Anti-hsa monoclonal antibodies and uses thereof

Abstract

The application provides an anti-HSA monoclonal antibody and application thereof, and relates to the technical field of immunoassay.The anti-HSA monoclonal antibody comprises a monoclonal antibody anti-HSA-mab4 and / or a monoclonal antibody anti-HSA-mab11.The monoclonal antibody anti-HSA-mab4 and the monoclonal antibody anti-HSA-mab11 have the characteristics of high purity, high sensitivity, high specificity and the ability to recognize different HSA antigen epitopes, can specifically recognize HSA in a respiratory tract nasal cavity sample, can also recognize HSA samples in feces, and further provide required raw materials for an immunoassay test strip product containing a trace amount of HSA samples, and can be widely applied to the preparation of detection products for detecting HSA antigens.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to an anti-HSA monoclonal antibody and its applications. Background Technology

[0002] Human serum albumin (HSA) is a protein abundant in human plasma, accounting for about half of all serum proteins. Produced in the liver, it is a monomeric, multi-domain macromolecule, a highly water-soluble, globular monomeric plasma protein with a relative molecular weight of 67 kDa, composed of 585 amino acid residues, one sulfhydryl group, and 17 disulfide bonds. HSA has a strong ligand-binding capacity, providing a reservoir and carrier for many endogenous and exogenous compounds. In fact, HSA represents a major carrier of fatty acids, influencing the pharmacokinetics of many drugs, providing metabolic modifications for some ligands, neutralizing potential toxicities, accounting for a large portion of the antioxidant capacity of human plasma, and exhibiting enzymatic activity. HSA is also a biomarker for many diseases, including cancer, rheumatoid arthritis, ischemia, postmenopausal obesity, severe acute graft-versus-host disease, and diseases requiring blood glucose monitoring.

[0003] In medicine, human serum albumin has a wide range of applications, including the treatment of hypovolemia, shock, burns, surgical blood loss, trauma, hemorrhage, extracorporeal circulation, acute respiratory distress syndrome, hemodialysis, acute liver failure, chronic liver disease, nutritional support, resuscitation, and hypoalbuminemia. Human serum albumin can also be used to help prolong the half-life of therapeutic proteins and peptides. In pharmaceutical formulations, human serum albumin is widely used as a protective agent, stabilizer, and excipient.

[0004] In recent years, colloidal gold immunochromatographic assays have achieved great success in point-of-care testing, but their quality control is often questioned by professionals. Strictly speaking, the quality control of conventional colloidal gold immunochromatographic assays can only indicate whether the reagent is ineffective and whether the test is valid. However, it cannot effectively control the quality of the sample. For example, even without adding a sample, adding a drop of physiological saline or diluent to the reaction well will produce a control band, thus rendering the quality control meaningless. In contrast, in nucleic acid testing, internal controls can determine whether the sample is normal and whether the testing process is successful. If the sample is unqualified, the internal control will not amplify, indicating that the test is invalid and ruling out false negatives caused by unqualified samples.

[0005] Colloidal gold reagents are problematic because substandard samples cannot be reflected in the quality control lines. The quality control lines for samples that were not collected or were insufficiently collected remain normal, easily leading to false negatives. This is especially true for self-test reagents, where improper user operation or difficult sampling (such as nasopharyngeal swabs) can easily cause false negatives due to substandard samples, reducing sensitivity. For heterogeneous samples (such as fecal samples), significant bias can occur due to sample size or individual differences; therefore, it is necessary to add quality control to the sample during immunochromatographic testing. HSA, the most abundant protein in human plasma, can be used to develop antibodies as an internal control reagent for detecting HSA-containing samples.

[0006] Therefore, it is both necessary and urgent to research and develop an HSA detection / quality control antibody to improve the detection rate and sensitivity of sample diagnostic products such as gold standard test strips.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] The primary objective of this invention is to provide an anti-HSA monoclonal antibody, which is characterized by high purity, high sensitivity, high specificity, and the ability to recognize different HSA antigenic epitopes. It can specifically recognize HSA in respiratory nasal cavity samples as well as HSA samples in feces, thereby providing the necessary raw materials for immunoassay strip products containing trace amounts of HSA samples.

[0009] A second objective of this invention is to provide an application of an anti-HSA monoclonal antibody.

[0010] The third objective of this invention is to provide a colloidal gold test strip with a double antibody sandwich method.

[0011] The fourth objective of this invention is to provide an HSA detection kit.

[0012] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0013] The present invention provides an anti-HSA monoclonal antibody, wherein the monoclonal antibody comprises the monoclonal antibody anti-HSA-mab4;

[0014] And / or, monoclonal antibody anti-HSA-mab11.

[0015] Furthermore, the monoclonal antibody anti-HSA-mab4 includes a monoclonal antibody light chain variable region and a monoclonal antibody heavy chain variable region, wherein:

[0016] The gene sequence encoding the light chain variable region of the monoclonal antibody anti-HSA-mab4 is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2;

[0017] The gene sequence encoding the heavy chain variable region of the monoclonal antibody anti-HSA-mab4 is shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.4.

[0018] Furthermore, the light chain variable region of the monoclonal antibody anti-HSA-mab4 includes CDR L1, CDR L2, and CDR L3;

[0019] The CDR L1, CDR L2, and CDR L3 in the light chain variable region of the monoclonal antibody anti-HSA-mab4 are, in order, amino acid residues 27-32, 50-52, and 91-101 from the N-terminus of SEQ ID NO.1 in the sequence listing.

[0020] Preferably, the heavy chain variable region of the monoclonal antibody anti-HSA-mab4 includes CDR H1, CDR H2, and CDRH3;

[0021] The CDR H1, CDR H2, and CDR H3 in the heavy chain variable region of the monoclonal antibody anti-HSA-mab4 are, in order, amino acid residues 26-34, 52-60, and 98-109 from the N-terminus of SEQ ID NO.3 in the sequence listing.

[0022] Furthermore, the monoclonal antibody anti-HSA-mab11 includes a monoclonal antibody light chain variable region and a monoclonal antibody heavy chain variable region, wherein:

[0023] The gene sequence encoding the light chain variable region of the monoclonal antibody anti-HSA-mab11 is shown in SEQ ID NO.5, and its amino acid sequence is shown in SEQ ID NO.6;

[0024] The gene sequence encoding the heavy chain variable region of the monoclonal antibody anti-HSA-mab11 is shown in SEQ ID NO.7, and its amino acid sequence is shown in SEQ ID NO.8.

[0025] Furthermore, the light chain variable region of the monoclonal antibody anti-HSA-mab11 includes CDR L1, CDR L2, and CDR L3;

[0026] The CDR L1, CDR L2, and CDR L3 in the light chain variable region of the monoclonal antibody anti-HSA-mab11 are, in order, amino acid residues 27-32, 50-52, and 89-103 from the N-terminus of SEQ ID NO.5 in the sequence listing.

[0027] Preferably, the heavy chain variable region of the monoclonal antibody anti-HSA-mab11 includes CDR H1, CDR H2, and CDRH3;

[0028] The CDR H1, CDR H2, and CDR H3 in the heavy chain variable region of the monoclonal antibody anti-HSA-mab11 are, in order, amino acid residues 26-34, 52-60, and 98-107 from the N-terminus of SEQ ID NO.7 in the sequence listing.

[0029] The above-mentioned anti-HSA monoclonal antibody provided by the present invention is used in the following (A) or (B):

[0030] (A) Preparation of detection products for HSA antigen;

[0031] (B) As a quality control preparation for immunological detection of samples containing HSA antigen;

[0032] Furthermore, the detection products include immunochromatographic detection reagents or kits, ELISA detection reagents or kits, immunomagnetic particle detection reagents or kits, immunofluorescence detection reagents or kits, or immunoblotting detection reagents or kits.

[0033] Furthermore, the test product is a colloidal gold test strip;

[0034] Preferably, the colloidal gold test strip is a double-antibody sandwich colloidal gold test strip.

[0035] The present invention provides a double-antibody sandwich colloidal gold test strip, which includes a substrate, a sample pad, a labeling and binding pad, an analytical membrane, and an absorbent pad.

[0036] The analytical membrane is coated with the monoclonal antibody anti-HSA-mab11, and the colloidal gold-labeled antibody on the labeled binding pad is the monoclonal antibody anti-HSA-mab4.

[0037] The present invention provides an HSA detection kit, the kit comprising the above-mentioned double-antibody sandwich colloidal gold test strip.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0039] The anti-HSA monoclonal antibodies provided by this invention include anti-HSA-mab4 and / or anti-HSA-mab11. These monoclonal antibodies, anti-HSA-mab4 and anti-HSA-mab11, possess high purity, high sensitivity, high specificity, and the ability to recognize different HSA antigen epitopes. They can specifically recognize HSA not only in respiratory nasal samples but also in fecal samples, thus providing the necessary raw materials for immunoassay strips containing trace amounts of HSA samples. These antibodies can be widely used in the preparation of detection products for HSA antigen detection, such as immunoblotting and immunofluorescence assays.

[0040] The anti-HSA monoclonal antibody provided by this invention can be widely used in the preparation of detection products for detecting HSA antigens, or as a quality control antibody in the preparation of detection products for immunological detection of samples containing HSA antigens.

[0041] The dual-antibody sandwich colloidal gold test strip provided by this invention includes a substrate, a sample pad, a labeled conjugate pad, an analytical membrane, and an absorbent pad. The analytical membrane is coated with the monoclonal antibody anti-HSA-mab11, and the colloidal gold-labeled antibody on the labeled conjugate pad is the monoclonal antibody anti-HSA-mab4. Verification has shown that when HSA-mab11 is used as the coating antibody in combination with HSA-mab4 as the labeling antibody, the dual-antibody sandwich colloidal gold test strip exhibits high specificity for HSA. It can specifically identify HSA in both respiratory nasal samples and fecal samples; moreover, it shows a clear gradient for different sample amounts.

[0042] The HSA detection kit provided by this invention includes the above-mentioned double-antibody sandwich colloidal gold test strip, which can specifically detect HSA. Detailed Implementation

[0043] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0045] According to one aspect of the present invention, an anti-HSA monoclonal antibody, said monoclonal antibody comprising the monoclonal antibody anti-HSA-mab4;

[0046] And / or, monoclonal antibody anti-HSA-mab11.

[0047] The anti-HSA monoclonal antibodies provided by this invention include anti-HSA-mab4 and / or anti-HSA-mab11. These monoclonal antibodies, anti-HSA-mab4 and anti-HSA-mab11, possess high purity, high sensitivity, high specificity, and the ability to recognize different HSA antigen epitopes. They can specifically recognize HSA not only in respiratory nasal samples but also in fecal samples, thus providing the necessary raw materials for immunoassay strips containing trace amounts of HSA samples. These antibodies can be widely used in the preparation of detection products for HSA antigen detection, such as immunoblotting and immunofluorescence assays.

[0048] In a preferred embodiment of the present invention, the monoclonal antibody anti-HSA-mab4 includes a monoclonal antibody light chain variable region and a monoclonal antibody heavy chain variable region, wherein:

[0049] The gene sequence encoding the light chain variable region of the monoclonal antibody anti-HSA-mab4 is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2;

[0050] The gene sequence encoding the heavy chain variable region of the monoclonal antibody anti-HSA-mab4 is shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.4.

[0051] As a preferred embodiment, the above-mentioned monoclonal antibody anti-HSA-mab4 contains both a light chain variable region and a heavy chain variable region.

[0052] In the preferred embodiment described above, the light chain variable region of the monoclonal antibody anti-HSA-mab4 includes CDRL1, CDR L2, and CDR L3; CDR L1, CDR L2, and CDR L3 in the light chain variable region of the monoclonal antibody anti-HSA-mab4 are, in order, amino acid residues 27-32, 50-52, and 91-101 from the N-terminus of SEQ ID NO.1 in the sequence listing.

[0053] The heavy chain variable region of the monoclonal antibody anti-HSA-mab4 includes CDR H1, CDR H2, and CDR H3; CDR H1, CDR H2, and CDR H3 in the heavy chain variable region of the monoclonal antibody anti-HSA-mab4 are, in order, amino acid residues 26-34, 52-60, and 98-109 from the N-terminus of SEQ ID NO.3 in the sequence listing.

[0054] It should be noted that the "variable region" of the aforementioned monoclonal antibody refers to the amino-terminal domain of the antibody's heavy or light chain that recognizes and binds to the antigen. The composition and arrangement of the amino acids in this region determine the antibody's specificity in recognizing the antigen. The heavy chain variable region can be referred to as "VH," and the light chain variable region as "VL." These domains are typically the most variable parts of the antibody and contain antigen-binding sites. The variable regions of both the heavy and light chains are each composed of three complementarity-determining regions (CDRs) (also known as hypervariable regions) connected by four framework regions (FRs). The scope of frames and CDRs has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest), E. Kabat et al., and Chothia. Any CDR determination method well known in the art, including combinations of methods, can identify CDRs of variable domains. CDRs in each chain are held together closely by FRs to form variable regions. Typically, the variable regions VL / VH of the heavy and light chains can be obtained by linking the following numbered CDRs with FRs in the following combination: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4.

[0055] In a preferred embodiment of the present invention, the monoclonal antibody anti-HSA-mab11 includes a monoclonal antibody light chain variable region and a monoclonal antibody heavy chain variable region, wherein:

[0056] The gene sequence encoding the light chain variable region of the monoclonal antibody anti-HSA-mab11 is shown in SEQ ID NO.5, and its amino acid sequence is shown in SEQ ID NO.6;

[0057] The gene sequence encoding the heavy chain variable region of the monoclonal antibody anti-HSA-mab11 is shown in SEQ ID NO.7, and its amino acid sequence is shown in SEQ ID NO.8.

[0058] As a preferred embodiment, the above-mentioned monoclonal antibody anti-HSA-mab11 contains both a light chain variable region and a heavy chain variable region.

[0059] In the preferred embodiment described above, the light chain variable region of the monoclonal antibody anti-HSA-mab11 includes CDRL1, CDR L2, and CDR L3;

[0060] The CDR L1, CDR L2, and CDR L3 in the light chain variable region of the monoclonal antibody anti-HSA-mab11 are, in order, amino acid residues 27-32, 50-52, and 89-103 from the N-terminus of SEQ ID NO.5 in the sequence listing.

[0061] Preferably, the heavy chain variable region of the monoclonal antibody anti-HSA-mab11 includes CDR H1, CDR H2, and CDRH3;

[0062] The CDR H1, CDR H2, and CDR H3 in the heavy chain variable region of the monoclonal antibody anti-HSA-mab11 are, in order, amino acid residues 26-34, 52-60, and 98-107 from the N-terminus of SEQ ID NO.7 in the sequence listing.

[0063] According to one aspect of the present invention, the above-described anti-HSA monoclonal antibody is used in either (A) or (B) as follows:

[0064] (A) Preparation of detection products for HSA antigen;

[0065] (B) As a quality control preparation for immunological detection of samples containing HSA antigen;

[0066] The anti-HSA monoclonal antibody provided by this invention can be widely used in the preparation of detection products for detecting HSA antigens, or as a quality control antibody in the preparation of detection products for immunological detection of samples containing HSA antigens.

[0067] In a preferred embodiment of the present invention, the detection product includes immunochromatographic detection reagents or kits, immunomagnetic particle detection reagents or kits, immunofluorescence detection reagents or kits, or immunoblotting detection reagents or kits.

[0068] Preferably, the test product is a colloidal gold test strip, and the colloidal gold test strip is a double-antibody sandwich colloidal gold test strip.

[0069] According to one aspect of the present invention, a double-antibody sandwich colloidal gold test strip includes a substrate, a sample pad, a labeling conjugate pad, an analytical membrane, and an absorbent pad.

[0070] The analytical membrane is coated with the monoclonal antibody anti-HSA-mab11, and the colloidal gold-labeled antibody on the labeled binding pad is the monoclonal antibody anti-HSA-mab4.

[0071] The dual-antibody sandwich colloidal gold test strip provided by this invention includes a substrate, a sample pad, a labeled conjugate pad, an analytical membrane, and an absorbent pad. The analytical membrane is coated with the monoclonal antibody anti-HSA-mab11, and the colloidal gold-labeled antibody on the labeled conjugate pad is the monoclonal antibody anti-HSA-mab4. Verification has shown that when HSA-mab11 is used as the coating antibody in combination with HSA-mab4 as the labeling antibody, the dual-antibody sandwich colloidal gold test strip exhibits high specificity for HSA. It can specifically identify HSA in both respiratory nasal samples and fecal samples; moreover, it shows a clear gradient for different sample amounts.

[0072] According to one aspect of the present invention, an HSA detection kit is provided, the kit comprising the above-described double-antibody sandwich colloidal gold test strip.

[0073] The HSA detection kit provided by the present invention includes the above-mentioned double antibody sandwich colloidal gold test strip, which can specifically detect HSA.

[0074] The technical solution of the present invention will be further described below with reference to the embodiments.

[0075] Example 1: Preparation of monoclonal antibody anti-HSA–mab4&11

[0076] Healthy male New Zealand white rabbits, aged 6 weeks, were selected and immunized according to a pre-specified immunization schedule, including:

[0077] Purchased rHSA (manufacturer: Shanghai Yuanye Biotechnology Co., Ltd.; Cat.S12018-500; Lot.J20HS174696) antigen was used as an immunogen to immunize New Zealand white rabbits.

[0078] Peripheral blood was collected from successfully immunized rabbits. PBMC cells were isolated from the rabbit peripheral blood using PBMC isolation technology, and then antigen-specific rabbit B cells were obtained using sorting technology. After in vitro culture of B cells, cell lines that could secrete antigen-specific antibodies were selected. mRNA was extracted, and antibody sequences were obtained. Recombinant expression was performed for a second round of screening. Positive clones were retained for sequencing verification to ensure that they were monoclonal antibodies.

[0079] (I) The experimental rabbits were immunized with rHSA antigen in stages.

[0080] The specific steps of animal immunization experiments include:

[0081] 1. Select two healthy 6-week-old New Zealand White rabbits (approximately 2.5 kg) and allow them to acclimatize to their new living environment. After a few days of stabilization, perform the first ear vein blood collection. Inactivate the collected blood at 37°C for 30 minutes, then incubate at 4°C overnight to allow coagulation and release serum. Centrifuge the coagulated blood at 3000 rpm / min for 15 minutes and collect the supernatant serum as a negative control.

[0082] 2. Inject each rabbit with 1 mL of emulsified antigen for each immunization. The antigen buffer solution must be free of chemicals harmful to rabbits. For the initial immunization, administer 500 μg of antigen to each rabbit, emulsified with Freund's complete adjuvant. Subsequent immunizations will administer 250 μg of antigen each time, using Freund's incomplete adjuvant.

[0083] 3. Vaccinate each rabbit at four sites (back and groin are acceptable), using 250 μl per site. Insert the needle at a 45-degree angle 1-2 cm subcutaneously, and leave it in place for a few seconds after injection to prevent antigen leakage. Vaccinate every two weeks, collecting blood 7-10 days after each vaccination, for a total of 3-5 vaccinations.

[0084] (II) Detection of serum titer:

[0085] Indirect ELISA method for detecting rabbit serum or plasma titers:

[0086] 1. Base plate coating: Dilute the antigen to 0.5 μg / ml with coating diluent, add 100 μl of the prepared coating solution to each well, and place in a 4℃ refrigerator for 24 h.

[0087] 2. After 24 hours, remove it from the refrigerator and place it at 37°C for 30 minutes to equilibrate. Then discard the liquid in the well. Wash the well with washing solution three times, for 3 minutes each time.

[0088] 3. Blocking the enzyme-labeled reaction wells: Add 200 μl of blocking buffer (1% BSA) to each well and incubate at 37°C for 120 min. After blocking, wash the wells three times with washing buffer for 3 min each time.

[0089] 4. Add the sample to be tested: Dilute the sample according to the required ratio, add 100 μl of the diluted sample to each enzyme-labeled reaction well, and incubate at 37℃ for 30 min; wash the wells three times with washing buffer for 3 min each time.

[0090] 5. Add enzyme-labeled antibody: Add the appropriate concentration of secondary antibody according to the instructions; incubate at 37℃ for 30 min, and wash with 50 μl per well as before.

[0091] 6. Add substrate solution: Add 100 μl of substrate per well and incubate at 37°C in the dark for 15–30 min.

[0092] 7. Termination of reaction: Add 50 μl of stop solution to each well to terminate the reaction, and measure the experimental results within 20 min.

[0093] Two New Zealand White rabbits, numbered R67 and R68 respectively, were immunized. After 4-5 immunizations, serum titer changes were measured. The data are shown in Table 1 below.

[0094] Table 1: Serum titer test data:

[0095]

[0096] (III) Screening for positive cells and performing recombinant detection:

[0097] Peripheral blood was collected from successfully immunized rabbits. PBMC cells were isolated from the rabbit peripheral blood using PBMC isolation technology, and then antigen-specific rabbit B cells were obtained using sorting technology. After in vitro culture of B cells, cell lines that could secrete antigen-specific antibodies were selected. Antibody sequences were extracted from the cell lines, and plasmids were constructed. High-throughput recombinant expression was performed in 293 cells, and the expression supernatant was screened for positive results by ELISA. Positive clones were selected, and plasmid single clones were picked and paired to select the optimal light and heavy chain plasmid pair. The plasmid was then amplified and transfected into 293 cells for small-scale production. The antibody was purified using a Protein A antibody purification column. The purified antibody had an ELISA titer >1:125000 and a purity >90%.

[0098] Two rounds of screening were completed in the rabbits. A total of 96 positive cell lines were screened out, and recombinant and subcloning were performed. Finally, 14 recombinant clones were screened out. The screening criterion was that the OD450 value of the expression supernatant was >1.

[0099] Table 2 below shows the titer data of some recombinant clone expression supernatants:

[0100] Table 2: Titer detection data of recombinant clone expression supernatant:

[0101]

[0102]

[0103]

[0104] (IV) Small-scale production and purification of antibodies

[0105] Fourteen pairs of recombinant clonal heavy-light chain plasmids were obtained through the above screening. After plasmid amplification, they were transfected into 293 cells for antibody production. After purification by Protein A affinity chromatography, a total of 12 antibodies were obtained. The antibody titer data are shown in Table 3 below:

[0106] Table 3: ELISA data on purified antibody titer

[0107]

[0108] Antibody titer assays showed that 12 monoclonal antibodies exhibited good specific binding ability to the HSA antigen, using molecular interaction systems. BLI assays detect antigen-antibody affinity and antibody sandwich pairing values.

[0109] Molecular interaction systems BLI detection method:

[0110] 1. Turn on the machine and prepare the necessary reagents and consumables for the experiment, as well as the antigens and antibodies for testing;

[0111] 2. Dilute the antigen to 10-30 μg / ml, and dilute the antibody to a concentration that allows it to bind to the antigen to saturation within 3 minutes;

[0112] 3. Open Octet BLIDiscovery software, create a new program, input sample information, and add the sample to the 96-well plate according to the information in the table, 200 μl per well;

[0113] 4. Select the appropriate sensor, set the file save path, and start the program;

[0114] 5. After the program finishes running, save and analyze the data. See Table 4 for details.

[0115] Table 4: Partial data on antibody target detection:

[0116]

[0117] Molecular Interaction Systems BLI assays were performed to detect antigen-antibody affinity, and six antibodies with good affinity were selected: HSA-mab1 (anti-HSA-mab1), mab4 (anti-HSA-mab4), mab5 (anti-HSA-mab5), mab8 (anti-HSA-mab8), mab10 (anti-HSA-mab10), and mab11 (anti-HSA-mab11).

[0118] Further use of molecular interaction systems BLI assays were performed to detect antigen epitopes and sandwich pairing effects. The results are shown in Table 4. The values ​​in the table represent the binding signals of the two antibodies recognizing different epitopes. The higher the binding signal, the greater the difference in epitopes recognized by the two antibodies. HSA-mab1 and mab10 antibodies target the same epitope, HSA-mab5 and mab11 antibodies target a second epitope, HSA-mab4 antibody targets a third epitope, and HSA-mab8 antibody targets a fourth epitope. Five antibodies (HSA-mab1, mab4, mab5, mab10, and mab11) with different affinities targeting different epitopes were selected for testing HSA colloidal gold products.

[0119] Example 2: Application of the monoclonal antibody anti-HSA-mab in products

[0120] Five antibodies were validated using an immunogold platform.

[0121] The specific experimental group used the above-mentioned experiments to obtain 5 antibodies, which were then labeled and coated with gold particles, and assembled into colloidal gold test strips. Four nasal swab samples were collected from two subjects (A and B), with two nasal swab samples taken from each subject. For sample 1, a sampling swab was rotated 5 times along the nasal cavity wall, and for sample 2, a sampling swab was rotated 1 time along the nasal cavity wall. The collected swabs were then processed with ID-1 extraction buffer and tested. The experimental data results are shown in the table below:

[0122] Table 5: Results of HSA rabbit monoclonal antibody colloidal gold application in nasal cavity samples:

[0123]

[0124] Note: In the table above, HSA-1 is the monoclonal antibody anti-HSA-mab1 in Example 1; HSA-2 is the monoclonal antibody anti-HSA-mab2 in Example 1; HSA-3 is the monoclonal antibody anti-HSA-mab3 in Example 1; HSA-4 is the monoclonal antibody anti-HSA-mab4 in Example 1; HSA-5 is the monoclonal antibody anti-HSA-mab5 in Example 1; HSA-6 is the monoclonal antibody anti-HSA-mab6 in Example 1; HSA-7 is the monoclonal antibody anti-HSA-mab7 in Example 1; HSA-8 is the monoclonal antibody anti-HSA-mab8 in Example 1; HSA-9 is the monoclonal antibody anti-HSA-mab9 in Example 1; HSA-10 is the monoclonal antibody anti-HSA-mab10 in Example 1; and HSA-11 is the monoclonal antibody anti-HSA-mab11 in Example 1. Samples A1 and B1 were obtained by rotating a sampling swab along the nasal cavity wall once; samples A5 and B5 were obtained by rotating a sampling swab along the nasal cavity wall five times.

[0125] The results showed that rabbit monoclonal antibodies targeting different epitopes were obtained by immunizing rabbits with HSA protein as an antigen. These antibodies could be used in combination with colloidal gold products and could detect HSA in random nasal swab samples. Among the different combinations, HSA-mab11 as the coating antibody combined with HSA-mab4 as the labeling antibody showed the best results, with high readings ≥G8 in all four samples. A1 and A5, and B1 and B5, represented different sample sizes, showed a clear gradient in readings.

[0126] When using the above combination 12 (NC membrane HSA-11, label pad HSA-4) to test fecal samples, the detection line reading also met the requirement of ≥G8. The experimental data results are shown in the table below:

[0127] Table 6: Results of HSA rabbit monoclonal antibody colloidal gold application in fecal samples:

[0128]

[0129] In summary, this application utilizes purchased HSA immunogen to immunize New Zealand white rabbits. After immunization, serum titers are tested. When the titers are high enough for antibody screening, peripheral blood cells (PBMCs) are extracted from the rabbits, and B cells are obtained through sorting. Highly specific B cell lines are then obtained through specific high-throughput screening. The heavy and light chain sequences of the antibody are then obtained through mRNA extraction, reverse transcription, and PCR. This sequence is recombined into an expression vector and transfected for expression. Through multiple screening steps, a high-purity, high-sensitivity, high-specificity monoclonal antibody against HSA, anti-HSA-mab4&11, recognizing different antigenic epitopes, is obtained.

[0130] This antibody can recognize HSA not only in respiratory nasal samples but also in fecal HSA samples, providing the necessary raw materials for immunoassay strips containing trace amounts of HSA. In this invention, after immunizing rabbits with the antigen, monoclonal antibodies against HSA can be screened out. The screened anti-HSA-mab 4 & 11 antibodies can recognize different antigenic epitopes and can be used for immunological detection such as immunoblotting and immunofluorescence. The two antibodies obtained have been verified to specifically recognize HSA.

[0131] Example 3: Sequence analysis of the heavy chain V region (VH) and light chain V region (VL) of the monoclonal antibody anti-HSA-mab4 and mab11.

[0132] (I) Analysis Methods:

[0133] 1. Design primers for amplifying the heavy chain V region (VH) and light chain V region (VL) genes during the recombination stage.

[0134] 2. Take the culture supernatant of B cells that are positive for ELISA, extract the mRNA from the cells using the magnetic bead method, use the mRNA as a template to reverse transcribe and synthesize the first strand of cDNA, and use the above amplification product as a template to PCR amplify the VH / VL gene of the antibody.

[0135] 3. Recover the heavy chain VH (approximately bp) and light chain VL (approximately bp) fragments of anti-HSA-mab4, and the heavy chain VH (approximately bp) and light chain VL (approximately bp) fragments of anti-HSA-mab11. After cloning and ligating into the vector, send the constructed plasmid to the company for sequencing.

[0136] 4. Then analyze the VH / VL gene sequences:

[0137] (II) Analysis Results:

[0138] (1) Monoclonal antibody anti-HSA-mab4:

[0139] 1. Light chain variable region:

[0140] The gene sequence encoding the light chain variable region of the monoclonal antibody anti-HSA-mab4 is shown in SEQ ID NO.1, as follows:

[0141] GCCATCGAAATGACCCAGACTCCAGCCTCCGTGTCTGAACCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGT CAGAACATTGGTAGTTGG TTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTAT TCTGCATCC ACTCTGACATCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGCTCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGT CAAACAGGTTATTTTAGTGATATTGATAGGACT TTCGGCGGAGGGACCGAGGTGGTGGTCAAA

[0142] Note: SEQ ID NO.1, where the underlined parts represent CDR L1, CDR L2 and CDR L3 in the variable region of the light chain of the monoclonal antibody anti-HSA-mab4, respectively.

[0143] The amino acid sequence of the light chain variable region of the monoclonal antibody anti-HSA-mab4 is shown in SEQ ID NO.2, as follows:

[0144] AIEMTQTPASVSEPVGGTVTIKCQAS QNIGSW LSWYQQKPGQPPKLLIY SAS TLTSGVPSRFSGSGSGTEFTLTISDLECADAATYYC QTGYDFSDIDRT FGGGTEVVVK

[0145] Note: SEQ ID NO.2, where the underlined parts represent CDR L1, CDR L2 and CDR L3 in the variable region of the light chain of the monoclonal antibody anti-HSA-mab4, respectively.

[0146] 2. Heavy chain variable region:

[0147] The gene sequence encoding the heavy chain variable region of the monoclonal antibody anti-HSA-mab4 is shown in SEQ ID NO.3, as follows:

[0148] CAGGAGCAGCTGGAGGAGTCCGGGGGAGGCCTGATCAAGCCTGAGGGATCCCTGACACTCACCTGCACAGCCTCT CGTTACGACCTCATTAGCAGCTACTAC ATTTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAATTGAGCGCATGC ATTCGGACTGGTGGCAGTGGTTACACT TACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAGCCTCGTCGACCGCGGTGACTCTGCAAATGACCAGGCTGACAGCCGCGGACACGGCCACCTATTTTTGT CAGGGATAT AGTCTTAGTTAGTGCTGATTTCAACGTC TGGGGCCCAGGCACCCTGGTCACCGTCTCCACA

[0149] Note: SEQ ID NO.3, where the underlined parts represent CDR H1, CDR H2, and CDR H3 in the variable region of the heavy chain of the monoclonal antibody anti-HSA-mab4, respectively.

[0150] The amino acid sequence of the heavy chain variable region of the monoclonal antibody anti-HSA-mab4 is shown in SEQ ID NO.4, as follows:

[0151] QEQLEESGGGLIKPEGSLTLTCTAS RYDLISSYY ICWVRQAPGKGLQLSAC IRTGGSGYT YYASWAKGRFTISKASSTAVTLQMTRLTAADTATYFC QGYSLSSADFNV WGPGTLVTVST

[0152] Note: SEQ ID NO.4, where the underlined parts represent CDR H1, CDR H2, and CDR H3 in the variable region of the heavy chain of the monoclonal antibody anti-HSA-mab4, respectively.

[0153] (2) Monoclonal antibody anti-HSA-mab11:

[0154] 1. Light chain variable region:

[0155] The gene sequence encoding the light chain variable region of the monoclonal antibody anti-HSA-mab11 is shown in SEQ ID NO.5, as follows:

[0156] GACATTGTGATGACCCAGACTCCAGCCTCCGTGTCTAAACCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGT CAGAGCATTAATATCTAC TTAAACTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTAC AAGGCATCC ACTCTGGCATCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGT CGAAGCAATTATGACAGTAGTCTTCATACTTACGGT GTCTATGCT TTCGGCGGAGGGACCGAGGTGGTGGTCAAA

[0157] Note: SEQ ID NO.5, where the underlined parts represent CDR L1, CDR L2 and CDR L3 in the variable region of the light chain of the monoclonal antibody anti-HSA-mab11, respectively.

[0158] The amino acid sequence of the light chain variable region of the monoclonal antibody anti-HSA-mab11 is shown in SEQ ID NO.6, and is as follows:

[0159] DIVMTQTPASVSKPVGGTVTIKCQAS QSINIY LNWYQQKPGQPPKRLIY CASH TLASGVPSRFSGSGSGTEFTLTISDLECADAATYYC RSNYDSSLHTYGVYA FGGGTEVVVK

[0160] Note: SEQ ID NO.6, where the underlined parts represent CDR L1, CDR L2 and CDR L3 in the variable region of the light chain of the monoclonal antibody anti-HSA-mab11, respectively.

[0161] 2. Heavy chain variable region:

[0162] The gene sequence encoding the heavy chain variable region of the monoclonal antibody anti-HSA-mab11 is shown in SEQ ID NO.7, as follows:

[0163] CAGGAGCAGCTGGAGGAGTCCGGGGGAGGCCTGGTCAAGCCTGAGGGATCCCTGACACTCACCTGCAAAGCCTCT CGATTCGACATCAGTGGCTACTCTCTAC ATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGC ATTTATGTTGGTAGTAGTGTTACAACT TACTACGCGACCTGGGCGAAAGGCCGGTTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGACAGCCGCGGACACGGCCACCTATTTCTGT GTGGCCGGT TATAGTTTTGGTAATGACATT TGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

[0164] Note: SEQ ID NO.7, where the underlined parts represent CDR H1, CDR H2, and CDR H3 in the variable region of the heavy chain of the monoclonal antibody anti-HSA-mab11, respectively.

[0165] The amino acid sequence of the heavy chain variable region of the monoclonal antibody anti-HSA-mab11 is shown in SEQ ID NO.8, as follows:

[0166] QEQLEESGGGLVKPEGSLTLTCKAS RFDISGYFY MCWVRQAPGKGLEWIAC IYVGSSVTT YYATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFC VAGYSFGNDI WGPGTLVTVSS

[0167] Note: SEQ ID NO.8, where the underlined parts represent CDR H1, CDR H2, and CDR H3 in the variable region of the heavy chain of the monoclonal antibody anti-HSA-mab11, respectively.

[0168] 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 combination of anti-HSA monoclonal antibodies, characterized in that, The anti-HSA monoclonal antibody combination consists of monoclonal antibody anti-HSA-mab4 and monoclonal antibody anti-HSA-mab11; The monoclonal antibody anti-HSA-mab4 includes a monoclonal antibody light chain variable region and a monoclonal antibody heavy chain variable region, wherein: The gene sequence encoding the light chain variable region of the monoclonal antibody anti-HSA-mab4 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; the gene sequence encoding the heavy chain variable region of the monoclonal antibody anti-HSA-mab4 is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.

4. The monoclonal antibody anti-HSA-mab11 includes a light chain variable region and a heavy chain variable region, wherein: The gene sequence encoding the light chain variable region of the monoclonal antibody anti-HSA-mab11 is shown in SEQ ID NO.5, and its amino acid sequence is shown in SEQ ID NO.6; the gene sequence encoding the heavy chain variable region of the monoclonal antibody anti-HSA-mab11 is shown in SEQ ID NO.7, and its amino acid sequence is shown in SEQ ID NO.

8.

2. The use of an anti-HSA monoclonal antibody combination according to claim 1 in either (A) or (B): (A) Preparation of detection products for HSA antigen; (B) As a quality control preparation for immunological detection of samples containing HSA antigen.

3. The application according to claim 2, characterized in that, The detection products include immunochromatographic detection reagents or kits, ELISA detection reagents or kits, immunomagnetic particle detection reagents or kits, immunofluorescence detection reagents or kits, or immunoblotting detection reagents or kits.

4. The application according to claim 3, characterized in that, The test product is a colloidal gold test strip.

5. The application according to claim 4, characterized in that, The colloidal gold test strip is a double-antibody sandwich colloidal gold test strip.

6. A double-antibody sandwich colloidal gold test strip, characterized in that, The dual-antibody sandwich colloidal gold test strip includes a substrate, a sample pad, a labeled binding pad, an analytical membrane, and an absorbent pad. The analytical membrane is coated with a monoclonal antibody, the amino acid sequence of which is shown in SEQ ID NO.6 and the amino acid sequence of which is shown in SEQ ID NO.

8. The colloidal gold-labeled antibody on the labeled binding pad is a monoclonal antibody, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.

4.

7. An HSA detection kit, characterized in that, The kit includes the double-antibody sandwich colloidal gold test strip as described in claim 6.

Images