Tetanus Toxoid (TT) carrier and diphtheria toxin nontoxic variant (CRM 197) carrier; the detection sensitivity can reach the Nake level, the detection sensitivity is improved, the operation is simple, and the specificity is strong.

Compared with the prior art, the invention has the following beneficial effects: the invention can be used for the diagnosis of the group B streptococcus capsular polysaccharide and the quantitative detection of the group III group B streptococcus capsular polysaccharide in the research and development process of the group B streptococcus vaccine and the combined vaccine, has strong specificity, can reach the Nake level of detection sensitivity, improves the detection sensitivity and is simple to operate.

The hybridoma cell strain preservation information provided by the invention is as follows:

hybridoma cell lines:

the classification is named as hybridoma cell strain WV-GBSIII-04;

preserving in China center for type culture Collection;

preservation number: CCTCCNO: c202028;

preservation time: 12 months 11 days 2020;

preservation address: chinese, university of martial arts, martial arts.

Drawings

FIG. 1 is a standard graph of Elisa.

Detailed Description

The following describes the technical scheme of the present invention in further detail with reference to the accompanying drawings and specific examples, but the present invention is not limited to the following technical scheme.

Example 1

1. Immunization of mice

Immune antigen: group III Streptococcus capsular polysaccharide and CRM197 conjugate stock solution;

immunization program and inoculation mode: 0. 14, 28 days, mice inguinal subcutaneously;

immunization dose: 1 ug/dose/needle;

mouse serum titer detection: an indirect Elisa method is adopted; taking 1ug/ml group III streptococcus capsular polysaccharide as a coating antigen, coating a 96-well ELISA plate at 100 ul/well, and refrigerating overnight at 2-8 ℃; after washing the plates, 100 ul/well of 5% nonfat dry milk solution was used and blocked at 37℃for 1h; after washing the plate, adding gradient diluted serum, and incubating for 1h at 37 ℃; after washing the plate, adding 100 ul/hole (working concentration 1:10000) of HRP-labeled goat anti-mouse IgG antibody, and incubating for 1h at 37 ℃; after washing the plate, adding TMB color development liquid into the plate for 100 ul/hole, and reacting for 20min; the reaction was terminated by adding 0.02mol/L sulfuric acid, and the result was analyzed by an ELISA reader OD450 nm.

Mice with higher serum titers (usually not lower than 1:10000 are required) are selected and boosted 3 days before fusion, and the antigen is group III-B streptococcus capsular polysaccharide, and the dosage is 50ug/0.5 ml/mouse, and the mice are injected intraperitoneally.

2. Cell fusion screening:

SP2/0 cell preparation:

the SP2/0 cells frozen by liquid nitrogen are subjected to resuscitative culture, passaged to a T75 bottle, cultured for 2-3 days, the cells grow to be full of 1 layer at the bottom of the bottle, and the liquid is changed 1 day before fusion, so that the cells are in the logarithmic phase and have good cell state.

During fusion, the T75 flask cells were collected in suspension in a 50ml centrifuge tube using 1640 culture medium, centrifuged at 1500rpm/min for 5min, the supernatant was discarded, and the cells were resuspended in 10ml1640 and then sampled and counted for further use.

Feeder cell preparation: the same strain of healthy Balb/c mice were used for peritoneal macrophages.

The preparation method comprises the following steps: mice were sacrificed by cervical removal and then soaked in 75% alcohol and sterilized for 3 minutes. In a biosafety cabinet, 1 cell culture plate is arrangedTearing the skin of the mouse in a box, exposing the peritoneum, slightly lifting the peritoneum of the mouse by holding a sterile small forceps with a left hand, injecting the culture solution into the abdominal cavity of the mouse by holding a 5ml disposable syringe with 3-5 ml1640 culture solution with a right hand, swaying the mouse to wash the abdominal cavity for about 1min, sucking out the flushing solution, adding Fetal Bovine Serum (FBS) into the culture medium at a ratio of 10% -20% (V/V), adding HAT into the culture medium at a ratio of 1% (V/V), mixing uniformly, paving a 96-well plate, 100 ul/well, and placing CO ₂ In an incubator, at 37.0deg.C, 5.0% CO ₂ And (5) culturing under the condition.

Typically, from 2 to 3 96-well plates can be prepared from 1 mouse.

Preparation of immune spleen cells:

mice immunized for 3 days with group III group B streptococcus capsular polysaccharide were sacrificed by cervical removal and then soaked in 75% alcohol and sterilized for 3 minutes. Placing in a biosafety cabinet, placing in a box with 1 cell culture plate, tearing open the skin of a mouse, exposing the peritoneum, holding a small sterile forceps with a left hand to slightly lift the peritoneum of the mouse, holding a small sterile scissors with a right hand to cut off the peritoneum, exposing the peritoneum, replacing a small sterile forceps with a left hand to clamp the spleen, replacing a sterile scissors with a right hand to remove fascia, taking out the spleen, placing into a cell filter screen, placing in a plate with 1640 culture solution, slightly squeezing the spleen with a disposable grinding rod, immersing the cells in the culture solution through the filter screen, repeatedly blowing and flushing with a pipette or gun head until no obvious tissue is formed, collecting spleen cell suspension in a 50ml centrifuge tube, centrifuging at 1500rpm/min for 5min, discarding supernatant, and sampling and counting the cells after resuspension with 10ml1640 for later use.

Cell fusion and HAT selection culture:

mixing the immune spleen cells and myeloma cells of the mice according to the proportion of 2:1-10:1 in a 50ml centrifuge tube, centrifuging at 1500rpm/min for 5min, discarding the supernatant, loosening the precipitated cells at the bottom of the light flick centrifuge tube, sucking 1ml of polyethylene glycol (PEG) with the temperature of 38.0 ℃ pre-heating by a 1min pipettor, slightly blowing into the cells at the bottom of the centrifuge tube within 1min, standing for 150 seconds under the water bath condition of 38.0 ℃, adding 5ml of 1640 culture solution to terminate the reaction within 1min, then adding the culture solution to 20ml,300g and 10min for centrifugation, and discarding the supernatant. The cells were washed 1 time repeatedly under the same conditions. The RPM1640 culture solution is used for re-suspending the cells according to the proportion of 10 to 20Adding Fetal Bovine Serum (FBS) into (V/V), adding HAT into (V/V) 1%, mixing, packaging into 96-well plate of existing feeder cells, 100-120 μl/well, adding CO ₂ 37.0deg.C, 5.0% CO in incubator ₂ And (5) culturing under the condition.

After fusion, the cells are selected and cultured in a culture medium containing 1% HAT, the liquid is changed for 1 time for 3-5 days, the growth of the fused cells can be observed for 7-14 days, and the cells are continuously cultured until the growth of the cells reaches about 20% of the bottom of the hole, so that fusion detection can be carried out.

Fusion detection:

the supernatant of the fusion cell culture in 96-well plates was taken and tested at 100 ul/well using the indirect Elisa method. Serum titers of mice were measured as before. Typically 2 assays are required.

The indirect enzyme-linked immunosorbent (ELISA) experiment was performed as follows:

diluting GBS III polysaccharide to 2 mug/mL, 100 mug/hole by using coating liquid, coating 96-hole ELISA plate at 4 ℃ overnight, and drying by beating 200 mug/Kong Xi plate 3 times by using PBST washing liquid in the next day; sealing with 5% skimmed milk, 200 μl/hole, sealing with 37 deg.C incubator for 1 hr, washing the plate 5 times, and drying; adding cell culture supernatant into the plate, incubating for 1h at 37 ℃ in an incubator with 100 mu L of each hole, washing the plate for 5 times, and beating to dry; adding goat anti-mouse IgG enzyme-labeled secondary antibody (1:10000) marked by horseradish peroxidase diluted with 0.01mol/L PBS (pH 7.4), incubating for 1h at 37 ℃ with 100 mu L/hole, washing the plate for 5 times, and beating to dry; then, 100. Mu.L of a developing solution was added to each well, developed in a dark place for 10 minutes, 100. Mu.L of a stop solution was added to each well, and the A450 value was measured.

Screening of hybridoma cell lines

Primary screening, namely, using GBS class III polysaccharide to coat an ELISA plate, detecting cell culture supernatant according to the indirect ELISA method, marking positive holes, changing liquid of all cloned cells, detecting once again after two days, using SP2/0 culture supernatant as negative control, judging holes with detection value/negative value of 2.1 as positive holes, screening out holes with positive detection values, using GBS six types of capsular polysaccharides of Ia, ib, II, III, IV and V respectively to coat the ELISA plate, performing positive hole specificity screening, and performing subsequent recloning and subcloning after specific positive holes which only react with III type polysaccharide but not react with other serotypes are screened out. During cloning, the above GBS class III polysaccharide, diphtheria toxin non-toxic variant (CRM 197) was used for screening.

5 96 well plates were primary screened for a total of 14 positive wells, see table 1; the 6 wells with relatively high OD values were selected from the 14 wells, further cloned and screened, and finally a cell line was stable and maintained in specificity by cloning and screening, as shown in Table 2. GBSIII-5B6-G3 cell line was deposited and subsequently studied and designated WV-GBSIII-04.

Cryopreservation and resuscitation of hybridoma cells

Cryopreservation of hybridoma cells: when the cells grow to be 80% -90% of the bottom of the culture bottle, blowing the cells to be fully suspended, centrifuging for 5min at 1500r/min, discarding the supernatant, re-suspending the cells by using cell freezing solution (30% fetal calf serum, 60% incomplete RPMI1640 culture solution, 10% DMSO) and adjusting the concentration to be 5 multiplied by 10 ⁶ ～1×10 ⁷ Split charging into freezing tube at 1 mL/tube, standing at-20deg.C for 1 hr after standing at 4deg.C for 1 hr, standing at-80deg.C for 1 hr, suspending overnight after transferring into liquid nitrogen tank, and storing in liquid nitrogen for a long time.

TABLE 1 initial detection OD ₄₅₀ Value of

TABLE 2 GBSII and GBSIII cell resuscitation titers and specific assays

EXAMPLE 2 monoclonal antibody specific detection

Test principle: the method comprises the steps of respectively coating GBS6 capsular polysaccharides, carrier proteins TT and CRM197 with corresponding concentrations by adopting an indirect ELISA method, respectively adding the same 1 strain of cell culture supernatant to combine with 6 antigens after sealing, adding an HRP-marked goat anti-mouse IgG antibody to react after washing the plates, finally developing color by using a TMB substrate system and stopping with dilute sulfuric acid, and detecting absorbance by using an enzyme-labeled instrument to judge the specificity of the cell strain to be detected.

The method comprises the following steps:

1. coating: GBS6 type capsular polysaccharides, ia, ib, II, III, IV and V were coated at 2ug/ml concentration, TT and CRM197 were coated at 1ug/ml concentration, 100 ul/well, and overnight in a refrigerator at 2-8deg.C.

2. The plate washer was used for 3 times, 200 ul/well of 5% nonfat dry milk solution was added, and the mixture was sealed in an incubator at 37℃for 1 hour.

3. The plate washer washes the plate 3 times, a proper amount of ELISA plate strips are buckled, 8 holes are needed for each cell culture supernatant, and 8 holes for negative control are arranged. Incubate for 1h at 37 ℃.

4. The plate was washed 3 times with a plate washer, and 100 ul/well (working concentration 1:10000) of HRP-labeled goat anti-mouse IgG antibody was added and incubated in an incubator at 37℃for 1h.

5. The plate is washed 3 times by a plate washing machine, 100 ul/hole of TMB color development liquid is added, and the color development is carried out for 20min in a dark place in an incubator at 37 ℃.

6. Adding 0.02mol/L dilute sulfuric acid to terminate the reaction, and performing OD (optical density) detection by an enzyme label instrument ₄₅₀ nm readings, analytical results are shown in Table 3.

TABLE 3 test results

As can be seen from table 3, the laboratory mab is GBSIII-type and does not combine with the other five types of capsular polysaccharides of GBS: ia. Ib, II, IV and V do not cross react with the common carrier proteins TT and CRM 197; the monoclonal antibody secreted by the GBSIII5B6-G3 (WV-GBSIII-04) hybridoma cell strain has better specificity.

Monoclonal antibody subclass identification

And detecting by using a commercial kit. The manufacturer: luoyang Baiolong laboratory materials center, cat: C060101. the class and subclass of monoclonal antibodies or specific affinity purified monoclonal antibodies in the mouse lymphocyte hybridoma culture supernatants are identified using a double antibody sandwich method. Coating a micro-pore plate with a secondary antibody of a common site of a mouse antibody, combining the micro-pore plate with the added mouse antibody in culture supernatant, adding HRP-marked anti-mouse antibodies of various classes and subclasses for respective reaction, finally developing color by using a TMB substrate system and stopping with dilute sulfuric acid, and detecting absorbance by using a microplate reader to judge the class or subclass of the detected monoclonal antibody.

The method comprises the following steps:

1. the kit stored at 2 to 8℃was first taken out and returned to room temperature (about 30 minutes), and then a 20X washing liquid was prepared as a working liquid with purified water (19 parts of purified water was added to each part of concentrated washing liquid).

2. Taking out the ELISA plate; and (3) a proper amount of ELISA strips are buckled, 6 holes are needed for each specimen, 6 holes are needed for positive control and 6 holes are needed for negative control. (the excess is kept in self-sealing bags, remembering to put into the desiccant)

3. The sample detection wells were each filled with 50 μl of sample diluent. Then 50 mu L of cell culture supernatant (or specific affinity purified antibody) is added into enzyme-labeled microwell plates, and 6 holes are added into each sample; positive and negative control wells were also 100. Mu.L per well with 6 wells each without the addition of the standard diluent. The plate was covered with a membrane and incubated at 37℃for 30 minutes.

4. The liquid in the plate is discarded, and after the plate is washed for 5 times, the plate is beaten dry or washed by a machine for 5 times on the water absorbing material without fiber. Then, 100 mu L of 6 enzyme-labeled secondary antibodies are respectively added into 6 holes of each specimen, and the same is true for each hole of the general positive control and the general negative control. Marking on the sample adding chart or the ELISA plate. The plate was covered with a membrane and incubated at 37℃for 30 minutes.

5. The liquid in the plate is sucked and discarded, and the plate is washed 5 times and then is beaten dry or washed 5 times on the water absorbing material without fiber. 50 mu L of each of the color reagent A and the color reagent B is added into each hole, and a new sealing plate film pasting plate is changed to develop color for 20 minutes at the temperature of 37 ℃ in the dark.

6. The kit has good specificity, the result can be observed by naked eyes, and the Ig class or subclass of the specimen can be known by looking at the enzyme-labeled secondary antibody corresponding to the hole with blue color. The Ig class or subclass of the specimen can be judged by referring to the two-wavelength measuring result of the enzyme-labeled secondary antibody corresponding to the high-value hole after the reaction stopping solution (50 mu L per hole) is used for stopping the reaction, wherein the Ig class or subclass of the specimen can be judged by referring to the two-wavelength measuring result of the enzyme-labeled secondary antibody corresponding to the high-value hole (the OD of the positive control is generally not less than 0.8, the OD of the negative control is generally not more than 0.15, the positive judging standard is that the OD of the sample is more than the OD+0.15, and the OD of the negative sample is lower than 0.05 by 0.05).

Table 4 test results

	1	2	3	4	5	6
							Antibody subclasses	IgG1	IgG2a	IgG2b	IgG3	IgM	IgA
Positive control	3.8429	3.993	4.0439	3.9721	3.8232	4.0438
							Negative control	0.0631	0.1642	0.1792	0.0394	0.0156	0.0593
GBSIII5B6-G3	0.0612	0.1514	0.1479	0.035	3.3049	0.0585

As can be seen from Table 4, the monoclonal antibodies secreted by the hybridoma cells in this example are of the IgM subtype.

Resuscitation of hybridoma cells: taking out the freezing tube from the liquid nitrogen tank, immediately putting into a 37 ℃ water bath for rapid melting, centrifuging for 5min at 1000r/min, discarding the supernatant, adding a culture solution containing 10% calf serum to resuspend cells, transferring into a culture flask, culturing in a 5% CO2 incubator at 37 ℃, returning the solution the next day, and continuing culturing.

EXAMPLE 3 preparation and potency determination of monoclonal antibodies (mouse ascites)

3.1 pretreatment of mice

3 BALB/c mice were taken from 8 weeks to 10 weeks, and 0.5 ml/c were injected with sterilized liquid paraffin, and used for inoculation of hybridoma cells after 7 days.

3.2 mice were inoculated with hybridoma cells

Blowing off the cells when the cells grow to 80% -90% of the bottom of the plate, centrifuging for 5min at 1200r/min, discarding the supernatant, and using RPMI-1640 medium suspended cells, counted, and cell number adjusted to 5×10 ⁵ ～l×10 ⁶ One week pre-treated mice, 0.5 mL/mL, were intraperitoneally injected. Note that the growth state of the mice is observed every day, and when the abdomen of the mice is obviously enlarged after about 7-10 days, ascites is extracted by a syringe, and the extraction can be continued at intervals of 1-2 days until the mice die.

3.3 isolation of monoclonal antibodies

Centrifuging the extracted ascites at 1200r/min for 5min each time, taking the supernatant, sub-packaging, and preserving at-20 ℃ to obtain the monoclonal antibody produced by the WV-GBSIII-04 cell strain.

Monoclonal antibody ascites potency detection

Detecting the titer of the monoclonal antibody by an indirect ELISA method, diluting GBS-type polysaccharide to 2 mug/mL by using a coating liquid, coating a 96-well ELISA plate at 4 ℃ overnight, using 200 mug/Kong Xi plate for 3 times by using PBST washing liquid in the next day, and performing beating drying; sealing with 5% skimmed milk, 200 μl/hole, sealing with 37 deg.C incubator for 1 hr, washing the plate 5 times, and drying; adding ascites monoclonal antibodies with different dilution factors into the plate, incubating for 1h in a temperature box with the temperature of 37 ℃ at 100 mu L per hole, washing the plate for 5 times, and beating to dry; adding horseradish peroxidase-labeled goat anti-mouse IgG enzyme-labeled secondary antibody (1:20000) diluted with 0.01mol/L PBS pH7.4, incubating for 1h at 37 ℃, washing the plate for 5 times, and drying; then, a developing solution was added in an amount of 100. Mu.L per well and developed in a dark place for 10 minutes, a stop solution was added in an amount of 100. Mu.L per well, and the A450 value was measured, and the positive (+) was obtained by setting the P/N value (positive well OD value/negative well OD value) to be equal to or higher than the set value, and the negative (-) was obtained by setting the negative (-) as shown in Table 5.

Table 5 test results

The titer of ascites prepared by the monoclonal cell can reach 1:102400 (10) ⁵ )。

EXAMPLE 4 use of monoclonal antibodies in competitive ELASA

GBS capsular polysaccharide type III: the technology center of Watson biotechnology Co-Ltd prepares group A meningococcal polysaccharide according to the three requirements of the pharmacopoeia of the people's republic of China, and the polysaccharide content is measured by a pharmacopoeia general rule 3103 phosphorus content measuring method and then is used as an internal reference.

4.1 configuration of the Main solution

Coating buffer (0.1 mol/L, pH9.6 HCl buffer): na2CO3.10H2O 0.86g; 30.586g of NaHCO; sterilized water was added to 200mL.

Dilution (0.01 mol/L, PBS buffer pH 7.4): 8g of NaCl; kcl0.2g; na2HPO4.12H2O2.9g; kh2po40.2g; sterilized water was added to 100mL.

Washing solution (0.01 mol/L, PBST pH 7.4): 1PBS buffer 000mL; tween-200.5mL.

Blocking solution (5% skimmed milk powder PBS dilution): 1g of skimmed milk powder; 20ml of LPBS dilution.

Color development liquid: 10mL of substrate solution; TMB0.5mL; H2O 232. Mu.L.

Stop solution (2 mol/LH2SO 4): 22.2mL of concentrated sulfuric acid (18 mol/L98%); sterilized water 177.8mL

4.2 establishment of Competition ELISA method and drawing of Standard Curve

According to the conditions of each group screened in 4.1, a competition ELISA method was established as follows.

Coating: group A neisseria meningitidis polysaccharide 2 μg/ml,100 μl per well, overnight at 4deg.C, antigen coated;

washing the plate: washing the plate for three times by a plate washing machine, and beating to dryness;

closing: sealing with 5% skimmed milk powder, 200 μl per well, 37deg.C, and 1 hr; the washing plate is arranged on the same plate;

competing: group a polysaccharide references were diluted 2-fold with PBS as: 1.25. Mu.g/ml, 0.625. Mu.g/ml, 0.3125. Mu.g/ml, 0.1563. Mu.g/ml, 0.0781. Mu.g/ml, 0.0391. Mu.g/ml, 0.0195. Mu.g/ml, 0.0098. Mu.g/ml, monoclonal antibody diluted at 1:1600, 50. Mu.L: adding 50 mu L of the mixture into the sealed plate, slightly shaking and uniformly mixing, and competing for 1h at 37 ℃; the washing plate is arranged on the same plate;

color development: 100 mu L of color development liquid is per hole, and color development is carried out at room temperature and in dark place for 10min;

and (3) terminating: after the color development is completed, 100 mu L of stop solution is used for stopping the reaction in each hole;

reading: the OD value of each well is read by a microplate reader at 450 nm.

TABLE 6 experimental results

As shown in Table 6, the logarithmic scale of GBSIII polysaccharide concentration is taken as the abscissa and OD is taken as the OD ₄₅₀ On the ordinate, log-Logit regression was performed and a standard curve was drawn. See FIG. 1, logarithmic scale of GBSIII polysaccharide concentration (sialic acid method), OD ₄₅₀ For Log-Log regression analysis on the ordinate, a standard curve was drawn, y= -1.0948x+3.6788, r2=0.9515, the linear range of the indirect competition ELISA method was 200ng-1500ng/ml, and samples detected with this standard curve were recovered within ±20%, as shown in table 7.

TABLE 7

The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A hybridoma cell line secreting anti-group III streptococcus capsular polysaccharide monoclonal antibodies, characterized in that said hybridoma cell line is designated WV-GBSIII-04, deposited with the chinese collection of typical cultures, accession number: cctccc NO: c202028, 12 months and 11 days of the preservation time 2020, wherein the preservation address is China, the university of Wuhan and Wuhan.

2. A monoclonal antibody directed against group III streptococcus capsular polysaccharides, wherein the monoclonal antibody is secreted by the hybridoma cell line of claim 1; the monoclonal antibodies are of the IgM subtype.

3. The monoclonal antibody against group III streptococcus capsular polysaccharide according to claim 2, wherein the monoclonal antibody is capable of specifically recognizing group III streptococcus capsular polysaccharide, group III streptococcus capsular polysaccharide conjugates and conjugate vaccines comprising group III streptococcus capsular polysaccharide.

4. The monoclonal antibody against group III streptococcus capsular polysaccharide according to claim 2, wherein the monoclonal antibody is obtained after immunization of mice with a combination of group III streptococcus capsular polysaccharide and diphtheria non-toxic variant protein.

5. Use of a monoclonal antibody according to claim 2 for the preparation of a product for detecting the capsular polysaccharide content of group III streptococcus B.

6. A test kit comprising the monoclonal antibody of claim 2.