Screening method of monoclonal antibody, ELISA (enzyme-linked immunosorbent assay) kit for screening monoclonal antibody and application of ELISA kit

Abstract

The invention provides a screening method of a monoclonal antibody, an ELISA kit for screening the monoclonal antibody and application of the ELISA kit, and relates to the technical field of biology. According to the screening method of the monoclonal antibody provided by the invention, the polypeptide and the carrier protein are coupled by utilizing the BMPS, so that not only is the problem of the coating efficiency of the polypeptide on an elisa plate solved, but also the polypeptide has the possibility of dissociating in a liquid phase due to the existence of the spacer arm, and the risk of polypeptide epitope shielding is greatly reduced; through a streptavidin-biotin system, it can be ensured that the polypeptide is completely free in a liquid phase, the risk that polypeptide epitopes are shielded is avoided, and the problem of ELISA false negative encountered in the high-throughput antibody screening process is greatly reduced.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for screening monoclonal antibodies, an ELISA kit for screening monoclonal antibodies, and their applications. Background Technology

[0002] In the development of monoclonal antibodies, peptides offer many advantages as antigens in animal immunization. For example, they allow for the selection of specific regions of target proteins for immunization. Furthermore, if recombinant proteins are difficult to express or purify, synthesizing peptides for immunization is an important salvage strategy. However, because peptide chains are typically 10-20 amino acids long and have small molecular weights, they are difficult to elicit a good immune response. Therefore, traditional techniques enhance immunogenicity by conjugating peptides to carrier proteins (such as keyhole limpet hemocyanin / KLH and bovine serum albumin / BSA).

[0003] Enzyme-linked immunosorbent assay (ELISA) is a crucial technique in monoclonal antibody screening. Using peptides as immunogens, it is typically performed via SMCC (Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) (spacer arm). When ELISA is performed after conjugating the peptide with the carrier protein KLH, the animal will simultaneously generate an immune response recognizing the peptide sequence, the conjugate SMCC, and the carrier protein KLH. Therefore, ELISA experiments require careful screening to identify only monoclonal antibodies that specifically recognize the peptide sequence. Generally, naked peptides are used for coating and detection directly. However, even slight changes in the physicochemical properties of the peptide (such as hydrophilicity / hydrophobicity and charge distribution) and coating conditions (pH, temperature, buffer composition) can lead to significant fluctuations in coating efficiency. Traditional methods use fixed concentrations (e.g., 1-10 μg / mL) and fixed times (e.g., overnight at 37°C) for coating, but the actual adsorption amount cannot be monitored in real time, resulting in large batch-to-batch variations and an increased risk of false negatives / positives.

[0004] Some laboratories employ carrier protein coupling to address the uncertainty in peptide coating efficiency. This involves coupling the peptide to a carrier protein (e.g., BSA) with a larger molecular weight, more binding sites, and minimal cross-linking with KLH via EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride). The adsorption capacity of the carrier protein allows for indirect coating. While this method can resolve the issue of inconsistent peptide coating efficiency, it still suffers from peptide epitope masking.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The primary objective of this invention is to provide a method for screening monoclonal antibodies to solve the aforementioned technical problems.

[0007] A second objective of this invention is to provide an ELISA kit for screening monoclonal antibodies.

[0008] A third objective of this invention is to provide the application of the above-described ELISA kit in monoclonal antibody screening.

[0009] To achieve the above objectives, the following technical solution is adopted:

[0010] In a first aspect, the present invention provides a method for screening monoclonal antibodies, which uses ELISA to screen monoclonal antibodies against peptides.

[0011] In ELISA, the polypeptide is coated on an enzyme-labeled plate, and the coating method includes at least one of the following a or b:

[0012] a. The polypeptide is coupled to a carrier protein via BMPS (3-maleimide propionic acid hydroxysuccinimide ester) before being coated onto an ELISA plate;

[0013] b. The ELISA plate is coated with streptavidin. The polypeptide is coupled to a biotin-labeled carrier protein via BMPS and then bound to the streptavidin on the ELISA plate via biotin.

[0014] As a further technical solution, the carrier protein includes BSA.

[0015] As a further technical solution, the biotin includes Sulfo-NHS-LC-LC-Biotin.

[0016] As a further technical solution, the ELISA includes indirect ELISA or competitive ELISA.

[0017] As a further technical solution, the indirect ELISA includes the following steps:

[0018] The polypeptide was coated onto an ELISA plate, blocked, and then the monoclonal antibody to be screened was added for the first incubation. After the first incubation, the reaction solution was removed, and then the enzyme-labeled secondary antibody was added for the second incubation. After the second incubation, the reaction solution was removed, and then TMB chromogenic solution was added for the colorimetric reaction. Monoclonal antibodies against the polypeptide were screened based on the colorimetric results.

[0019] In a second aspect, the present invention provides an ELISA kit for screening monoclonal antibodies, the ELISA kit comprising: an enzyme-labeled plate coated with a polypeptide;

[0020] The coating method includes at least one of the following a or b:

[0021] a. The polypeptide is coupled to a carrier protein via BMPS before being coated onto an ELISA plate;

[0022] b. The ELISA plate is coated with streptavidin. The polypeptide is coupled to a biotin-labeled carrier protein via BMPS and then bound to the streptavidin on the ELISA plate via biotin.

[0023] As a further technical solution, the carrier protein includes BSA.

[0024] As a further technical solution, the biotin includes Sulfo-NHS-LC-LC-Biotin.

[0025] As a further technical solution, the ELISA kit also includes washing solution, blocking solution, enzyme-labeled secondary antibody, and colorimetric solution.

[0026] Thirdly, the present invention provides the application of the above-mentioned ELISA kit in monoclonal antibody screening.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The monoclonal antibody screening method provided by this invention uses BMPS to couple peptides to carrier proteins, which not only solves the problem of peptide coating efficiency on ELISA plates, but also allows peptides to remain free in the liquid phase due to the presence of spacer arms, greatly reducing the risk of peptide epitope masking. Through the streptavidin-biotin system, it can be ensured that the peptides are 100% free in the liquid phase, avoiding the risk of peptide epitope masking and greatly reducing the false negative problem encountered in high-throughput antibody screening. Attached Figure Description

[0029] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 WB test results;

[0031] Figure 2From top to bottom, the diagram shows the binding of naked peptide, Peptide-BSA (EDC Linker), Peptide-BSA (BMPS Linker), and Peptide-BSA-Biotin (BMPS Linker) on an ELISA plate. Detailed Implementation

[0032] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0033] In a first aspect, the present invention provides a method for screening monoclonal antibodies, which uses ELISA to screen monoclonal antibodies against peptides.

[0034] In ELISA, the polypeptide is coated on an enzyme-labeled plate, and the coating method includes at least one of the following a or b:

[0035] a. The polypeptide is coupled to a carrier protein via BMPS before being coated onto an ELISA plate;

[0036] b. The ELISA plate is coated with streptavidin. The polypeptide is coupled to a biotin-labeled carrier protein via BMPS and then bound to the streptavidin on the ELISA plate via biotin.

[0037] The monoclonal antibody screening method provided by this invention uses BMPS to couple peptides to carrier proteins, which not only solves the problem of peptide coating efficiency on ELISA plates, but also allows peptides to remain free in the liquid phase due to the presence of spacer arms, greatly reducing the risk of peptide epitope masking. Through the streptavidin-biotin system, it can be ensured that the peptides are 100% free in the liquid phase, avoiding the risk of peptide epitope masking and greatly reducing the false negative problem encountered in high-throughput antibody screening.

[0038] In some alternative embodiments, the carrier protein includes, but is not limited to, BSA, or other carrier proteins well known to those skilled in the art.

[0039] In some alternative embodiments, the biotin comprises Sulfo-NHS-LC-LC-Biotin.

[0040] Biotin is coated onto the carrier protein by covalently linking the Sulfo-NHS group to the primary amine group of the carrier protein.

[0041] In some alternative implementations, the ELISA includes an indirect ELISA or a competing ELISA.

[0042] In some alternative implementations, the indirect ELISA includes the following steps:

[0043] The polypeptide was coated onto an ELISA plate, blocked, and then the monoclonal antibody to be screened was added for the first incubation. After the first incubation, the reaction solution was removed, and then the enzyme-labeled secondary antibody was added for the second incubation. After the second incubation, the reaction solution was removed, and then TMB chromogenic solution was added for the colorimetric reaction. Monoclonal antibodies against the polypeptide were screened based on the colorimetric results.

[0044] In a second aspect, the present invention provides an ELISA kit for screening monoclonal antibodies, the ELISA kit comprising: an enzyme-labeled plate coated with a polypeptide;

[0045] The coating method includes at least one of the following a or b:

[0046] a. The polypeptide is coupled to a carrier protein via BMPS before being coated onto an ELISA plate;

[0047] b. The ELISA plate is coated with streptavidin. The polypeptide is coupled to a biotin-labeled carrier protein via BMPS and then bound to the streptavidin on the ELISA plate via biotin.

[0048] This kit is highly sensitive and can achieve high-throughput screening of monoclonal antibodies against antipeptides.

[0049] In some alternative embodiments, the carrier protein includes, but is not limited to, BSA, or other carrier proteins well known to those skilled in the art.

[0050] In some alternative embodiments, the biotin comprises Sulfo-NHS-LC-LC-Biotin.

[0051] Biotin is coated onto the carrier protein by covalently linking the Sulfo-NHS group to the primary amine group of the carrier protein.

[0052] In some alternative implementations, the ELISA kit further includes a washing buffer, a blocking buffer, an enzyme-labeled secondary antibody, and a colorimetric solution.

[0053] Thirdly, the present invention provides the application of the above-mentioned ELISA kit in monoclonal antibody screening.

[0054] The kit provided by this invention has high throughput and can be used for efficient screening of monoclonal antibodies against peptides.

[0055] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0056] Example 1: Peptide EDC conjugation of BSA

[0057] The experimental procedure involved coupling the peptide to the carrier protein BSA using the EDC method (without spacers). Stable coating efficiency was achieved by leveraging the adsorption capacity of the carrier protein. The resulting product was designated as Peptide-BSA (EDC Linker).

[0058] Reagents: Naked peptide (peptide sequence: EDDDEESEAQGPKC (SEQ ID NO.1), unless otherwise specified, this peptide will be used in the following experiments), 0.1M MES solution (pH 4.5-5.4), 1×PBS (pH 7.4), DMSO (50mM cell culture grade), BSA, EDC, DMSO, 5mM EDTA aqueous solution, UP water, 10% SLS solution, 8M urea, 0.5M NaOH, 0.5M HCl.

[0059] Consumables: 15mL and 50mL centrifuge tubes, various sizes of pipette tips, 1.5mL EP tubes, and 30KD ultrafiltration tubes.

[0060] Equipment: vortex mixer, electronic analytical balance, pipettes of various sizes, 4℃ refrigerator, magnetic stirrer, dialysis clamp.

[0061] Step a): Remove the peptide, EDC, and BSA to be cross-linked from the refrigerator, allow them to equilibrate at room temperature for 30 minutes, and then accurately weigh 1.0 mg of peptide, 1.5 mg of BSA, and 10 mg of EDC using an electronic analytical balance.

[0062] Step b): The feed ratio for this experiment is a mass ratio. In a 0.1M MES system, the mass ratio is peptide:BSA:EDC = 1:1.5:1. In a urea system dissolved in 1xPBS or 1xPBS, the mass ratio is peptide:BSA:EDC = 1:1.5:10.

[0063] Step c): Add the weighed peptide to 50 μL of DMSO, then add 0.1 M MES solution to bring the volume to 250 μL. At this point, the peptide concentration should be 4 mg / mL.

[0064] Step d): Dissolve 1.5 mg of weighed BSA in 300 μL of 0.1 M MES solution. At this point, the concentration of BSA is 5 mg / mL.

[0065] Step e): Dissolve 10 mg of EDC in 1 mL of fresh UP water. At this point, the EDC concentration is 10 mg / mL.

[0066] Step f): First, mix the clarified peptide solution with the BSA solution using a vortex mixer. Add 100 μL of EDC solution to the mixture, mix thoroughly, and let stand at room temperature for 2 hours.

[0067] Step g): During the reaction, monitor the reaction frequently and invert the tube several times to mix. After the reaction is complete, transfer the mixture to a 30KD ultrafiltration tube, fill it with 1×PBS, and replace the buffer three times. Centrifuge to remove uncoupled peptides and finally quantify the result (1 mg of peptide is concentrated to about 1 mL, with an error of ±0.01 mL).

[0068] Example 2: Peptide BMPS conjugation with BSA

[0069] The experimental procedure used the BMPS method (spacer arm) The peptide is coupled to the carrier protein BSA, and the adsorption capacity of the carrier protein achieves stable coating efficiency. In addition, the spacer arm of a certain length between BSA and the peptide can avoid steric hindrance to the binding of the antibody to the peptide epitope to a certain extent, reducing the risk of peptide epitope masking. The prepared product is denoted as Peptide-BSA (BMPS Linker).

[0070] Reagents: naked peptide, BSA, BMPS, DMSO, 1×PBS (pH=7.4).

[0071] Consumables: 15mL and 50mL imported centrifuge tubes, 1.5mL EP tubes, pipette tips, 30KD ultrafiltration tubes.

[0072] Equipment: Electronic analytical balance, pipette, 4℃ freezer, -20℃ freezer, sample mixer.

[0073] Step a): Dissolve a certain amount of polypeptide, such as (1 mg), in a certain amount of DMSO, such as (20 uL).

[0074] Step b): Weigh a certain amount of BSA (peptide:BSA = 1mg:1mg) and dissolve it in 1×PBS to a concentration of 5mg / mL.

[0075] Step c): Weigh a certain amount of BMPS and dissolve it completely in DMSO. The concentration of BMPS is 30 mg / mL (1 mg of BSA requires 0.375 mg of BMPS).

[0076] Step d): Add the BMPS solution to the BSA solution while gently shaking, and react at room temperature for 1.5 hours.

[0077] Step e): Add the activated BSA solution to a 30KD ultrafiltration tube, fill it with 1×PBS, replace the buffer three times, and then transfer it to a 1.5mL EP tube.

[0078] Step f): Add the dissolved peptide to the solution and react at room temperature for 1.5 h. Then transfer the solution to a 30 kDa ultrafiltration tube, fill it with 1×PBS, replace the buffer three times, centrifuge to remove uncoupled peptides, and finally quantify the solution (1 mg of peptide is concentrated to about 1 mL, with an error of ±0.01 mL).

[0079] Example 3: Indirect ELISA Detection Scheme

[0080] The screening of monoclonal antibody supernatant was performed using an indirect ELISA method. We coated the supernatant with Peptide (immunogen naked peptide), Peptide-BSA (EDC Linker), Peptide-BSA (BMPS Linker), and Irrelevant Peptide-BSA (BMPS Linker, an unrelated peptide, i.e., prepared using another unrelated peptide according to the Peptide-BSA (BMPS Linker) preparation method) respectively to screen the monoclonal supernatant.

[0081] Reagents: Goat anti-Rabbit IgG-HRP (Huaan Biotechnology: HA1001), TMB substrate (Sigma: T2885), Tris (Shanghai Sangon Biotech: A501492), glycine (Shanghai Sangon Biotech: GB0235), BSA (Shanghai Sangon Biotech: A500023-0100), Tween-20 (Shanghai Sangon Biotech: A600560), NaHCO3 (Shanghai Sangon Biotech: A610482-0500); Na2CO3, Na2HPO4·12H2O, NaH2PO4·2H2O, citric acid, glycerol, DMSO, and concentrated sulfuric acid were purchased from Hangzhou Shuangmu Chemical; hydrogen peroxide and EDTA were purchased from Shanghai Sangon Biotech, domestically produced analytical grade.

[0082] Consumables: Microplate (Hangzhou Shengyou).

[0083] Equipment: Electric thermostatic incubator (Shanghai Senxin: DRP-9162), ELISA reader (MD: Cmax plus).

[0084] Step a): Coating: Dilute the naked peptide, Peptide-BSA (EDC Linker), Peptide-BSA (BMPS Linker), and Irrelevant Peptide-BSA (BMPS Linker, unrelated peptide) to 1 μg / mL with coating buffer, add 50 μL / well to the microplate, seal, and coat overnight at 4°C. The connection methods of the naked peptide, Peptide-BSA (EDC Linker), and Peptide-BSA (BMPS Linker) to the microplate are as follows... Figure 2 As shown.

[0085] Step b): Blocking: Shake off the liquid in the wells, add 1% BSA / TBS to the microplate at a rate of 100 μL / well, and place it in a 37°C thermostatic incubator for 1 hour for blocking.

[0086] Step c): Sample addition: After removing the liquid from the wells, add 50 μL / well of 1:10 diluted eukaryotic expression supernatant to the microplate. Seal the plate and incubate at 37°C for 45 min.

[0087] Step d): Add secondary antibody: Shake off the primary antibody mixture, add washing buffer (1×TBST) to the microplate at a rate of 180 μL / well, and wash the microplate twice. Dilute Goat anti-Rabbit IgG-HRP to the working concentration (1:15000) with 1% BSA, add 50 μL / well to the microplate, cap it, and incubate at 37°C for 30 min.

[0088] Step e): Color development, termination and reading: Discard the liquid in the wells, add washing buffer to the microplate at a rate of 180 μL / well, and wash the microplate 3 times; add 50 μL of freshly prepared TMB chromogenic substrate to each reaction well, and incubate at 37°C for 10 min; then add 50 μL / well of stop solution to terminate the reaction, and measure the OD value at 450 nm on the microplate reader.

[0089] Table 1

[0090]

[0091]

[0092]

[0093] Note: The numbers in the table refer to the hole positions, and the data below the hole position is the OD. 450The nm readings represent monoclonal antibodies secreted by a different cell line in each well. Since this is the ELISA verification stage in the monoclonal screening process, negative / weak positive / strong positive results will appear, indicating whether a specific clone was screened and the strength of the positive result. The same strain is present in the same well. "+" indicates a serum positive control, and "-" indicates a water negative control.

[0094] Indirect ELISA data (Table 1) showed that the naked peptide coating efficiency for MR240528 was very low. While serum as a positive control provided some identification, no positive results were detected in the supernatant. Coating with Peptide-BSA (EDC Linker) resolved the low coating efficiency issue, but the number of detected positive clones was still lower than with Peptide-BSA (BMPS Linker) coating. This demonstrates that the spacer arm between the peptide and carrier protein is crucial, as it can reduce steric hindrance-related binding issues between the peptide epitope and the antibody. Furthermore, since the conjugated antigen-contrast peptide contains both conjugating agent and BSA, cross-detection with Irrelevant Peptide-BSA (BMPS Linker, an unrelated peptide) was used to exclude non-specific clones to determine the specificity of the detected positive ELISA clones.

[0095] Taking 1C5, 1C7, 1E12, and 1F2 as examples, 1C7 and 1E12 showed no positive results using naked peptide / peptide-BSA (EDC Linker) / Irrelevant Peptide-BSA (BMPS Linker), but Peptide-BMPS-BSA was positive. To further rule out the risk of false positives, Western blotting (WB) was used to validate these four clones. A mixture of GAPDH (glyceraldehyde-3-phosphate dehydrogenase, 36 kDa) and HSP90 (heat shock protein 90, 90 kDa) was used as the internal control, and both showed the target band. The WB membrane quality control was satisfactory. The PC (positive control) used serum-purified antibody, which showed the target band around 55 kDa. The results indicate that clones 1C7 and 1E12 both showed the target band on Neuro-2a (positive lysis buffer). Figure 1 This demonstrates the specificity of the clone. Clones 1C7 and 1E12, which tested negative using naked peptide / Peptide-BSA (EDC Linker), and clones 1C5 and 1F2, which tested positive, exhibit the same Western blotting specificity. This further illustrates that the Peptide-BSA (BMPS Linker) coupling method, with its spacer arm, greatly exposes the peptide epitopes and reduces the risk of false negatives.

[0096] To eliminate the error of a single project, this invention selected some peptide projects for verification. The clone screening and comparison in this way also confirmed the importance of peptide coating efficiency and peptide epitope exposure for peptide projects.

[0097] Of course, for peptide projects, the peptide coating after BMPS conjugation still cannot fully guarantee the exposure of peptide epitopes, because there are still great uncertainties in the coating process. For example, both the carrier protein end and the peptide end are bound to the ELISA plate, which may result in potential peptide epitope obscuration. Therefore, we subsequently designed a sandwich ELISA detection scheme using streptavidin-biotin.

[0098] The binding of streptavidin to biotin is one of the strongest known non-covalent interactions. Streptavidin is a tetrameric protein composed of four identical subunits, each of which can bind one biotin molecule. By modifying the ELISA detection protocol, it is possible to better ensure that the peptide epitope is fully exposed in the liquid phase.

[0099] Example 4: Crosslinking Biotin using the Peptide BMPS Method

[0100] The experimental protocol used was via Sulfo-NHS-LC-LC-Biotin (spacer arm) After coupling the BSA, the BMPS method (spacer arm) is then used. The peptide is coupled to BSA-Biotin, and the naked peptide is indirectly bound to the ELISA plate using the streptavidin-biotin system, achieving liquid binding between the naked peptide and the antibody. The resulting product is designated Peptide-BSA-Biotin (BMPS Linker).

[0101] Reagents: naked peptide, BSA, BMPS, DMSO, 1×PBS (pH=7.4), Sulfo-NHS-LC-LC-Biotin.

[0102] Consumables: 15mL and 50mL imported centrifuge tubes, 1.5mL EP tubes, pipette tips, 30KD ultrafiltration tubes.

[0103] Equipment: Electronic analytical balance, pipette, 4℃ freezer, -20℃ freezer, sample mixer.

[0104] Step a): Dissolve a certain amount of polypeptide, such as (1 mg), in a certain amount of DMSO, such as (20 uL).

[0105] Step b): Weigh a certain amount of BSA (peptide:BSA = 1mg:1mg) and dissolve it in 1×PBS to 5mg / mL.

[0106] Step c): Weigh a certain amount of BMPS and dissolve it completely in DMSO. The concentration of BMPS is 30 mg / mL (1 mg of BSA requires 0.375 mg of BMPS).

[0107] Step d): Weigh a certain amount of Sulfo-NHS-LC-LC-Biotin and dissolve it completely in 1×PBS to prepare a final concentration of 10 mg / mL (1 mg of BSA requires 0.5 mg of Sulfo-NHS-LC-LC-Biotin).

[0108] Step e): Add BMPS and Sulfo-NHS-LC-LC-Biotin solution to BSA solution while gently shaking, and react at room temperature for 1.5 h.

[0109] Step f): Add the activated BSA solution to a 30KD ultrafiltration tube, fill it with 1×PBS, replace the buffer three times, and then transfer it to an EP tube.

[0110] Step g): Add the dissolved peptide to the solution and react at room temperature for 1.5 h. Then transfer the solution to a 30 kDa ultrafiltration tube, fill it with 1×PBS, replace the buffer three times, centrifuge to remove uncrosslinked peptides, and finally quantify the solution (1 mg of peptide is concentrated to about 1 mL, with an error of ±0.01 mL).

[0111] Example 5 Sandwich ELISA Detection Solution

[0112] Streptavidin was coated onto an ELISA plate and blocked with BSA. Then, a certain concentration of Peptide-BSA-Biotin (BMPS Linker) was added. Through the binding of streptavidin and biotin, the naked peptide could be free in the liquid phase through the spacer arm of BMPS and could bind well with the antibody.

[0113] Reagents: Streptavidin, Goat anti-Rabbit IgG-HRP (Huaan Biotechnology: HA1001), TMB substrate (Sigma: T2885), Tris (Shanghai Sangon Biotech: A501492), glycine (Shanghai Sangon Biotech: GB0235), BSA (Shanghai Sangon Biotech: A500023-0100), Tween-20 (Shanghai Sangon Biotech: A600560), NaHCO3 (Shanghai Sangon Biotech: A610482-0500); Na2CO3, Na2HPO4·12H2O, NaH2PO4·2H2O, citric acid, glycerol, DMSO, and concentrated sulfuric acid were purchased from Hangzhou Shuangmu Chemical; hydrogen peroxide and EDTA were purchased from Shanghai Sangon Biotech, domestically produced analytical grade.

[0114] Consumables: Microplate (Hangzhou Shengyou).

[0115] Equipment: Electric thermostatic incubator (Shanghai Senxin: DRP-9162), ELISA reader (MD: Cmax plus).

[0116] Step a): Coating: Dilute streptavidin to 1 μg / mL with coating buffer, add 50 μL / well to the microplate, cover and coat overnight at 4°C.

[0117] Step b): Blocking: Shake off the liquid in the wells, add 1% BSA / TBS to the microplate at a rate of 100 μL / well, and place it in a 37°C thermostatic incubator for 1 hour for blocking.

[0118] Step c): Sample addition: After removing the liquid from the wells, dilute Peptide-BSA-Biotin (BMPS Linker) to 1 μg / mL and add 50 μL / well to the microplate. Seal the plate and incubate at 37°C for 45 min. The linkage structure of Peptide-BSA-Biotin (BMPS Linker) on the microplate is shown below. Figure 2 As shown.

[0119] Step d): Sample addition: After removing the liquid from the wells, add 50 μL / well of 1:10 diluted eukaryotic expression supernatant to the microplate. Seal the plate and incubate at 37°C for 45 min.

[0120] Step e): Add secondary antibody: Shake off the primary antibody mixture, add washing buffer (1×TBST) to the microplate at a rate of 180 μL / well, and wash the microplate twice. Dilute Goat anti-Rabbit IgG-HRP to the working concentration (1:15000) with 1% BSA, add 50 μL / well to the microplate, cap it, and incubate at 37°C for 30 min.

[0121] Step f): Color development, termination and reading: Discard the liquid in the wells, add washing buffer to the microplate at a rate of 180 μL / well, and wash the microplate 3 times; add 50 μL of freshly prepared TMB chromogenic substrate to each reaction well, and incubate at 37°C for 10 min; then add 50 μL / well of stop solution to terminate the reaction, and measure the OD value at 450 nm on the microplate reader.

[0122] Table 2

[0123]

[0124]

[0125] Note: The strains at the same well positions are the same as those in Table 1. "+" indicates a serum positive control, and "-" indicates a water negative control.

[0126] The sandwich ELISA data (Table 2) show that the results of the streptavidin-biotin system and the peptide-BSA (BMPSLinker) coating are similar, proving that the streptavidin-biotin ELISA detection system is suitable for high-throughput screening of peptide projects and can perfectly avoid potential peptide epitope occlusion.

[0127] As the data above shows, naked peptide coating has coating efficiency issues, and there is a risk of epitope obscuring after binding to the ELISA plate. While Peptide-BSA (EDC Linker) solves the peptide coating efficiency problem, the lack of a spacer arm in the coupling agent means that if an epitope is present near the crosslinking site, it may affect antibody binding. Additionally, the peptide may be directly coated on the ELISA plate. Peptide-BSA (BMPS Linker) solves both the peptide coating efficiency problem and, due to the presence of the spacer arm, allows the peptide to remain free in the liquid phase, greatly reducing the risk of peptide epitope obscuring. Finally, the streptavidin-biotin system ensures that the peptide is definitely free in the liquid phase, virtually eliminating the risk of peptide epitope obscuring and significantly reducing false negatives encountered in high-throughput antibody screening using ELISA.

[0128] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for screening monoclonal antibodies, characterized in that, Monoclonal antibodies against anti-peptides were screened using ELISA. In ELISA, the polypeptide is coated on an enzyme-labeled plate, and the coating method includes at least one of the following a or b: a. The polypeptide is coupled to a carrier protein via BMPS before being coated onto an ELISA plate; b. The ELISA plate is coated with streptavidin. The polypeptide is coupled to a biotin-labeled carrier protein via BMPS and then bound to the streptavidin on the ELISA plate via biotin.

2. The method for screening monoclonal antibodies according to claim 1, characterized in that, The carrier protein includes BSA.

3. The method for screening monoclonal antibodies according to claim 1, characterized in that, The biotin includes Sulfo-NHS-LC-LC-Biotin.

4. The method for screening monoclonal antibodies according to claim 1, characterized in that, The ELISA includes indirect ELISA or competitive ELISA.

5. The method for screening monoclonal antibodies according to claim 4, characterized in that, The indirect ELISA includes the following steps: The polypeptide was coated onto an ELISA plate, blocked, and then the monoclonal antibody to be screened was added for the first incubation. After the first incubation, the reaction solution was removed, and then the enzyme-labeled secondary antibody was added for the second incubation. After the second incubation, the reaction solution was removed, and then TMB chromogenic solution was added for the colorimetric reaction. Monoclonal antibodies against the polypeptide were screened based on the colorimetric results.

6. An ELISA kit for screening monoclonal antibodies, characterized in that, The ELISA kit includes: an enzyme-labeled plate coated with peptides; The coating method includes at least one of the following a or b: a. The polypeptide is coupled to a carrier protein via BMPS before being coated onto an ELISA plate; b. The ELISA plate is coated with streptavidin. The polypeptide is coupled to a biotin-labeled carrier protein via BMPS and then bound to the streptavidin on the ELISA plate via biotin.

7. The ELISA kit according to claim 6, characterized in that, The carrier protein includes BSA.

8. The ELISA kit according to claim 6, characterized in that, The biotin includes Sulfo-NHS-LC-LC-Biotin.

9. The ELISA kit according to claim 6, characterized in that, The ELISA kit also includes washing solution, blocking solution, enzyme-labeled secondary antibody, and colorimetric solution.

10. The use of the ELISA kit according to any one of claims 6-9 in monoclonal antibody screening.

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