WO2023245801A1 - Anti-candida mannan monoclonal antibody and use thereof - Google Patents

Abstract

Provided in the present application are an anti-candida mannan monoclonal antibody and the use thereof. The monoclonal antibody is a rabbit-derived monoclonal antibody, has multiple antigen recognition sites, good specificity and high affinity, and has an important application value in the preparation of candida detection products.

Description

An anti-Candida mannan monoclonal antibody and its application Technical field

The invention belongs to the field of biotechnology, and specifically relates to an anti-Candida mannan monoclonal antibody and its application.

Background technique

Candida albicans is a clinically isolated opportunistic human pathogenic fungus. Especially in patients with downregulated immunity, such as organ transplant patients, HIV-infected patients, etc., it can cause a wide range of superficial and Deep systemic infections include the oral cavity, female vagina, etc., causing thrush, vaginitis, etc. It can also invade epidermis and endothelial cells, enter the bloodstream, and reach internal organs, such as kidneys, brain, etc., causing sepsis, which can even lead to death in severe cases.

In recent years, there has been a significant increase in Candida albicans infections. Infections caused by Candida rank fourth among blood-borne infections and third among catheter-related infections. Even with the use of antifungal drugs, the mortality rate remains. Up to 40%. Systemic Candida infection does not have typical clinical symptoms, and there is currently a lack of specific early diagnosis methods for Candida infection.

At present, the gold standard for detecting Candida infection is blood culture. However, the disease progresses rapidly. Traditional culture methods have low positive detection and long culture cycles, which often lead to missed diagnosis and misdiagnosis, leading to delayed disease progression. Therefore, it is of great significance to develop a highly specific antibody that can be used for Candida detection.

Monoclonal antibodies are highly uniform antibodies produced by a single B cell clone and only target a specific antigenic epitope. They are called monoclonal antibodies. Usually prepared using hybridoma technology. Hybridoma antibody technology is based on cell fusion technology, which fuses sensitized B cells with the ability to secrete specific antibodies and myeloma cells with unlimited reproduction ability into B cell hybrids. tumor. By cultivating a single hybridoma cell with this characteristic into a cell population, a specific antibody, that is, a monoclonal antibody, can be prepared against an antigenic epitope.

CN105866409A discloses a Candida mannan antigen immunodetection kit, which includes a Mn-coated enzyme-labeled plate and an anti-Mn polyclonal enzyme-labeled antibody. The kit coats Mn on an enzyme plate, competes the sample to be tested or standard antigen with the coated antigen for binding to limited antibody binding sites, and then reacts with the substrate through horseradish peroxidase for color development. , draw a standard curve based on the Mn standard substance, and calculate the concentration value of the antigen to be detected based on the standard curve. The test kit has good sensitivity and specificity, is simple and easy to operate, and detects quickly and sensitively. But because polyclonal antibodies detect a range of epitopes on a protein, they are subject to a higher risk of batch-to-batch variation than monoclonal antibodies.

Currently, the most widely used monoclonal antibodies are mouse monoclonal antibodies, but mouse monoclonal antibodies have problems with weak affinity and poor specificity. Rabbit monoclonal antibody technology has developed rapidly in recent years. Compared with mouse monoclonal antibodies, rabbit monoclonal antibodies have the ability to recognize more sites and have stronger affinity and specificity.

Immunoassay technology is a method that utilizes the specific binding reaction between antigens and antibodies to achieve qualitative or quantitative detection of antigens or antibodies by detecting markers labeled on the reactants. According to the different labeling substances, it is divided into enzyme-linked immunoassay technology (Enzyme-Linked Immunosorbant Assay, ELISA), immunofluorescence detection technology, chemiluminescence immunoassay technology, immune microsphere technology, immunocolloidal gold technology, etc. Among them, 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.

Antibodies with excellent performance are the basis of immunoassay technology. Only with good antibodies can immunoassay kits with excellent performance be developed. Therefore, providing an anti-Candida mannan monoclonal antibody that recognizes more sites, has stronger affinity and specificity is of great significance in Candida detection.

Contents of the invention

In view of the shortcomings of the existing technology, the purpose of the present invention is to provide an anti-Candida mannan monoclonal antibody and its application. The anti-Candida mannan monoclonal antibody is a rabbit-derived monoclonal antibody, which has the characteristics of multiple antigen recognition sites, good specificity, and high affinity. It solves the practical application problems of mouse-derived monoclonal antibodies and provides Candida Provides new solutions for bacterial detection, diagnosis, prevention and treatment.

In order to achieve the purpose of this invention, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides an anti-Candida mannan monoclonal antibody, which includes a heavy chain variable region and a light chain variable region;

The heavy chain variable region includes the heavy chain CDR3 shown in SEQ ID NO.3;

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

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

Preferably, the light chain variable region also includes the light chain CDR1 shown in SEQ ID NO.4 and the 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 in the present invention has many original recognition sites, good specificity and high affinity; the monoclonal antibody is a rabbit-derived monoclonal antibody and has many antigen recognition sites; the monoclonal antibody does not interact with other carbohydrates. (Including capsular polysaccharide, galactomannan, lipopolysaccharide, peptidoglycan, 1,3-β-D-glucan and BSA) cross-reaction and strong specificity; the screened monoclonal antibodies have strong specificity for Mn antigen It has strong binding ability and high affinity, and its titer for Mn antigen reaches 1:1280000 (OD value>0.5).

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

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

SEQ ID NO.7：

SEQ ID NO.8：

Preferably, the anti-Candida mannan monoclonal antibody also includes any one or a combination of at least two of rabbit-derived IgG1, IgG2, IgG3 or IgG4 constant regions, preferably rabbit-derived IgG1 constant region.

The present invention uses New Zealand big-eared rabbits as experimental animals to prepare monoclonal antibodies, which overcomes the defect of weak affinity of mouse-derived antibodies; Candida mannan is used as an antigen for immunization, and the obtained spleen cells undergo cell fusion with myeloma cells. A rabbit-derived monoclonal antibody was obtained with good stability and strong affinity for Candida mannan. The monoclonal antibody of the present invention has good specificity and strong affinity with mannan, but does not cross-react with other sugars such as galactomannan, capsular polysaccharide, lipopolysaccharide, etc., and can be potentially used in rosary beads. Antigen detection of bacteria, detection and identification of clinical samples of Candida infection, etc.

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

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

SEQ ID NO.9：

SEQ ID NO.10：

In a third aspect, the present invention provides an expression vector, which includes the nucleic acid molecule described in the second aspect.

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

Preferably, the host cells include 293F cells or CHO cells.

In a fifth aspect, the present invention provides a pharmaceutical composition comprising the anti-Candida mannan monoclonal antibody described in the first aspect.

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

In a sixth aspect, the present invention provides a kit for detecting Candida, which includes the anti-Candida mannan monoclonal antibody described in the first aspect.

Preferably, the kit for detecting Candida also includes any one or a combination of at least two of a positive control substance, a negative control substance, an antibody diluent, a chromogenic solution, a stop solution, a blocking solution or a washing solution.

In a seventh aspect, the present invention provides the anti-Candida mannan monoclonal antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the host cell described in the fourth aspect, or The use of any one or a combination of at least two of the pharmaceutical compositions described in the fifth aspect in the preparation of candida infection treatment drugs and/or detection products.

Compared with the existing technology, the present invention has the following beneficial effects:

The monoclonal antibody in the present invention has many original recognition sites, good specificity and high affinity; the monoclonal antibody is a rabbit-derived monoclonal antibody and has many antigen recognition sites; the monoclonal antibody does not interact with other sugars (including Capsular polysaccharide, galactomannan, lipopolysaccharide, peptidoglycan, 1,3-β-D-glucan and BSA) cross-react with strong specificity; the screened monoclonal antibodies have strong resistance to Mn antigen The binding ability and affinity are high, and the titer for Mn antigen reaches 1:1280000 (OD value>0.5).

Description of the drawings

Figure 1 is a diagram of the SDS-PAGE electrophoresis results of the monoclonal antibody in Example 6.

Figure 2 is the detection result of the affinity activity of the monoclonal antibody in Example 7.

Figure 3 shows the cross-reaction results in Example 8.

Detailed ways

The technical solution of the present invention will be further described below through specific implementations. Those skilled in the art should understand that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field shall be followed, or the product instructions shall be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased through regular channels.

Example 1 Antigen preparation

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

(1) Candida cell culture and Candida mannan crude extraction

Inoculate Candida into Sabouraud liquid medium (each liter of medium contains 1% peptone and 4% D-glucose), and culture it on a 30°C shaker at 200rpm for about 42 hours. When the pH value of the culture medium drops to 4.2-4.5 Stop the culture when the temperature is reached; inactivate the resulting culture solution in a blast drying oven at 80°C for 2 hours, collect the cells by centrifugation, and wash the collected cells with physiological saline; resuspend the cells in sodium citrate solution and autoclave at 121°C for 90 minutes. , repeated twice.

Centrifuge at 4°C and 16000g for 15 minutes to collect the supernatant, perform alcohol precipitation, and collect the precipitate; redissolve the precipitate with deionized water, and perform CTAB treatment; centrifuge at 4°C and 16000g for 15 minutes to collect the supernatant, and adjust the pH of the supernatant to 8.8 , a precipitate precipitated; centrifuge at 4°C and 16000g for 15 minutes to collect the precipitate, and redissolve it with glacial acetic acid. The reconstituted solution is subjected to alcohol precipitation, and the precipitate is collected. The precipitate is the crude Candida mannan (Mn), and the precipitate is reconstituted with deionized water. , dialysis, and the dialysis product is filtered through a 0.22 μm filter membrane, and the obtained product is the crude Candida mannan product.

(2) Further purify the obtained Candida mannan crude extract. The purification steps are as follows:

Candida mannan crude extract → GE high-throughput separation → gel exclusion device optimization → gradient elution → staged collection of components → retentate → dialysis desalting → sugar concentration determination → concentration → sugar concentration determination → purity determination → Pure Candida mannan antigen was obtained.

Example 2 Animal Immunization

Mix Mn antigen and Freund's complete adjuvant in equal volumes to an appropriate volume. After sufficient emulsification, perform subcutaneous injection into New Zealand big-eared rabbits at multiple points, and the immune dose for each rabbit is controlled at 0.1-0.8 mg. Three days before immunization, ear blood from New Zealand big-eared rabbits was collected, and the serum was separated as a negative control. Immunize once every 2 weeks after the initial immunization, and the method is the same as the first time. A total of 6 immunizations were administered.

Select New Zealand big-eared white rabbits of appropriate age and weighing about 1.5 kilograms, and raise them in a standard animal room for 3 days. If there are no abnormalities, vaccination will begin. Take 30 μg of the Mn antigen prepared above, add it to 0.5 mL of autoclaved physiological saline, and mix thoroughly with a micro vortex shaker. Then, push and pull it with 0.5 mL of Freund's complete adjuvant with a syringe to fully emulsify, and then immunize New Zealand big-eared rabbits by subcutaneous injection at multiple points on the back.

Two weeks later, take 30 μg of Mn antigen, add it to 0.5 mL of sterilized physiological saline, mix thoroughly with a micro vortex shaker, and then push and pull with 0.5 mL of Freund's incomplete adjuvant with a syringe to fully emulsify and perform booster immunization. . Thereafter, booster immunizations were performed every other week, for a total of six immunizations. Starting from the third immunization, 200-500 μL of blood from the ear margin of New Zealand white rabbits was taken one week after the immunization to measure its potency and affinity. Spleens were harvested after the last immunization.

The specific immunization steps are as follows:

(1) First week: For the first immunization, dilute the Mn antigen to 1 mg/mL with physiological saline, and fully emulsify it with Freund's complete adjuvant 1:1. Put the two solutions into two syringes respectively without generating bubbles. Connect the two syringes, gradually mix the antigen diluent and the adjuvant from slow to fast, and finally make the mixture into a milky white water-in-oil emulsion, and then perform multi-point subcutaneous immunization on the back, with a dose of 1 mL per animal.

(2) Third week: To strengthen the immunity, the Mn antigen is diluted to 0.5 mg/mL with physiological saline and fully emulsified with Freund's incomplete adjuvant at 1:1. The immunization dose is 1 mL per animal.

(3) The fifth week: Boost immunity. The immunization method is the same as the third week. The immunization dose is 1mL per animal.

(4) Week 6: The first serum titer test. Take 200-500 μL of ear marginal venous blood from the immunized New Zealand big-eared rabbits, let it stand for 30 minutes, then centrifuge to separate the serum, and measure the antiserum titer.

(5) Seventh week: strengthen immunity, the immunization method is the same as step (3).

(6) Eighth week: Second serum titer test, same as step (4).

(7) Week 9: Boost immunity, the immunization method is the same as step (3).

(8) Week 10: The third serum titer test, the same as step (4).

(9) Week 11: Strengthen immunity, the immunization method is the same as step (3).

Antiserum titer determination method:

Use Mn antigen as the coating antigen with a coating concentration of 100ng/well and coat at 37°C for 2 hours. Then add blocking solution prepared with 3% BSA and incubate at 37°C for 1 hour. Shake off the liquid and dry it for later use. Then carry out gradient dilution of the prepared antibody serum and add it to the enzyme plate. Add 100 μL to each well and incubate at 37°C for 1 hour. Then wash it three times with PBST and let it stand for 3 minutes each time. Then use HRP-labeled goat anti-rabbit IgG as the Enzyme-labeled antibody, diluted 1:5000, add 100 μL to each well, incubate at 37°C for 1 hour, then wash three times with PBST and let stand for 40 seconds each time. Add 100 μL TMB to each well, incubate at 37°C for 15 min to develop color, then add 50 μL stop solution to each well to end the reaction, and detect the OD ₄₅₀ value on a microplate reader.

Example 3 Preparation of rabbit monoclonal antibodies

The titer of the prepared rabbit antiserum is tested. If the titer is qualified, the spleens of qualified New Zealand big-eared rabbits are used for cell fusion to prepare monoclonal hybridoma cell lines. The specific preparation method is as follows:

(1) Preparation of immune spleen cells

The immunized New Zealand big-eared rabbits were sacrificed, and the spleens were removed under aseptic conditions. Wash once with cell culture medium, grind, pass through a stainless steel mesh, wash twice with cell culture medium, and centrifuge to obtain cells.

(2) Cell fusion

Take SP2/0 myeloma cells in the logarithmic phase, mix them with the obtained spleen cells, wash once with cell culture medium without fetal bovine serum, centrifuge, discard the supernatant, add polyethylene glycol solution and keep the temperature at 37°C for about 90 seconds. , terminate the reaction with cell culture medium without fetal bovine serum, centrifuge, resuspend with HAT selection culture medium containing 20% fetal bovine serum, add the cells to a 96-well plate, and place them in a cell culture incubator for culture. The culture temperature is 37°C and the _CO2 content is 5.0%.

(3) Cell monocloning and screening

Dilute the well-growing cells in the 96-well plate to 1-3 cells/mL with cell culture medium, add them to the 96-well plate, and place them in a cell culture incubator for culture. The culture temperature is 37°C and the _CO2 content is 5.0%. Each cell line is numbered separately, and the cell lines whose culture medium supernatant is positive are selected for expansion and culture. Finally, hybridoma cell lines were obtained.

(4) Screen hybridoma cells

The obtained hybridoma cells were screened by ELISA method to find up to 10 wells of polyclonal antibody cell lines targeting Candida mannan antigen. The cell lines can produce specific monoclonal antibodies with high affinity against the respective antigens.

After the cell fusion step, there are two parental cells and three randomly fused cells in the culture medium. In order to obtain a hybridoma cell line that can secrete the target antibody, the successfully fused hybridoma cells must be separated from the numerous cells. B lymphocytes cannot survive in vitro for a long time. Only myeloma cells and their own fused cells need to be removed. Therefore, the fused cells need to be cultured in HAT culture medium to selectively retain hybridoma cells.

The growth of the cells can be observed on the 5th day after fusion. On the 10th to 14th day, the indirect ELISA method can be used to detect the cell culture supernatant and screen positive hybridoma cell lines for cloning culture. The positive hybridoma cells were cloned and cultured using the limiting dilution method. The positive hybridoma cells with the strongest detection result titer were expanded until the cell positivity rate reached 100% to determine the strain. Use ELISA to measure the potency of the culture supernatant of the established hybridoma cell line, and freeze the expanded cultured monoclonal hybridoma cell line in liquid nitrogen.

Conduct specificity and affinity tests on the hybridoma cell supernatants, select hybridoma cells suitable for pairing, recombinantly express the selected four-well polyclonal hybridoma cell lines to obtain monoclonal antibodies, and use the purified antibodies obtained Sandwich method matching was performed to establish a sandwich detection system for Candida mannan antigen, and finally the best paired monoclonal antibodies were obtained.

Example 4 Rabbit monoclonal antibody sequence determination

(1) Isolate total RNA from hybridoma cells

After the hybridoma cells are homogenized, TRIzol is added, and the homogenate can be divided into a transparent upper aqueous layer (containing RNA), a phase interface, and a red lower organic layer (containing DNA and protein). The RNA is then precipitated from the aqueous layer using isopropyl alcohol. DNA was precipitated from the organic layer using ethanol. Proteins were precipitated from the phenol-ethanol supernatant using isopropanol precipitation. Wash the precipitated RNA to remove impurities and resuspend it for later use.

(2) Reverse transcribe total RNA into cDNA

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

(3) Rapid amplification of cDNA ends (RACE)

Antibody fragments of heavy and light chains were amplified according to the standard operating procedure of Rapid Amplification of cDNA Ends (RACE). The amplified antibody fragments were individually cloned into standard cloning vectors. Perform colony PCR to screen for clones with the correct size insert. The antibody sequence is obtained.

The nucleotide sequence encoding the heavy chain variable region of the anti-Candida mannan monoclonal antibody includes the sequence shown in SEQ ID NO. 9; the light chain encoding the anti-Candida mannan monoclonal antibody may The nucleotide sequence of the variable region includes the 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 and expression

(1) Construction of expression vector for monoclonal antibodies

Based on the rabbit monoclonal antibody sequence determination results, appropriate enzyme cutting sites were set to perform gene synthesis on the heavy chain and light chain respectively; the gene synthesis products were connected to the pcDNA3.4 expression vector respectively.

(2) Expression and purification of monoclonal antibodies

Expression of monoclonal antibodies:

(a) Preparation before transfection: cell counting and passage processing to ensure that the cells have the conditions for transfection;

(b) Cell transfection: After shaking the cell solution, use a pipette to aseptically absorb 50 μL of cell suspension, add 950 μL of culture medium to dilute it 20 times, and use a cell counting board to count cells. The viable cell density is approximately (4.5-5.5 )×10 ⁶ cells/mL, the cell viability should be 95-99%;

(c) Prepare transfection reagent and plasmid mixture, and incubate at room temperature for 20 minutes;

(d) Slowly add the transfection reagent into the culture medium of the cells to be transfected, and place the shake flask in a cell culture shaker at 37.0±0.2°C, 120 rpm and 8% _CO2 for culture;

(e) On the 2nd day after transfection (18 to 22 hours after transfection, day 1), add the feed material to the shake flask, and shake the shake flask gently during the addition process; place the shake flask at 37.0 Cultivate in a cell culture shaker at ±0.2°C, 120 rpm and 8% _CO2 , and continue culturing for 3-5 days.

(3) Purification of monoclonal antibodies

(a) Cell culture medium harvest: Centrifuge to collect the cell culture medium supernatant, and use ProteinABeads for antibody purification;

(b) After the purification is completed, the antibody concentration is measured.

Example 6 Determination of molecular weight of monoclonal antibodies

Use SDS-PAGE electrophoresis to identify the molecular weight of monoclonal antibodies. The loading volume of each electrophoresis lane is 5 μg. At the same time, a series of known molecular weight standards are used as a reference (marker). First, electrophoresis is performed at 90V for 20 minutes, and then at 140V until the After all the indicator has run out, remove the gel and stain it with Coomassie Brilliant Blue. After staining, the molecular weight of the monoclonal antibody is analyzed on the gel. Figure 1 shows the SDS-PAGE electrophoresis results of monoclonal antibodies. It can be seen from Figure 1 that the protein heavy and light chains of monoclonal antibodies are mainly distributed around 55kDa and 25kDa. In Figure 1, lane M ₁ is a marker, and lane 1 is a single marker. Cloning antibodies.

Example 7 Using ELISA method to detect the affinity activity (titer) of monoclonal antibodies against Candida mannan (Mn)

Affinity activity detection, the steps for detecting affinity activity are as follows:

Dilute the Mn antigen with PBS to 1ng/μL, add 100μL per well to a 96-well microplate, and coat at 37°C for 2 hours; discard the supernatant, wash the plate three times with 0.01M PBST, and prepare a solution containing 3% in PBST. Add 100 μL of BSA blocking solution to each well and block for 2 hours at 37°C; discard the supernatant and wash 5 times with PBST. The purified and concentrated antibodies are gradient diluted from 1:1000 to 1:2560000. Add each well, add 100 μL to each well, and incubate at 37°C for 1 hour; discard the antibody diluent, wash 6 times with PBST, dilute HRP-labeled goat anti-rabbit IgG (secondary antibody) with 1:5000 blocking solution, add 100 μL of secondary antibody to each well, Incubate at 37°C for 1 hour; discard the secondary antibody dilution, wash 6 times with PBST, add 100 μL of TMB to each well, and place at 37°C for 15 min in the dark; add 50 μL of 1M dilute sulfuric acid to each well to terminate the reaction, and measure the absorbance at 450 nm.

The test results of the affinity activity of monoclonal antibodies are shown in Table 1 and Figure 2. When the OD value remains unchanged, the greater the dilution factor of the monoclonal antibody, the stronger the binding ability of the antigen-antibody. The screened monoclonal antibodies The cloned antibody has strong binding ability to Mn antigen, and the titer of Mn antigen reaches 1:1280000 (OD value>0.5).

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 Using ELISA method to detect the specificity of monoclonal antibodies

The specificity of monoclonal antibodies is detected through cross-reaction. The steps of cross-reaction are as follows:

The enzyme plate was coated with mannan, galactomannan, capsular polysaccharide, lipopolysaccharide, peptidoglycan, 1,3-β-D-glucan and BSA respectively, and the coating amount per well was 50ng/ hole; dilute the monoclonal antibody to 10ng/mL, add it to each microplate, add 100μL to each well, and incubate at 37°C for 1 hour; after washing, add 100μL of secondary antibody (HRP-labeled goat anti-rabbit IgG) to each well, and incubate at 37°C. Incubate for 0.5h; add TMB after washing, incubate at 37°C for 15min, and terminate reading. The cross-reaction results are shown in Figure 3. The results show that the obtained monoclonal antibody does not cross-react with other sugars and has strong specificity.

In summary, the present invention provides an anti-Candida mannan monoclonal antibody. The monoclonal antibody is a rabbit-derived monoclonal antibody with multiple antigen recognition sites, good specificity, and high affinity. It solves the problem of mouse-derived monoclonal antibodies. There are problems in the practical application of antibodies. The anti-Candida mannan monoclonal antibody has important application value in preparing products for Candida detection.

The applicant declares that the above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the technical field should understand that any person skilled in the technical field will not use the invention disclosed in the present invention. Within the technical scope, changes or substitutions that can be easily imagined fall within the protection scope and disclosure scope of the present invention.

Claims (10)

An anti-Candida mannan monoclonal antibody, characterized in that the anti-Candida mannan monoclonal antibody includes a heavy chain variable region and a light chain variable region; The heavy chain variable region includes the heavy chain CDR3 shown in SEQ ID NO.3; The light chain variable region includes the light chain CDR3 shown in SEQ ID NO. 6. The anti-Candida mannan monoclonal antibody according to claim 1, wherein the heavy chain variable region also includes the heavy chain CDR1 shown in SEQ ID NO.1 and the heavy chain CDR1 shown in SEQ ID NO.2 Heavy chain CDR2; The light chain variable region also includes the light chain CDR1 shown in SEQ ID NO.4 and the light chain CDR2 shown in SEQ ID NO.5. The anti-Candida mannan monoclonal antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region includes the sequence shown in SEQ ID NO.7; The amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO. 8. The anti-Candida mannan monoclonal antibody according to claim 1, characterized in that the anti-Candida mannan monoclonal antibody further includes any one of rabbit-derived IgG1, IgG2, IgG3 or IgG4 constant region Or a combination of at least two, preferably rabbit-derived IgG1 constant region. A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the anti-Candida mannan monoclonal antibody according to any one of claims 1-4; The nucleotide sequence encoding the heavy chain variable region of the anti-Candida mannan monoclonal antibody includes the sequence shown in SEQ ID NO. 9, and the light chain encoding the anti-Candida mannan monoclonal antibody may The nucleotide sequence of the variable region includes the sequence shown in SEQ ID NO.10. An expression vector, characterized in that the expression vector includes the nucleic acid molecule of claim 5. A host cell, characterized in that the host cell contains at least one copy of the expression vector of claim 6, or the nucleic acid molecule of claim 5 is integrated into the genome of the host cell; The host cells include 293F cells or CHO cells. A pharmaceutical composition, characterized in that the pharmaceutical composition includes the anti-Candida mannan monoclonal antibody according to any one of claims 1-4; The pharmaceutical composition also includes any one or a combination of at least two of pharmaceutically acceptable carriers, excipients or diluents. A kit for detecting Candida, characterized in that the kit for detecting Candida includes the anti-Candida mannan monoclonal antibody according to any one of claims 1-4; The kit for detecting Candida also includes any one or a combination of at least two of a positive control substance, a negative control substance, an antibody diluent, a chromogenic solution, a stop solution, a blocking solution or a washing solution. The anti-Candida mannan monoclonal antibody of any one of claims 1-4, the nucleic acid molecule of claim 5, the expression vector of claim 6, the host cell of claim 7, or the Application of any one or a combination of at least two of the pharmaceutical compositions described in claim 8 in the preparation of drugs and/or detection products for treating candida infection.

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