CN114933652B

CN114933652B - Anti-candida mannan monoclonal antibody and application thereof

Info

Publication number: CN114933652B
Application number: CN202210728744.XA
Authority: CN
Inventors: 付成华; 魏新宇; 翟栓柱; 盛长忠; 粟艳; 周泽奇
Original assignee: Dynamiker Biotechnology Tianjin Co Ltd
Current assignee: Dynamiker Biotechnology Tianjin Co Ltd
Priority date: 2022-06-24
Filing date: 2022-06-24
Publication date: 2023-03-28
Anticipated expiration: 2042-06-24
Also published as: WO2023245801A1; CN114933652A

Abstract

The invention provides an anti-candida mannan monoclonal antibody and application thereof, wherein the anti-candida mannan monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises a heavy chain CDR3 shown by SEQ ID NO.3, and the light chain variable region comprises a light chain CDR3 shown by SEQ ID NO. 6. The monoclonal antibody is a rabbit source monoclonal antibody, has multiple antigen recognition sites, good specificity and high affinity, solves the problem of the mouse source monoclonal antibody in the aspect of practical application, and has important application value in preparing candida detection products.

Description

Anti-candida mannan monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an anti-candida mannan monoclonal antibody and application thereof.

Background

Candida albicans (Candida albicans) is a clinically isolated opportunistic human pathogenic fungus, and particularly in immune-down-regulated patients, such as organ transplant patients, HIV infectors and the like, can cause wide superficial and deep systemic infections, infection parts including oral cavities, female vaginas and the like, cause thrush, vaginitis and the like, and can also invade epidermis and endothelial cells into blood to reach internal organs, such as kidneys, brains and the like, cause septicemia and serious death.

In recent years, candida albicans infection has a significantly increased tendency, and the candida caused infection is located at the 4 th site in blood-borne infection and at the 3 rd site in catheter-related infection, and even if antifungal drugs are used, the mortality rate of the candida caused infection is still as high as 40%. There are no typical clinical symptoms of systemic candida infection, and there is currently no specific early diagnostic tool for candida infection.

At present, the gold standard for detecting candida infection is blood culture, however, the disease condition of the disease develops rapidly, the positive detection rate of the traditional culture method is low, the culture period is long, and missed diagnosis and misdiagnosis are often caused, so that the disease condition is delayed. Therefore, the development of an antibody with strong specificity which can be used for detecting candida is of great significance.

Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone, directed against only a particular epitope, and are referred to as monoclonal antibodies. Usually, hybridoma (hybridoma) antibody technology is used to prepare hybridoma, which is a method of fusing a sensitized B cell having the ability to secrete a specific antibody and a myeloma cell having an unlimited proliferation ability into a B cell hybridoma based on cell fusion technology. By culturing a single hybridoma having such a characteristic into a cell population, a monoclonal antibody, which is a specific antibody against one epitope, can be produced.

CN105866409A discloses a candida mannan antigen immunodetection kit, which comprises an enzyme label plate coated with Mn and an anti-Mn polyclonal enzyme labeled antibody. The kit comprises the steps of coating Mn on an enzyme label plate, competitively binding a sample to be detected or a standard antigen and the coated antigen to a limited antibody binding site, carrying out chromogenic reaction on the sample to be detected or the standard antigen and a substrate through horseradish peroxidase, drawing a standard curve according to a Mn standard substance, and calculating the concentration value of the antigen to be detected according to the standard curve. The test kit has good sensitivity and specificity, simple and easy operation, and quick and sensitive detection. However, since polyclonal antibodies can detect a range of epitopes on a protein, they are at higher risk of inter-batch variation than monoclonal antibodies.

At present, the most widely used monoclonal antibody is a murine monoclonal antibody, but the murine monoclonal antibody has the problems of weak affinity and poor specificity. The rabbit monoclonal antibody technology has developed rapidly in recent years, and compared with a mouse monoclonal antibody, the rabbit monoclonal antibody has the advantages of recognition of more sites, stronger affinity and specificity.

The immunoassay technique is a method for qualitatively or quantitatively detecting an antigen or an antibody by detecting a marker labeled on a reactant by utilizing a specific binding reaction between the antigen and the antibody. According to the difference of the labeled substances, enzyme-Linked immunosorbent Assay (ELISA), immunofluorescence Assay, chemiluminescence immunoassay, immunomicrosphere, immunocolloidal gold, etc. are classified. The chemiluminescence immunoassay technology has the characteristics of simple operation, high sensitivity and short detection time, and is widely used in clinical detection and scientific research.

The antibody with excellent performance is the basis of the immunoassay technology, and the immunoassay kit with excellent performance can be developed only if the antibody is good. Therefore, the provision of an anti-candida mannan monoclonal antibody which recognizes more sites and has stronger affinity and specificity has important significance in candida detection.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide an anti-candida mannan monoclonal antibody and application thereof. The anti-candida mannan monoclonal antibody is a rabbit-derived monoclonal antibody, has the characteristics of multiple antigen recognition sites, good specificity and high affinity, solves the problem of practical application of the mouse-derived monoclonal antibody, and provides a new scheme for candida detection, diagnosis, prevention and treatment.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides an anti-candida mannan monoclonal antibody, comprising a heavy chain variable region and a light chain variable region;

the heavy chain variable region comprises a heavy chain CDR3 shown in SEQ ID NO. 3;

the light chain variable region comprises light chain CDR3 shown in SEQ ID NO. 6.

Preferably, the heavy chain variable region further comprises heavy chain CDR1 shown in SEQ ID NO.1 and heavy chain CDR2 shown in SEQ ID NO. 2.

Preferably, the light chain variable region further comprises light chain CDR1 shown in SEQ ID NO.4 and light chain CDR2 shown in SEQ ID NO. 5.

SEQ ID NO.1：SGYDMC。

SEQ ID NO.2：CSGNSGNTYYASWAKG。

SEQ ID NO.3：GILDTGWTRLDL。

SEQ ID NO.4：QASKSVANTDLA。

SEQ ID NO.5：GASNLAS。

SEQ ID NO.6：LGGYSGGSDNA。

The monoclonal antibody has multiple original recognition sites, good specificity and high affinity; the monoclonal antibody is a rabbit-derived monoclonal antibody, and has a plurality of antigen recognition sites; the monoclonal antibody does not generate cross reaction with other saccharides (including capsular polysaccharide, galactomannan, lipopolysaccharide, peptidoglycan, 1,3-beta-D-glucan and BSA), and has strong specificity; the screened monoclonal antibody has stronger binding capacity and high affinity to Mn antigen, and the titer to the Mn antigen reaches 1.

Preferably, the amino acid sequence of the heavy chain variable region comprises the sequence shown in SEQ ID NO. 7.

Preferably, the amino acid sequence of the light chain variable region comprises the sequence shown in SEQ ID NO. 8.

SEQ ID NO.7：

METGLRWLLLVAVLKGVQCQSLEESGGGLVKPGASLTLTCKASGFSFSSGYDMCWVRQAPGKGLEWIACSGNSGNTYYASWAKGQFTISKTSTTVTLQMTSLTAADTATYFCARGILDTGWTRLDLWGQGTLVTVSS。

SEQ ID NO.8：

MDTRAPTQLLGLLLLWLPGATFAAVLTQTPSPVSAAVGGTVSINCQASKSVANTDLAWYQQKPGQRPKLLIYGASNLASGVPSRFKGSGSGTQFTLTISDVQCADAATYYCLGGYSGGSDNAFGGGTEVVVK。

Preferably, the anti-candida mannan monoclonal antibody further comprises any one of or a combination of at least two of rabbit-derived IgG1, igG2, igG3 or IgG4 constant regions, preferably rabbit-derived IgG1 constant regions.

The invention takes the New Zealand big ear rabbit as the experimental animal to prepare the monoclonal antibody, thus overcoming the defect of weak affinity of the mouse source antibody; the rabbit-derived monoclonal antibody is obtained by taking candida mannan as an antigen for immunization and performing cell fusion on the obtained spleen cells and myeloma cells, has good stability and strong affinity to the candida mannan. The monoclonal antibody has good specificity and strong affinity with mannan, does not generate cross reaction with other saccharides such as galactomannan, capsular polysaccharide, lipopolysaccharide and the like, and can be potentially applied to antigen detection of candida, detection and identification of clinical samples infected by candida and the like.

In a second aspect, the present invention provides a nucleic acid molecule encoding the anti-candida mannan monoclonal antibody of the first aspect.

Preferably, the nucleotide sequence encoding the heavy chain variable region of the anti-candida mannan monoclonal antibody comprises a sequence shown in SEQ ID NO.9, and the nucleotide sequence encoding the light chain variable region of the anti-candida mannan monoclonal antibody comprises a sequence shown in SEQ ID NO. 10.

SEQ ID NO.9：

atggagactgggctgcgctggcttctcctggtcgctgtgctcaaaggtgtccagtgtcagtcattagaggagtccgggggaggcctggtcaagcctggggcatccctgacactcacctgcaaagcctctggattctccttcagtagcggctacgacatgtgctgggtccgccaggctccagggaagggactagagtggatcgcatgttctggtaatagtggtaacacttactacgcgagctgggcgaaaggccaattcaccatctccaaaacctcgaccacggtgactctgcaaatgaccagtctgacagccgcggacacggccacctatttctgtgcgagaggtattcttgatactggttggactcggttggatctctggggccagggcaccctggtcaccgtctcctca。

SEQ ID NO.10：

atggacacgagggcccccactcagctgctggggctcctgctgctctggctcccaggtgccacatttgccgccgtgctgacccagactccatctcccgtgtctgcagctgtgggaggcacagtcagcatcaattgccaggccagtaagagtgttgctaataccgacttagcctggtatcagcagaaaccagggcagcgtcccaagctcctgatctatggtgcatccaatctggcatctggggtcccatcgcggttcaaaggcagtggatctgggacacagttcactctcaccatcagcgacgtgcagtgtgccgatgctgccacttactactgtctaggcggttatagtggtggtagtgataatgctttcggcggagggaccgaggtggtggtcaaa。

In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule of the second aspect.

In a fourth aspect, the present invention provides a host cell comprising at least one copy of the expression vector of the third aspect or having integrated into its genome the nucleic acid molecule of the second aspect.

Preferably, the host cell comprises a 293F cell or a CHO cell.

In a fifth aspect, the present invention provides a pharmaceutical composition comprising the anti-candida mannan monoclonal antibody of the first aspect.

Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

In a sixth aspect, the invention provides a candida detection kit, which comprises the anti-candida mannan monoclonal antibody of the first aspect.

Preferably, the kit for detecting candida also comprises any one of a positive control, a negative control, an antibody diluent, a developing solution, a stopping solution, a confining solution or a washing solution or the combination of at least two of the positive control, the negative control, the antibody diluent, the developing solution, the stopping solution and the confining solution.

In a seventh aspect, the present invention provides the use of any one or a combination of at least two of the anti-candida mannan monoclonal antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the host cell of the fourth aspect, or the pharmaceutical composition of the fifth aspect, in the preparation of a drug for treating and/or detecting candida infections.

Compared with the prior art, the invention has the following beneficial effects:

Drawings

FIG. 1 is a diagram showing the results of SDS-PAGE electrophoresis of a monoclonal antibody in example 6.

FIG. 2 shows the results of measurement of affinity activity of monoclonal antibodies in example 7.

FIG. 3 shows the results of the cross-reaction in example 8.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

EXAMPLE 1 antigen preparation

The preparation method of candida mannan antigen (Mn) is as follows:

(1) Thallus culture of candida and crude extraction of candida mannan

Inoculating Candida in Sabouraud's liquid culture medium (containing 1% peptone, 4%D-glucose per liter of culture medium), culturing at 30 deg.C in shaker at 200rpm for about 42h, and stopping culturing when pH of culture solution is reduced to 4.2-4.5; inactivating the obtained culture solution for 2h by using an air-blast drying oven at the temperature of 80 ℃, centrifugally collecting thalli, and cleaning the collected thalli by using normal saline; the cells were resuspended in sodium citrate solution and autoclaved at 121 ℃ for 90min, repeated twice.

Centrifuging at 4 deg.C and 16000g for 15min, collecting supernatant, precipitating with ethanol, and collecting precipitate; re-dissolving the precipitate with deionized water, and performing CTAB treatment; centrifuging at 4 deg.C and 16000g for 15min, collecting supernatant, adjusting pH of the supernatant to 8.8, and precipitating; centrifuging at 4 deg.C and 16000g for 15min, collecting precipitate, re-dissolving with glacial acetic acid, precipitating with ethanol, collecting precipitate, i.e. crude Candida mannan (Mn), re-dissolving with deionized water, dialyzing, filtering dialysis product with 0.22 μm filter membrane, and collecting the product.

(2) The crude candida mannan extract obtained was further purified according to the following steps:

the method comprises the steps of crude candida mannan extract → GE high-throughput separation → optimization of a gel exclusion device → gradient elution → component stage collection → trapped fluid → dialysis desalination → sugar concentration determination → concentration → sugar concentration determination → purity determination → obtaining of pure candida mannan antigen.

Example 2 animal immunization

The Mn antigen was mixed with freund's complete adjuvant in equal volumes to the appropriate volume. After full emulsification, the New Zealand big ear rabbits are injected subcutaneously in multiple points, and the immune dose of each rabbit is controlled to be 0.1-0.8mg. Ear blood of New Zealand big ear rabbits was taken 3 days before immunization, and serum was separated to serve as a negative control. Immunization was performed 1 time every 2 weeks after the initial immunization, and the method was the same as that of 1 st time. The total immunization was 6 times.

New Zealand big ear white rabbits with proper age and weight of about 1.5 kg are selected and raised in a standard animal house for 3 days, and if no abnormal condition exists, the rabbits are immunized. Adding 30 mu g of the prepared Mn antigen into 0.5mL of autoclaved physiological saline, and fully and uniformly mixing by using a micro vortex oscillator. Then the vaccine is mutually pushed and pulled with a 0.5mL Freund's complete adjuvant by an injector, and is fully emulsified, and subcutaneous multipoint injection immunization is carried out on the back of New Zealand big ear rabbits.

After two weeks, 30 mu g of Mn antigen is taken and added into 0.5mL of sterilized physiological saline, and is fully and uniformly mixed by a micro vortex oscillator, and then the mixture is mutually pushed and pulled by an injector with 0.5mL of Freund's incomplete adjuvant, and is fully emulsified for strengthening immunity. Thereafter, six booster immunizations were performed every other week. And starting from the third immunization, taking 200-500 mu L of new Zealand big ear white rabbit ear vein blood after one week of immunization, and measuring the titer and the affinity. Spleens were harvested after the last immunization.

The specific immunization steps are as follows:

(1) In the first week: the primary immunization comprises the steps of diluting the Mn antigen to 1mg/mL by using physiological saline, fully emulsifying the Mn antigen with Freund's complete adjuvant 1:l, respectively filling the two solutions into two injectors without generating air bubbles, butting the two injectors, gradually mixing the antigen diluent and the adjuvant from slow to fast, finally preparing the mixed solution into milky water-in-oil emulsion, and then carrying out subcutaneous multipoint immunization on the back with the dose of one l mL.

(2) In the third week: the immunization is strengthened, the Mn antigen is diluted to 0.5mg/mL by using physiological saline and fully emulsified with Freund incomplete adjuvant 1:1, and the immunization dose is 1mL each.

(3) In the fifth week: the immunization was performed in the same manner as in the third week at an immunization dose of 1mL each.

(4) In the sixth week: first serum titer test. Taking 200-500 μ L of ear marginal venous blood of immunized New Zealand big ear rabbit, standing for 30min, centrifuging to separate serum, and determining antiserum titer.

(5) In the seventh week: and (4) boosting the immunity in the same way as the step (3).

(6) And in the eighth week: and (5) carrying out secondary serum titer detection in the same step (4).

(7) In the ninth week: and (4) boosting the immunity in the same way as the step (3).

(8) In the tenth week: and (5) carrying out serum titer detection for the third time in the same step (4).

(9) In the eleventh week: and (4) boosting the immunity in the same way as the step (3).

The antiserum titer determination method comprises the following steps:

coating Mn antigen as coating antigen at a concentration of 100ng/well for 2h at 37 deg.C, adding blocking solution prepared by 3% BSA, incubating at 37 deg.C for 1h, shaking off the solution, and oven drying. Then the prepared antibody serum is diluted in a gradient way, added into an enzyme label plate, added with 100 mu L of each hole, incubated for 1h at 37 ℃, then washed with PBST for three times, kept stand for 3min each time,then, HRP-labeled goat anti-rabbit IgG was used as an enzyme-labeled antibody, and the dilution was 1. Adding 100 μ L TMB into each well, incubating and developing at 37 deg.C for 15min, adding 50 μ L stop solution into each well to terminate reaction, and detecting OD with microplate reader ₄₅₀ The value is obtained.

EXAMPLE 3 preparation of rabbit mAb

Performing titer detection on the prepared rabbit antiserum, and respectively performing cell fusion on the spleen of the qualified New Zealand big ear rabbit under the condition of qualified titer to prepare a monoclonal hybridoma cell strain, wherein the specific preparation method is as follows:

(1) Preparation of immune spleen cells

The immunized New Zealand big ear rabbits were sacrificed and the spleens were removed under aseptic conditions. Washing with cell culture solution for 1 time, grinding, sieving with stainless steel sieve, washing with cell culture solution for 2 times, and centrifuging to obtain cells.

(2) Cell fusion

Taking SP2/0 myeloma cells in logarithmic phase, mixing with the obtained spleen cells, washing with a cell culture solution without fetal calf serum once, centrifuging, removing supernatant, adding a polyethylene glycol solution, keeping the temperature at 37 ℃ for about 90s, terminating the reaction with the cell culture solution without fetal calf serum, centrifuging, resuspending with HAT selection culture solution containing 20% fetal calf serum, adding the cells into a 96-well plate, and culturing in a cell culture box. The culture temperature was 37 ℃ and CO ₂ The content is 5.0%.

(3) Cell monoclonality and screening

Cells with good growth state in a 96-well plate are diluted to 1-3 cells/mL by using a cell culture solution, added into the 96-well plate, and placed into a cell culture box for culture. The culture temperature is 37 ℃ and CO ₂ The content is 5.0%. Each cell line is numbered respectively, and the cell lines with positive culture solution supernatant are selected for amplification culture. Finally obtaining the hybridoma cell strain.

(4) Screening for hybridoma cells

The obtained hybridoma cells were screened by ELISA method to find a polyclonal antibody cell line against candida mannan antigens in up to 10 wells, which produced specific monoclonal antibodies with high affinity for the respective antigens.

After the cell fusion step, two parent cells and three randomly fused cells are present in the culture medium, and in order to obtain a hybridoma cell line capable of secreting the target antibody, the successfully fused hybridoma cells must be separated from a plurality of cells. B lymphocytes cannot survive in vitro for a long period of time, and only myeloma cells and their own fused cells need to be removed, and therefore, it is necessary to culture the fused cells in HAT medium, selectively retaining hybridoma cells.

The growth condition of the cells can be observed on the 5 th day after fusion, and the cell culture supernatant can be detected by adopting an indirect ELISA method and positive hybridoma cell strains can be screened for cloning culture on the 10 th to 14 th days. And cloning and culturing the positive hybridoma cells by adopting a limiting dilution method. And (3) expanding the positive hybridoma cells with the strongest detection result titer to a fixed strain when the cell positive rate reaches 100%. The titer of culture supernatant of the fixed hybridoma cell line was measured by ELISA, and the expanded monoclonal hybridoma cell line was cryopreserved in liquid nitrogen.

And (3) testing the specificity and affinity of the supernatant of the hybridoma cells, selecting the hybridoma cells suitable for pairing from the supernatant, performing recombinant expression on the selected four-hole polyclonal hybridoma cell strain to obtain a monoclonal antibody, performing sandwich method pairing on the obtained purified antibody, establishing a sandwich method detection system of the candida mannan antigen, and finally respectively obtaining the best paired monoclonal antibodies.

Example 4 sequence determination of Rabbit monoclonal antibodies

(1) Isolation of Total RNA from hybridoma cells

After homogenization of the hybridoma cells, TRIzol was added and the homogenate separated into a clear upper aqueous layer (containing RNA), an interphase, and a red lower organic layer (containing DNA and protein). The RNA was then precipitated from the aqueous layer with isopropanol. The DNA was precipitated from the organic layer with ethanol. Proteins were precipitated from phenol-ethanol supernatant using isopropanol precipitation. The precipitated RNA is washed, impurities are removed, and the RNA is resuspended for later use.

(2) Reverse transcription of Total RNA into cDNA

A cDNA single strand which is complementary with the RNA template is synthesized on the 3' -end of tRNA by taking dNTP as a substrate, RNA as a template and tRNA as a primer according to the 5' → 3' direction, and forms an RNA-cDNA hybrid with the RNA template. Then, the RNA strand is hydrolyzed under the action of reverse transcriptase, and the second DNA strand is synthesized by using cDNA as template. To this end, total RNA is reverse transcribed into cDNA.

(3) Rapid Amplification of CDNA Ends (RACE)

Antibody fragments of the heavy and light chains were amplified according to standard procedures for Rapid Amplification of CDNA Ends (RACE). The amplified antibody fragments were cloned into standard cloning vectors, respectively. Colony PCR was performed to screen clones with inserts of the correct size. The antibody sequence was obtained.

The nucleotide sequence of the heavy chain variable region of the anti-candida mannan monoclonal antibody comprises a sequence shown in SEQ ID NO. 9; the nucleotide sequence of the variable region of the light chain of the anti-candida mannan monoclonal antibody comprises a sequence shown in SEQ ID NO. 10.

SEQ ID NO.9：

SEQ ID NO.10：

Example 5 construction of monoclonal antibody expression vector and purification of expression

(1) Construction of expression vectors for monoclonal antibodies

Setting appropriate enzyme cutting sites according to the sequence determination result of the rabbit monoclonal antibody, and performing gene synthesis on the heavy chain and the light chain respectively; the gene synthesis products were ligated to pcDNA3.4 expression vectors, respectively.

(2) Expression and purification of monoclonal antibodies

Expression of monoclonal antibodies:

(a) Preparation before transfection: counting and subculturing cells to ensure that the cells have transfection conditions;

(b) Cell transfection: after shaking the cell fluid, 50. Mu.L of the cell suspension was aseptically aspirated by a pipette, diluted 20-fold with 950. Mu.L of the medium, and the cells were counted using a cell counting plate at a viable cell density of about (4.5-5.5). Times.10 ⁶ cells/mL, the cell viability should be 95-99%;

(c) Preparing a transfection reagent and a plasmid mixture, and incubating for 20min at room temperature;

(d) Slowly adding the transfection reagent to the cell culture medium to be transfected, shaking the flask at 37.0 + -0.2 deg.C, 120rpm and 8% CO ₂ Culturing in the cell culture shaker;

(e) On day 2 post-transfection (18 to 22 hours post-transfection, day 1), the feed was added to the shake flask and the flask was gently shaken during the addition; the shake flask was placed at 37.0. + -. 0.2 ℃ at 120rpm and 8% CO ₂ The cells are cultured in the shaking table for 3-5 days.

(3) Purification of monoclonal antibodies

(a) Harvesting of cell culture solution: centrifuging to collect cell culture liquid supernatant, and purifying the antibody by using Protein A Beads;

(b) Antibody concentration measurements were performed after purification was complete.

EXAMPLE 6 monoclonal antibody molecular weight determination

The molecular weight of the monoclonal antibody is identified by SDS-PAGE electrophoresis, the sample addition amount per channel is 5 mug, and meanwhile, a standard series of known molecular weights is used as a reference (marker), the monoclonal antibody is firstly electrophoresed for 20min under the voltage of 90V and then electrophoresed under the voltage of 140V until the indicators are allThe gel was removed, stained with Coomassie Brilliant blue, and the stained gel was analyzed for molecular weight of the monoclonal antibody. FIG. 1 is a SDS-PAGE result of the monoclonal antibody, and it can be seen from FIG. 1 that the heavy and light chains of the monoclonal antibody protein are mainly distributed at about 55kDa and 25kDa, and lane M in FIG. 1 ₁ Marker, lane 1 monoclonal antibody.

Example 7 detection of the affinity Activity (potency) of monoclonal antibodies against Candida mannan (Mn) Using ELISA method

And (3) detecting the affinity activity, wherein the steps of detecting the affinity activity are as follows:

diluting the Mn antigen to 1 ng/mu L with PBS, adding 100 mu L of the Mn antigen into a 96-well enzyme label plate per well, and coating for 2h at 37 ℃; discard the supernatant, wash the plate 3 times with 0.01M PBST, prepare a blocking solution containing 3% BSA using PBST, add 100. Mu.L per well, block for 2h at 37 ℃; discard the supernatant, wash 5 times with PBST, perform gradient dilution of the purified and concentrated antibody from 1; discarding the antibody diluent, washing with PBST for 6 times, diluting HRP-labeled goat anti-rabbit IgG (secondary antibody) with 1; discarding the secondary antibody diluent, washing with PBST for 6 times, adding 100 μ L TMB into each well, and standing at 37 deg.C in dark for 15min; the reaction was stopped by adding 50. Mu.L of 1M dilute sulfuric acid to each well, and the absorbance was measured at 450 nm.

The results of the detection of the affinity activity of the monoclonal antibody are shown in table 1 and fig. 2, and under the condition that the OD value is kept unchanged, the larger the dilution factor of the monoclonal antibody is, the stronger the binding capacity of the antigen-antibody is, the stronger the binding capacity of the selected monoclonal antibody to the Mn antigen is, and the titer to the Mn antigen is 1.

TABLE 1

Dilution factor	OD value
		1:10000	4.215
1:20000	4.139
		1:40000	4.063
1:80000	3.975
		1:160000	3.579
1:320000	2.654
		1:640000	1.928
1:1280000	0.962

Example 8 detection of specificity of monoclonal antibody by ELISA method

The specificity of the monoclonal antibody was detected by cross-reaction, which was performed as follows:

respectively coating the enzyme label plate with mannan, galactomannan, capsular polysaccharide, lipopolysaccharide, peptidoglycan, 1,3-beta-D-glucan and BSA, wherein the coating amount of each hole is 50 ng/hole; diluting the monoclonal antibody to 10ng/mL, adding the diluted monoclonal antibody into each enzyme label plate, adding 100 mu L of monoclonal antibody into each hole, and incubating for 1h at 37 ℃; after washing, 100. Mu.L of secondary antibody (HRP-labeled goat anti-rabbit IgG) was added to each well and incubated at 37 ℃ for 0.5h; after washing, TMB was added and incubation was carried out at 37 ℃ for 15min, and the reading was terminated. The results of the cross-reaction are shown in FIG. 3, which indicates that the obtained monoclonal antibody does not cross-react with other saccharides and has strong specificity.

In conclusion, the invention provides the anti-candida mannan monoclonal antibody, which is a rabbit-derived monoclonal antibody, has multiple antigen recognition sites, good specificity and high affinity, solves the problem of the mouse-derived monoclonal antibody in the aspect of practical application, and has important application value in preparation of candida detection products.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.

Sequence listing

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<120> anti-candida mannan monoclonal antibody and application thereof

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85 90 95

Thr Ile Ser Asp Val Gln Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Leu

100 105 110

Gly Gly Tyr Ser Gly Gly Ser Asp Asn Ala Phe Gly Gly Gly Thr Glu

115 120 125

Val Val Val Lys

130

<210> 9

<211> 411

<212> DNA

<213> Artificial sequence

<400> 9

atggagactg ggctgcgctg gcttctcctg gtcgctgtgc tcaaaggtgt ccagtgtcag 60

tcattagagg agtccggggg aggcctggtc aagcctgggg catccctgac actcacctgc 120

aaagcctctg gattctcctt cagtagcggc tacgacatgt gctgggtccg ccaggctcca 180

gggaagggac tagagtggat cgcatgttct ggtaatagtg gtaacactta ctacgcgagc 240

tgggcgaaag gccaattcac catctccaaa acctcgacca cggtgactct gcaaatgacc 300

agtctgacag ccgcggacac ggccacctat ttctgtgcga gaggtattct tgatactggt 360

tggactcggt tggatctctg gggccagggc accctggtca ccgtctcctc a 411

<210> 10

<211> 396

<212> DNA

<213> Artificial sequence

<400> 10

atggacacga gggcccccac tcagctgctg gggctcctgc tgctctggct cccaggtgcc 60

acatttgccg ccgtgctgac ccagactcca tctcccgtgt ctgcagctgt gggaggcaca 120

gtcagcatca attgccaggc cagtaagagt gttgctaata ccgacttagc ctggtatcag 180

cagaaaccag ggcagcgtcc caagctcctg atctatggtg catccaatct ggcatctggg 240

gtcccatcgc ggttcaaagg cagtggatct gggacacagt tcactctcac catcagcgac 300

gtgcagtgtg ccgatgctgc cacttactac tgtctaggcg gttatagtgg tggtagtgat 360

aatgctttcg gcggagggac cgaggtggtg gtcaaa 396

Claims

1. The anti-candida mannan monoclonal antibody is characterized by comprising a heavy chain variable region and a light chain variable region;

the heavy chain variable region comprises a heavy chain CDR3 shown in SEQ ID NO.3, a heavy chain CDR1 shown in SEQ ID NO.1 and a heavy chain CDR2 shown in SEQ ID NO. 2;

the light chain variable region comprises a light chain CDR3 shown in SEQ ID NO. 6; the light chain variable region further comprises a light chain CDR1 shown in SEQ ID NO.4 and a light chain CDR2 shown in SEQ ID NO. 5.

2. The anti-candida mannan monoclonal antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 7;

the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8.

3. The anti-candida mannan monoclonal antibody according to claim 1, further comprising any one of rabbit IgG1, igG2, igG3 or IgG4 constant regions or a combination of at least two thereof.

4. The anti-candida mannan monoclonal antibody according to claim 3, wherein the constant region of the anti-candida mannan monoclonal antibody is a rabbit-derived IgG1 constant region.

5. A nucleic acid molecule encoding the anti-candida mannan monoclonal antibody of any one of claims 1-4;

the nucleotide sequence of the heavy chain variable region of the anti-candida mannan monoclonal antibody is encoded to comprise a sequence shown in SEQ ID NO.9, and the nucleotide sequence of the light chain variable region of the anti-candida mannan monoclonal antibody is encoded to comprise a sequence shown in SEQ ID NO. 10.

6. An expression vector comprising the nucleic acid molecule of claim 5.

7. A host cell comprising at least one copy of the expression vector of claim 6, or having the nucleic acid molecule of claim 5 integrated into the genome of the host cell;

the host cell includes 293F cells or CHO cells.

8. A pharmaceutical composition comprising the anti-candida mannan monoclonal antibody of any one of claims 1-4;

the pharmaceutical composition further comprises any one or a combination of at least two of pharmaceutically acceptable carriers, excipients or diluents.

9. A candida detection kit, comprising the anti-candida mannan monoclonal antibody according to any one of claims 1 to 4;

the candida detection kit further comprises any one or combination of at least two of a positive control substance, a negative control substance, an antibody diluent, a developing solution, a stopping solution, a confining solution or a washing solution.

10. Use of any one or a combination of at least two of the anti-candida mannan monoclonal antibody according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, the host cell according to claim 7 or the pharmaceutical composition according to claim 8 for the preparation of a drug for the treatment and/or detection of candida infectious diseases.