CN109880805B

CN109880805B - Cryptococcus resistant capsular polysaccharide monoclonal antibody and preparation and application of hybridoma cell strain thereof

Info

Publication number: CN109880805B
Application number: CN201910230634.9A
Authority: CN
Inventors: 苑庆华; 何永胜; 周跃辉; 王玉芳; 臧丹戎; 黄炎彬; 王兴
Original assignee: Tianjin Xinuo Biological Pharmaceutical Co ltd
Current assignee: Tianjin Xinuo Biological Pharmaceutical Co ltd
Priority date: 2019-03-26
Filing date: 2019-03-26
Publication date: 2022-09-06
Anticipated expiration: 2039-03-26
Also published as: CN109880805A

Abstract

The invention relates to a cryptococcus resistant capsular polysaccharide hybridoma cell strain and a cryptococcus resistant capsular polysaccharide monoclonal antibody secreted by the cryptococcus resistant hybridoma cell strain, wherein the antibody can be specifically combined with cryptococcus capsular polysaccharide and can be used for in vitro detection of cryptococcus infection, the antibody titer is over million, and the cryptococcus resistant hybridoma cell strain has excellent affinity and good specific binding capacity; the detection limit of the colloidal gold labeled immunodiagnostic reagent developed by taking the antibody as a raw material is 0.5ng/ml, and the colloidal gold labeled immunodiagnostic reagent has better performances in all aspects of sensitivity, specificity, stability and the antibody.

Description

Monoclonal antibody for resisting cryptococcus capsular polysaccharide and preparation and application of hybridoma cell strain thereof

Technical Field

The invention belongs to the field of monoclonal antibodies, and particularly relates to a cryptococcus resistant capsular polysaccharide monoclonal antibody and preparation and application of a hybridoma cell strain thereof.

Background

Immunodiagnostic reagents are the most rapidly developing sub-field of in vitro diagnostic reagents, and use specific binding between antigens and antibodies for qualitative or quantitative detection.

In recent years, with the clinical application of broad-spectrum antibiotics, glucocorticoids, antitumor drugs and immunosuppressants, the development of various invasive diagnosis and treatment technologies and the increasing number of HIV-1 infected people have increased, and the incidence rate of deep fungal infection also increases year by year. Cryptococcus is one of the common pathogens of deep fungal infections in clinic, and cryptococcus causing human infections are mainly cryptococcus neoformans and cryptococcus gatherens, and mainly cause cryptococcal meningitis and cryptococcosis pneumococci. Capsular polysaccharide is the extracellular structure of cryptococcus, is an important toxicological factor and is a biomarker of cryptococcus infection. Therefore, the detection of the cryptococcus capsular polysaccharide has important significance for the clinical auxiliary diagnosis of cryptococcus infection.

Currently, the methods for clinically detecting cryptococcus mainly include a culture method, an ink staining microscopy method, a colloidal gold method and the like. Wherein, the culture method is the 'gold standard' for detecting cryptococcus, the detection needs 3-10 days, the sensitivity is low, the time consumption is long, and the delayed treatment is easy to cause; the cerebrospinal fluid and ink smear can diagnose the cryptococcus meningitis early and quickly, but the specificity and the sensitivity of the diagnosis depend on the technical level of a tester, and the detection rate is low; the latex agglutination method is complicated in operation. Compared with other methods, the colloidal gold method is rapid, simple and convenient, high in accuracy and good in specificity, and is more and more popular in clinical detection and medical research. Only one rapid immunoassay product is used for clinical detection of cryptococcus on the market, namely a cryptococcus antigen detection kit (colloidal gold immunochromatography) of the American Immuno-mycology company, and the price is expensive. Therefore, the development of a cryptococcus capsular polysaccharide rapid diagnosis product with independent intellectual property rights has important significance for early diagnosis and timely treatment of cryptococcus infection and improvement of the medical health level in China.

The development of the cryptococcus capsular polysaccharide diagnostic reagent by the colloidal gold method firstly needs to extract cryptococcus capsular polysaccharide antigen, and after the high-purity antigen is obtained, antigen modification is needed, so that the bottleneck that the cryptococcus capsular polysaccharide antigen is weak in immunogenicity as a hapten is broken through, a good immune reaction is stimulated in a mouse body, and then a hybridoma technology is adopted to screen an antibody with high affinity and specificity for the development of related in-vitro diagnostic reagents.

Disclosure of Invention

In order to solve the technical problems, the invention provides an anti-cryptococcus capsular polysaccharide monoclonal antibody, a hybridoma cell strain secreting the antibody, a kit containing the monoclonal antibody or the hybridoma cell strain and application of the monoclonal antibody or the hybridoma cell strain in detection of cryptococcus capsular polysaccharide.

The technical scheme adopted by the invention is as follows: an anti-cryptococcus capsular polysaccharide monoclonal antibody hybridoma cell strain is named as 1G10, and the preservation number is CGMCCNo: 17082.

a monoclonal antibody of cryptococcus capsular polysaccharide comprises a heavy chain variable region sequence and a light chain variable region sequence, wherein the CDR region of the heavy chain variable region sequence comprises three sequences as shown in SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, and the CDR region of the light chain variable region sequence comprises three sequences as shown in SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6;

the CDR regions of the heavy chain variable region sequences comprise three sequences:

SEQ ID NO:1GFSFSSYFMS(CDRH1)

SEQ ID NO:2AIDIIGGNTYYPDIVKG(CDRH2)

SEQ ID NO:3RDGNHGWFFDV(CDRH3)

the light chain variable region sequence comprises three sequences in the CDR region:

SEQ ID NO:4RSSQSLVHSNGNTFLH(CDRL1)

SEQ ID NO:5KVSNRFS(CDRL2)

SEQ ID NO:6SQSTHVPWT(CDRL3)

preferably, the heavy chain variable region sequence is set forth in SEQ ID NO 7 and the light chain variable region sequence is set forth in SEQ ID NO 8.

SEQ ID NO:7

QVKLQESGGGLVKLGGSLKLSCAASGFSFSSYFMSWVRQSAEKRLELVAAIDIIGGNTYYPDIVKGRFTISRDNAKNTLYLQMTSLRSEDTSLYYCARRDGNHGWFFDVWGAGTTVTVSS

SEQ ID NO:8

MKLPVRLLVMMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTFLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKRADAAPTV

The monoclonal antibody against cryptococcus capsular polysaccharide is secreted by hybridoma cell strain 1G 10.

A nucleotide molecule comprising a nucleotide sequence encoding an anti-cryptococcus capsular polysaccharide monoclonal antibody;

preferably, the nucleotide sequence of the variable region of the heavy chain of the cryptococcus polysaccharide monoclonal antibody coded by the nucleotide molecule is shown as SEQ ID NO. 9, and the nucleotide sequence of the variable region of the light chain of the cryptococcus capsular polysaccharide monoclonal antibody coded by the nucleotide molecule is shown as SEQ ID NO. 10.

SEQ ID NO:9

CAGGTGAAGCTGCAGGAGTCTGGGGGAGGCTTAGTGAAACTTGGAGGGTCCTGAAACTCTCCTGTGCAGCCTCTGGATTCTCTTTCAGTAGCTATTTCATGTCTTGGGTTCGCCAGAGTGCAGAGAAGAGGCTGGAGTTGGTCGCAGCCATTGATATTATTGGTGGTAATACCTACTATCCAGACATTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTATACCTGCAAATGACCAGTCTGAGGTCTGAGGACACATCCTTGTATTACTGTGCAAGACGGGATGGTAACCACGGCTGGTTCTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTC

SEQ ID NO:10

ATGAAGTTGCCTGTTAGGCTGTTGGTGATGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTTTTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCC

The application of the monoclonal antibody hybridoma cell strain or monoclonal antibody against cryptococcus capsular polysaccharide in a detection kit.

The kit comprises an anti-cryptococcus capsular polysaccharide monoclonal antibody, and is based on immunoassay.

Preferably, the kit is a colloidal gold immunoassay kit, a chemiluminescent kit, a radioimmunoassay kit, an enzyme linked immunosorbent kit or a fluoroimmunoassay kit or the kit is a microfluidic chip;

preferably, the kit is a colloidal gold immunoassay kit.

Preferably, detecting a pathogenic cryptococcus bacterium;

preferably, the cryptococcus capsular polysaccharide antigen is detected.

A preparation method of a cryptococcus capsulatus capsular polysaccharide detection card by a colloidal gold method is used for preparing an immune colloidal gold test strip by a double-antibody sandwich method, and the preparation method comprises the following steps:

step (2) adding 0.1M K into colloidal gold solution while stirring ₂ CO ₃ Adjusting the pH value of the solution, adding an anti-cryptococcus capsular polysaccharide monoclonal antibody, stirring, adding a 10% bovine serum albumin solution and 2% PEG20000, stirring, centrifuging at a low speed to obtain a supernatant, centrifuging at a high speed to obtain a precipitate, and diluting to constant volume with a colloidal gold heavy suspension to form a gold-labeled antibody;

spraying the gold-labeled antibody on a glass cellulose membrane, and drying to prepare a gold-labeled pad;

adding a 1% thimerosal sodium solution into the three-way cryptococcus resistant capsular polysaccharide monoclonal antibody, uniformly mixing to form a detection line coating solution, adding PBS (phosphate buffer solution) and a 1% thimerosal sodium solution into goat anti-mouse IgG (immunoglobulin G), uniformly mixing to form a quality control line coating solution, scratching the quality control line coating solution and the detection line coating solution on a nitrocellulose membrane, and drying to obtain a coating film;

step four, sticking the coating film on a bottom plate, lapping the gold label pad and absorbent paper with the coating film, laminating and cutting to obtain the cryptococcus capsulatus capsular polysaccharide detection card by a colloidal gold method;

preferably, the pH value is adjusted to 9.5 in the first step;

preferably, in the first step, the low-speed centrifugation is 1000-2000 rpm, preferably 1500 rpm, and the high-speed centrifugation is 10000-150000 rpm, preferably 11000 rpm;

preferably, the drying temperature in the second step and the third step is 37 ℃, and the drying humidity is less than 30%.

The preparation method of the anti-cryptococcus capsular polysaccharide monoclonal antibody hybridoma cell strain comprises the steps of extracting cryptococcus capsular polysaccharide through a CTAB method, purifying to obtain a cryptococcus capsular polysaccharide coupled carrier, immunizing a mouse with the cryptococcus capsular polysaccharide coupled with the carrier, fusing cells, and performing cell subcloning through a limiting dilution method to obtain the hybridoma cell strain 1G10 stably secreting the specific anti-cryptococcus capsular polysaccharide monoclonal antibody.

The invention has the advantages and positive effects that:

firstly, the antibody titer is over million, the antibody has excellent affinity, has no cross reaction with (1,3) -beta-D glucan, Candida albicans mannan and aspergillus galactomannan, and has good specific binding capacity;

secondly, the detection limit of the colloidal gold labeled immunodiagnostic reagent developed by taking the antibody as a raw material is 0.5ng/ml, and the comparison result of 1834 clinical samples and imported reagents shows that the sensitivity is 98.8 percent and the specificity is 98.6 percent;

through systematic evaluation, including antibody subtype and titer, kit sensitivity, specificity and stability, the antibody has better performance in all aspects, thereby being suitable for being used as an immunodiagnostic reagent for preparing an in vitro diagnosis kit;

in a word, the monoclonal antibody of the invention shows good performance in various aspects such as antibody titer, specificity, detection effect and the like, so that the invention provides the monoclonal antibody of cryptococcus capsular polysaccharide with outstanding comprehensive capacity, which can be used as an in vitro diagnostic reagent.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of antibody 1G10 of the present invention.

Detailed Description

The following provides a detailed description of the inventive arrangements.

The invention relates to a monoclonal antibody hybridoma cell strain for resisting cryptococcus capsular polysaccharide, which is a biological material named as 1G10 and belongs to hybridoma cells, wherein the microorganism preservation number is CGMCCNo: 17082 the collection place is China general microbiological culture Collection center, and the collection date is 2018, 12 months and 19 days, and the detection result is survival.

An anti-cryptococcus capsular polysaccharide monoclonal antibody secreted by the hybridoma cell strain 1G10 comprises a heavy chain variable region sequence and a light chain variable region sequence;

SEQ ID NO:1GFSFSSYFMS(CDRH1)

SEQ ID NO:2AIDIIGGNTYYPDIVKG(CDRH2)

SEQ ID NO:3RDGNHGWFFDV(CDRH3)

SEQ ID NO:4RSSQSLVHSNGNTFLH(CDRL1)

SEQ ID NO:5KVSNRFS(CDRL2)

SEQ ID NO:6SQSTHVPWT(CDRL3)

the heavy chain variable region sequence is shown as SEQ ID NO. 7, and the light chain variable region sequence is shown as SEQ ID NO. 8.

SEQ ID NO:7

SEQ ID NO:8

A nucleotide molecule comprising a nucleotide sequence encoding an anti-cryptococcus polysaccharide monoclonal antibody.

Wherein the nucleotide sequence of the heavy chain variable region of the cryptococcus polysaccharide monoclonal antibody is shown as SEQ ID NO. 9, and the nucleotide sequence of the light chain variable region of the cryptococcus polysaccharide monoclonal antibody is shown as SEQ ID NO. 10.

SEQ ID NO:9

SEQ ID NO:10

The monoclonal antibody against cryptococcus capsular polysaccharide has better performance in all aspects through systematic evaluation, including evaluation on antibody subtype and titer, kit sensitivity, specificity and stability, so that the monoclonal antibody against cryptococcus capsular polysaccharide is suitable for being used as an immunodiagnostic reagent for preparing an in vitro diagnosis kit. Can be made into a colloidal gold immunoassay kit, a chemiluminescence kit, a radioimmunoassay kit, an enzyme linked immunosorbent assay kit or a fluorescence immunoassay kit, or a microfluid chip; the prepared kit can detect cryptococcus capsular polysaccharide antigen, and the detection pathogen is cryptococcus.

The cryptococcus capsular polysaccharide detection card is prepared by using a colloidal gold method, and the immune colloidal gold test strip by using a double-antibody sandwich method is prepared by the following steps:

and step four, attaching the coating film on a bottom plate, overlapping the gold label pad and absorbent paper with the coating film, laminating, and cutting to obtain the cryptococcus capsulatus capsular polysaccharide detection card by the colloidal gold method.

The preparation method comprises the following steps:

step A, adding 0.1M K into colloidal gold solution while stirring ₂ CO ₃ Adjusting the pH value to 9.5, adding 2mg/mL anti-cryptococcus capsular polysaccharide monoclonal antibody, stirring, adding 10% bovine serum albumin solution and 2% PEG20000, stirring, centrifuging at 4 ℃ at low speed of 2000 r/min, discarding the precipitate, taking the supernatant, centrifuging the obtained supernatant at 4 ℃ at high speed of 10000 r/min, discarding the supernatant, centrifuging the precipitate at constant volume by using colloidal gold heavy suspension to form the gold-labeled antibody;

step two, cutting the glass cellulose membrane into strips, spraying the gold-labeled antibody on the glass cellulose membrane, and drying for 2 hours at the drying temperature of 37 ℃ and the drying humidity of less than 30 percent to prepare a gold-labeled pad;

adding a 1% thimerosal sodium solution into a three-way 2mg/mL anti-cryptococcus capsular polysaccharide monoclonal antibody, shaking and uniformly mixing to form a detection line coating solution, adding PBS (phosphate buffer solution) and a 1% thimerosal sodium solution into 20mg/mL goat anti-mouse IgG (immunoglobulin G), uniformly mixing to form a quality control line coating solution, scribing the quality control line coating solution and the detection line coating solution on a nitrocellulose membrane, keeping the distance between a quality control line and the detection line by 5mm, drying at the temperature of 37 ℃, and drying at the drying humidity of less than 30% for 16 hours to obtain a coating film;

and step four, cutting the absorbent paper into strips, attaching the coating film to the bottom plate, overlapping the gold mark pad and the absorbent paper with the coating film, laminating, and cutting to obtain the cryptococcus capsular polysaccharide detection card by the colloidal gold method.

The present invention will now be described in detail with reference to specific embodiments and examples, which are set forth to illustrate, but are not to be construed as limiting the invention.

Example 1: preparation method of monoclonal antibody against cryptococcus capsular polysaccharide

1. Polysaccharide extraction

Culturing Cryptococcus (ATCC 208821) in liquid Sabouraud's medium, culturing at 20-25 deg.C for 3-4 days, inactivating bacterial liquid, centrifuging, collecting supernatant, adding sodium acetate to the supernatant to final concentration of 5%, and adjusting pH to 5.0; adding 3 times volume of anhydrous ethanol, and standing at 4 deg.C overnight to obtain creamy precipitate at the bottom; removing supernatant, precipitating to obtain cryptococcus capsular polysaccharide, air drying the precipitate, weighing (or calculating mass by concentration), dissolving the precipitate with 0.2M NaCl, dialyzing with 50KD dialysis bag for 24 hr, and suspending and concentrating at 4 deg.C; slowly adding CTAB accounting for 0.3 percent of the mass of the polysaccharide precipitate, standing, centrifuging, washing the obtained precipitate twice by 10 percent ethanol, dissolving the precipitate in 1M NaCl, adding 2.5 times of absolute ethanol by volume until the precipitate appears, standing, centrifuging, and precipitating the obtained precipitate again by ethanol to obtain the refined cryptococcus capsular polysaccharide. Dissolving refined cryptococcus capsular polysaccharide with 2M NaCl, stirring to dissolve the precipitate like glycerol, dialyzing for 24 hr to remove CTAB, dialyzing with distilled water for 1d, and measuring related data.

2. Coupled carriers

Taking 2ml (concentration is 1mg/ml) of refined cryptococcus capsular polysaccharide NaCl solution, and adjusting the pH value to 5.8; adding 100ul of acetonitrile-dissolved CDAP (concentration is 10mg/ml), and mixing; after 30s, p H to 8.1 is adjusted by triethylamine, and the reaction is carried out for 2.5min at room temperature; adding 100ul of ADH solution (with the concentration of 50mg/ml), and maintaining p H at 8.05-8.15 by using 0.1mol/L HCl for reaction for 2 hours; after the reaction is finished, putting the solution into a dialysis bag, dialyzing with normal saline at 4 ℃ overnight, and then measuring the derivatization degree; the carrier protein is dissolved to 2mg/ml, according to the mass polysaccharide: uniformly mixing the carrier at a ratio of 1:3, adjusting the pH value to 5.6, adding an EDAC solution under the ice bath condition until the final concentration is 0.5M, adjusting the pH value to 5.6, and reacting at room temperature for 3 hours; dialyzing for 1d at 4 ℃ with 0.2M sodium chloride after the reaction is finished; purifying the conjugate by a sephadex column; equilibrate the column with 0.2M sodium chloride; carrying out centrifugal treatment on the sample at 5000 rpm for 10min, and loading the sample with the loading amount of 3 ml; eluting with 0.2M sodium chloride as mobile phase, and connecting the eluate tube by tube to obtain 1.5 ml/tube cryptococcus capsular polysaccharide coupling carrier; the polysaccharide (phenol-sulfuric acid method) and protein concentration (BCA method) were measured tube by tube. And (4) drawing a curve to determine the coupling effect, carrying out SDS-PAGE identification, and calculating the coupling ratio.

3. Immunization of mice

Using cryptococcus capsular polysaccharide coupling carrier to immunize female Balb/c mice of about 6 weeks of age, and preparing antibody, wherein the content of saccharide in the coupling is 0.1mg/ml, and the content of polysaccharide: the protein coupling ratio is 1: 3; dividing into 3 groups according to the immune dose, wherein each group comprises 3 mice; calculated according to the polysaccharide content, the first group of immunity dose is 1 ug/mouse, the first group of immunity dose is 2.5 ug/mouse, and the first group of immunity dose is 10 ug/mouse. First-stage immunization, taking a proper amount of conjugate, diluting the conjugate to 300ul by distilled water, adding 300ul of equivalent Freund's complete adjuvant, emulsifying uniformly, and injecting the immunized mouse at multiple points subcutaneously; two weeks later, the same dose of the two-stage immunization was taken, i.e., the mice were immunized by intraperitoneal injection, and the two-stage immunization was performed once more after two weeks, i.e., the mice were immunized by intraperitoneal injection, and after 7 days, blood was collected from the rat tail, and the serum titer of the mice was measured by ELISA. The method comprises the following specific steps: GXM 5ug/ml, 100 ul/well, 4 ℃ overnight coated ELISA plate, spin-dried, PBST washing 3 times. 5% of skim milk powder, 200 ul/hole, and sealing at 37 ℃ for 2 h. Collecting blood from mouse tail, 3000 rpm, centrifuging, collecting serum, and diluting with PBS at a ratio of 1:1000 to 1:512000 for use. Spin-dry, PBST wash 3 times, 1:1000 times add PBS diluted primary antibody, 100 ul/hole, 37 degrees, 1 h. Spin-drying, washing with PBST for 3 times, adding goat-anti-mouse secondary antibody diluted by 1:6000 times of PBS (100 ul/hole), 37 degrees, and 45 min. Spin-drying, washing with PBST for 5 times, adding 100ul TMB/well, developing at 37 deg.C for 10min, terminating, and reading.

4. Cell fusion

Three days before fusion, mice are subjected to boosting immunization, the inoculation amount is the same as that of the previous immunization, and adjuvant is not added, and intraperitoneal injection is carried out. Preparing feeder layer cells one day before fusion, taking Balb/c 1 mouse with age of 6-8 weeks, taking eyeball to exsanguinate, dislocating cervical vertebra to death, placing in 75% alcohol for sterilization for 5min, fixing on a disc, and aseptically cutting off abdominal skin in a super clean bench. The HAT selection medium (10 ml) was aspirated by a sterile syringe, injected into the abdominal cavity of a mouse, gently kneaded with an alcohol cotton ball, and then the medium was withdrawn. Added to 40ml of HAT culture solution, and spread in 4 96-well cell culture plates, 100. mu.L/well, 37 ℃ and cultured in a 5% CO2 cell culture box. Myeloma cells (Sp2/0 cells) were recovered one week before fusion, cultured in PRMI-1640 medium containing 10% fetal bovine serum, and subcultured at 37 ℃ in a 5% CO2 incubator. The cells in the logarithmic growth phase were collected into a centrifuge tube, counted, and diluted to 107 cells/ml for use. Collecting Balb/c mice with 3 days of enhanced immunity, picking eyeball, bleeding to prepare positive serum, removing cervical vertebra, killing, sterilizing with 75% alcohol for 5min, aseptically taking out spleen from ultra-clean bench, washing in aseptic petri dish for several times, and stripping connective tissue. Placing spleen on microporous copper net, adding fresh RPMI-1640 culture solution, sucking the culture solution with syringe, injecting into spleen section, blowing down splenocytes, repeating for several times, and lightly grinding the rest spleen with the inner plug of syringe until there is no obvious red tissue block. And gently blowing and beating the splenocyte suspension in the plate, transferring the splenocyte suspension into a 50ml centrifuge tube, centrifuging for 5min at 1000r/min, collecting splenocytes, and counting for later use. Mixing splenocytes of the immunized mice with Sp2/0 cells according to the cell number of 10:1, adding the mixture into a 50mL centrifuge tube, centrifuging the mixture for 5min at 1000r/min, discarding the supernatant, gently rubbing the mixture at the palm to fully mix the two cells, placing the centrifuge tube into a 100mL blue-covered bottle, filling hot water at 37 ℃ into the blue-covered bottle, dropwise adding preheated 1mL DMSO/PEG into the fusion tube within 1min, and slowly and quickly rotating the centrifuge tube while adding. Then, the reaction was terminated by immediately adding the blood-free RPMI-1640 culture medium, 1ml for the first minute, 2ml for the second minute, 3ml for the third minute and 4ml for the fourth minute. Carrying out water bath at 37 ℃ for 5min, centrifuging at 800r/min for 5min, discarding the supernatant, suspending the precipitate with HAT, mixing uniformly into 40ml HAT selection culture solution containing 20% calf serum preheated at 37 ℃, spreading into 96-well cell plates with feeder cells, culturing at 100 mu L/well, and placing the culture plates into a 37 ℃ and 5% CO2 incubator for culture. After 7d, the cell plates will be half-exchanged with fresh HAT medium and 10 days later with HT medium. The positive cells in the 96-well plate were subcloned by limiting dilution: firstly, preparing feeder layer cells according to the method, taking hybridoma cells to be cloned for cell counting, diluting the cells to 5-8 cells/ml by using an HT culture medium, adding the cells into a 96-well cell plate with the laid feeder cells at 100 mu L/well, cloning one 96-well cell plate for each hybridoma cell, and culturing in a 5% CO2 cell culture box at 37 ℃. Counting the number of clones in the cell hole after about 5 days, marking, changing the culture medium after 7 days, and detecting when the cells are fully paved at 1/3-1/2 of the whole hole bottom. After 2-3 times of cloning, when all cell wells of the 96-well plate are positive, performing expanded culture, fixing the strains and freezing and storing. And (5) carrying out expanded culture on the hybridoma cells of the positive detection determination strains and freezing and storing. The specific process is as follows: the hybridoma cells which grow vigorously and are in good state are gently blown down from the cell bottle by using the anti-free blood-free DMEM, and are centrifuged at 1000r/min for 5min, and the supernatant is discarded. Adding freezing medium (containing 40% RPMI-1640 culture medium, 50% fetal bovine serum, and 10% DMSO), blowing out cells, and packaging into cell freezing tube. And (4) placing the freezing tube into a freezing box, placing the freezing box in a refrigerator at the temperature of-70 ℃, transferring the freezing tube into liquid nitrogen after one day, and recording.

5. Preparation of ascites

Taking 10-12 weeks old female Balb/c mice, injecting sterile liquid paraffin into abdominal cavity, 0.5 mL/mouse, 7 days later, injecting hybridoma cells cultured to logarithmic phase by abdominal cavity, and 5 multiplied by 10 ⁶ One cell/one. The observation is carried out every day, about 7-10 days, after the abdomen of the mouse is obviously raised, the skin of the abdomen is disinfected by a 75% alcohol cotton ball, the abdominal cavity is punctured by a 16-gauge needle, and the ascites is collected. And collecting the ascites again after the ascites is regenerated and accumulated. Centrifuging the collected ascites at 3000r/min for 10min, collecting the intermediate clear part, filtering with filter paper, and packaging at-70 deg.C.

6. Antibody purification

The purification of ascites by the octanoic acid-ammonium sulfate method comprises the following steps: taking 3ml (n) of ascites, centrifuging 1000g, taking a clear part, adding 6ml (n x 2) of acetic acid-sodium acetate buffer solution, uniformly mixing, adjusting the pH to 4.5, stirring and slowly adding 99ul of n-octanoic acid (n x 33), standing for 1-2h at 4 ℃, 12000g, centrifuging for 10min, taking a supernatant, discarding a precipitate, filtering the supernatant by using filter paper, adding 10XPBS with one tenth of the volume of the solution, adjusting the pH to 7.4, slowly adding supersaturated ammonium sulfate with the same volume (protein can be seen to be separated out when 2/3 is added), standing for 1h at 4 ℃, 10000g, centrifuging for 20min, discarding the supernatant, reserving the precipitate, re-dissolving the precipitate to 3ml (n) by using PBS, dialyzing in a 50kd dialysis bag and PBS at 4 ℃ for overnight.

7. Antibody subclass identification

The subclass identification of the monoclonal antibody was performed by capture ELISA according to the instruction of SIGMA kit, which is as follows: diluting the monoclonal antibody subclass identification reagent 1:1000, adding into an enzyme-labeled hole, incubating at 100 mu L/hole for 1h at 37 ℃; PBST is washed for three times and patted dry; diluting the antibody 1:1000 times, adding the sample, incubating at 37 ℃ for 1h at a concentration of 100 mu L/hole; PBST is washed for three times and patted dry; diluting HRP enzyme-labeled goat anti-mouse IgG secondary antibody at a ratio of 1:6000, adding the sample, incubating at 100 mu L/hole for 30min at room temperature; developing for 10-20 min. And the subclass type of the monoclonal antibody is determined by that the OD450 reading value is obviously higher than that of the subclass reagent added in other wells. The antibody subtype of antibody 1G10 was IgG 1.

8. Antibody titer determination

The antibody titer after purification is measured by adopting an indirect ELISA method, and the method comprises the following steps: GXM is respectively diluted to 5ng/ml and 100 ul/hole, meanwhile, a non-coating control is set up, coating is carried out overnight at 4 ℃, spin-drying and PBST washing are carried out for 3 times; 5% of skim milk powder, 200 ul/hole, and sealing at 37 ℃ for 2 h; spin-dry, wash 3 times with PBST, add 1:1000 times diluted antibody (concentration 1mg/ml), total 12 gradients, set up no coating control 100 ul/well, 37 ℃, 1 h. Spin-drying, washing with PBST for 3 times, adding goat-anti-mouse secondary antibody diluted by 1:6000 times of PBS (100 ul/well), 37 deg.C, and 45 min. Spin-drying, PBST washing for 5 times, adding 100ul TMB/hole, developing at 37 deg.C for 10min, terminating, and reading. The antibody is diluted to 1mg/ml after purification, and the titer reaches more than 1: 1280000.

9. Antibody purity and molecular weight identification

Performing antibody molecular weight and purity identification by an SDS-PAGE method; preparing glue, wherein the separation glue accounts for 12 percent, and the concentration glue accounts for 5 percent; preparing a sample, mixing 20ul of sample and 20ul of buffer, uniformly mixing, and boiling for 3 min; each hole is loaded with 20ul, and a protein pre-staining Marker contrast is set; 80 volts for 30min and 120 volts for 2 h; after electrophoresis, placing into a Coomassie brilliant blue solution for dyeing; decolorizing for 5min each for 3 times; the purified monoclonal antibody was identified by SDS-PAGE to have clear bands without any impurity, as shown in FIG. 1, with clear bands at 50kDa and 25 kDa.

Example 2 preparation of colloidal gold Immunity test paper strip

The monoclonal antibody of cryptococcus capsular polysaccharide is labeled by colloidal gold to prepare the immune colloidal gold test strip by a double-antibody sandwich method, which is named as cryptococcus capsular polysaccharide test card (colloidal gold method), and the specific process is as follows:

step one preparation of gold-labeled antibody

50mL of colloidal gold solution is measured and added into a beaker which is sterilized and dried, the beaker is placed on an electric heating magnetic stirrer for stirring at the speed of 500-600rpm, and 0.1M K is added while stirring ₂ CO ₃ Adjusting the pH of the solution to 9.5; marking: adding 0.3mL (2mg/mL) of monoclonal antibody of cryptococcus capsular polysaccharide, stirring for 1h at the same rotation speed; and (3) sealing: adding 5mL of 10% Bovine Serum Albumin (BSA) solution and 5mL of 2% PEG200002.5ml, stirring at the same speed for 1 h; low-speed centrifugation: centrifuging at 4 deg.C for 30min at 1500 rpm, removing precipitate, and collecting supernatant; high-speed centrifugation: centrifuging the supernatant at 11000 r/min at 4 ℃ for 50min, discarding the supernatant, and metering the volume of the precipitate to 5mL by using colloidal gold heavy suspension.

Step two gold label pad preparation

Cutting the glass cellulose film by a numerical control strip cutting machine, wherein the specification is 20mm multiplied by 300mm multiplied by 20 strips; taking 5mL of gold-labeled antibody; spraying the gold-labeled antibody on the glass cellulose membrane by using a three-dimensional plane dot membrane gold spraying instrument, calling the program 1, wherein the speed is 100mm/sec, the gold spraying amount is 8.0ul/cm, and the gold spraying pressure is 1.0kg/cm ² The length of the metal spraying is 300mm, and the total number of the metal spraying is 20; drying for 2h in an electric heating constant temperature incubator at 37 ℃ and humidity of less than 30%.

Step three coating film preparation

Preparing a detection line coating solution: adding 0.5mL of cryptococcus resistant capsular polysaccharide monoclonal antibody (2mg/mL) into 10uL of 1% thimerosal sodium solution, shaking for 2min by using a micro mixer, and fully shaking and uniformly mixing; preparing a quality control line coating solution: adding 0.4mL PBS and 10uL 1% thimerosal sodium solution into 0.1mL goat anti-mouse IgG (20mg/mL), shaking for 2min by using a micro mixer, and fully shaking and uniformly mixing; marking the coating liquid of a quality control line and the coating liquid of a detection line on a nitrocellulose membrane (NC membrane) by using a three-dimensional plane dot membrane metal spraying instrument, calling a program 0, marking the length of the membrane at 300mm, marking the quality control line at a position 12mm away from the top end of the membrane, marking the detection line at a position 17mm away from the top end of the membrane, and keeping the distance between the quality control line and the detection line at 5mm, wherein the quality control line is a No. 1 pump, the detection line is a No. 3 pump, the speed is 100mm/sec, the film marking amount is 0.8 uL/cm. Marking the positions of a quality control line (C line) and a detection line (T line) by using a pencil; drying in an electric heating constant temperature incubator at 37 deg.C and humidity less than 30% for 16 h.

Step four card making

Cutting the absorbent paper by a numerical control slitter, wherein the specification is 20mm multiplied by 300mm multiplied by 20 strips; assembling: pasting the coated NC film on a bottom plate, ensuring the edge to be aligned, pasting the prepared gold label pad and the water absorption paper to manufacture a large plate, wherein the lamination parameters are as follows: the gold label pad is overlapped with an NC film for 2mm, and the absorbent paper is overlapped with an NC film for 5 mm; slitting: and cutting the assembled large board into test strips with the width of 3.0mm by using a microcomputer automatic cutting machine. Card installation: the cut test strips were assembled into intact plastic cards and pressed with a press. Bag sealing: putting 1 card and 1 drier into the aluminium foil bag together, sealing with a sealing machine, sealing width 2 mm.

Example 3 comparison of clinical samples with colloidal gold immunotest strips

The cryptococcus capsular polysaccharide detection card (colloidal gold method) prepared in example 2 is used as an examination reagent for clinical tests, a cryptococcus antigen detection kit (colloidal gold immunochromatography) of the American Immuno-mycology company is selected as a contrast reagent, the group serum and cerebrospinal fluid samples are detected by a blind method, the detection results are statistically analyzed, and the consistency of the detection results of the test strip and the contrast reagent is inspected. In the cerebrospinal fluid sample, the number of cases with consistent positive results of the examination reagents and the contrast reagents is 296 cases, the number of cases with positive detection results of only the examination reagents is 18 cases, the number of cases with positive detection results of only the contrast reagents is 4 cases, the number of cases with consistent negative results is 464 cases, the detection results of the examination reagents and the contrast reagents have the positive coincidence rate of 98.67%, the negative coincidence rate of 96.27%, the total coincidence rate of 97.19% and the Kappa coefficient of 0.9410. In the serum sample, the number of cases with consistent positive results of the examination reagents and the comparison reagents is 105, the number of cases with positive results of only the examination reagents is 2, the number of cases with positive results of only the comparison reagents is 1, the number of cases with consistent negative results is 964, the positive coincidence rate of the examination reagents and the comparison reagents is 99.06%, the negative coincidence rate is 99.79%, the total coincidence rate is 99.72%, and the Kappa coefficient is 0.9844. The Kappa coefficients of the cerebrospinal fluid sample and the serum sample are both greater than 0.75 and are highly consistent, and the two systems are considered to be equivalent, so that the cryptococcus capsular polysaccharide detection card (colloidal gold method) prepared by the method has a good clinical application value.

Example 4 sequencing of the amino acid sequence of monoclonal antibodies to cryptococcus capsular polysaccharide

1) Total RNA extraction, single-stranded cDNA synthesis:

total RNA from 1G10 hybridoma cell line was extracted by Trizol (kit from Invitrogen) and reversed to a cDNA library using M-MLV reverse transcriptase (from Invitrogen).

Heavy chain framework region upstream primer

P1：5’SAGGTGMAGCTKCASSARTCWGG3’(SEQ ID NO:11)

Heavy chain variable region downstream primer

P2：5’TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA3’(SEQ ID NO:12)

Light chain leader peptide upstream primer

P3：5’ATGGATTTTCAAGTGCAGATTTTCAG3’(SEQ ID NO:13)

Light chain variable region downstream primer

P4：5’GGATACAGTTGGTGCAGCATCAGCCCGTTT3’(SEQ ID NO:14)

A PCR reaction system (50. mu.l) was prepared as follows:

2 μ l of cDNA; 2. mu.l of upstream primer (10. mu.M); 2. mu.l of downstream primer (10. mu.M); dNTP mix 2. mu.l; pfu DNA polymerase (5U/. mu.l) 1. mu.l; 5 mul of 10 Xpfu Buffer II; ddH2O make up to 50. mu.l.

Reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; the following cycle was repeated 30 times: 45s at 95 ℃, 45s at 60 ℃ and 45s at 72 ℃; finally, extension was carried out at 72 ℃ for 10 min.

The VL and VH fragments were separated and recovered by agarose gel electrophoresis. The recovered VL and VH fragments were ligated with pMD19-T (simple) vector (Takara) in the following system:

VL PCR product/VH PCR product each 70ng, pMD19-T (simple) vector 1. mu.l, Solution I ligation reaction 5. mu.l; ddH2O was made up to 10. mu.l and ligated overnight at 4 ℃.

The ligation products were transformed into E.coli DH5 alpha competent bacteria, cultured overnight at 37 ℃, single colonies were picked up, shaken at 37 ℃ for 2 hours, and then subjected to PCR identification of bacterial liquid, using cDNA of the corresponding antibody as a positive control. The reaction system (25. mu.l) was prepared as follows:

bacterial liquid: 1 μ l, forward primer (10 μ M): 1 mul; downstream primer (10 μ M): 1 mul; dNTP mix (2.5 Mm each) 2. mu.l; taq DNA polymerase (5U/. mu.l): 0.5 mul; 10 × Taq Buffer (Mg2+ plus): 2.5 mul; water was added to 25. mu.l. The reaction conditions were as before.

The PCR positive clone of the selected strain is expanded and cultured, and the positive clone plasmid is extracted by a plasmid extraction kit (Takara company) and is checked and sequenced. At least 5 clone samples were tested per chain of each antibody until the sequencing results were identical for at least three samples. The heavy chain and light chain variable region sequences of the antibody 1G10 are successfully cloned and aligned to meet the sequence characteristics of the variable region of a typical antibody.

SEQ ID NO:1GFSFSSYFMS(CDRH1)

SEQ ID NO:2AIDIIGGNTYYPDIVKG(CDRH2)

SEQ ID NO:3RDGNHGWFFDV(CDRH3)

SEQ ID NO:4RSSQSLVHSNGNTFLH(CDRL1)

SEQ ID NO:5KVSNRFS(CDRL2)

SEQ ID NO:6SQSTHVPWT(CDRL3)

SEQ ID NO:7

SEQ ID NO:8

The nucleotide sequence of the heavy chain variable region of the cryptococcus polysaccharide monoclonal antibody is shown as SEQ ID NO. 9, and the nucleotide sequence of the light chain variable region of the cryptococcus polysaccharide monoclonal antibody is shown as SEQ ID NO. 10.

SEQ ID NO:9

SEQ ID NO:10

While one embodiment of the present invention has been described in detail, the description is only a preferred embodiment of the present invention and should not be taken as limiting the scope of the invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.

Sequence listing

<110> Tianjin Xinuo biomedical Co., Ltd

<120> monoclonal antibody of cryptococcus resistant capsular polysaccharide and preparation and application of hybridoma cell strain thereof

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tgcaaatgac cagtctgagg tctgaggaca catccttgta ttactgtgca agacgggatg 300

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tcttgcagat ctagtcagag ccttgtacac agtaatggaa acaccttttt acattggtac 180

ctgcagaagc caggccagtc tccaaagctc ctgatctaca aagtttccaa ccgattttct 240

ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 300

agagtggagg ctgaggatct gggagtttat ttctgctctc aaagtacaca tgttccgtgg 360

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Claims

1. A hybridoma cell strain secreting monoclonal antibodies against cryptococcus capsulatus capsular polysaccharide is characterized in that: is named as 1G10, and the preservation number is CGMCCNo: 17082.

2. an anti-cryptococcus capsular polysaccharide monoclonal antibody, which is characterized in that: the sequences include a heavy chain variable region sequence comprising CDRH1 shown in SEQ ID NO:1, CDRH2 shown in SEQ ID NO:2 and CDRH3 shown in SEQ ID NO:3 and a light chain variable region sequence comprising CDRL1 shown in SEQ ID NO:4, CDRL2 shown in SEQ ID NO:5 and CDRL3 shown in SEQ ID NO: 6.

3. The monoclonal antibody against a cryptococcus capsular polysaccharide according to claim 2, characterized in that: the heavy chain variable region sequence is shown as SEQ ID NO. 7, and the light chain variable region sequence is shown as SEQ ID NO. 8.

4. The monoclonal antibody against cryptococcus capsular polysaccharide according to claim 2, characterized in that: secreted by the hybridoma cell line 1G10 of claim 1.

5. A kit comprising an anti-cryptococcus capsular polysaccharide monoclonal antibody according to any one of claims 2-4, wherein: the kit is based on immunoassay.

6. The kit of claim 5, wherein the monoclonal antibody against a capsular polysaccharide of cryptococcus is selected from the group consisting of: the kit is a colloidal gold immunoassay kit, a chemiluminescence kit, a radioimmunoassay kit, an enzyme linked immunosorbent assay kit or a fluorescence immunoassay kit or the kit is a microfluid chip.

7. The anti-cryptococcus capsular polysaccharide monoclonal antibody kit of claim 6, characterized in that: the kit is a colloidal gold immunoassay kit.

8. The anti-cryptococcus capsular polysaccharide monoclonal antibody kit of claim 6, characterized in that: used for detecting pathogenic cryptococcus.

9. The anti-cryptococcus capsular polysaccharide monoclonal antibody kit of claim 8, characterized in that: used for detecting cryptococcus capsular polysaccharide antigen.