CN118667000A - Anti-HSA monoclonal antibody and application thereof - Google Patents

Abstract

The invention provides an anti-HSA monoclonal antibody and application thereof, and relates to the technical field of immunodetection. The anti-HSA monoclonal antibody comprises monoclonal antibody anti-HSA-mab4 and/or monoclonal antibody anti-HSA-mab11. The monoclonal antibodies anti-HSA-mab4 and anti-HSA-mab11 have the characteristics of high purity, high sensitivity and high specificity and can identify different HSA antigen epitopes, not only can specifically identify HSA in nasal samples of respiratory tracts, but also can identify HSA samples in feces, further provides a required raw material for immune test strip products containing trace HSA samples, and can be widely applied to preparing detection products for detecting the HSA antigens.

Description

Anti-HSA monoclonal antibody and its application

Technical Field

The present invention relates to the technical field of immunoassay, and in particular to an anti-HSA monoclonal antibody and application thereof.

Background Art

Human serum albumin (HSA) is a protein in human plasma, which is abundant and accounts for about half of serum proteins. It is produced in the liver and is a monomeric multi-domain macromolecule. It is a highly water-soluble globular monomeric plasma protein with a relative molecular weight of 67KDa, consisting of 585 amino acid residues, a thiol group and 17 disulfide bonds. HSA has a strong ligand binding ability and provides a storage reservoir and carrier for many endogenous and exogenous compounds. In fact, HSA represents the main carrier of fatty acids, affects the pharmacokinetics of many drugs, provides metabolic modifications of some ligands, renders potential toxins harmless, accounts for most of the antioxidant capacity of human plasma, and exhibits enzymatic effects. HSA is also a biomarker for many diseases, including cancer, rheumatoid arthritis, ischemia, postmenopausal obesity, severe acute graft-versus-host disease, and diseases that require blood glucose monitoring.

In medicine, human serum albumin has a wide range of uses, 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 extend the half-life of therapeutic proteins and peptides. In formulations, human serum albumin is widely used as a protective agent, stabilizer and excipient.

In recent years, colloidal gold chromatography immunoassay reagents have achieved great success in instant testing, but their quality control is often questioned by professionals. Strictly speaking, the quality control of conventional colloidal gold immunochromatographic detection reagents can only indicate whether the reagents are invalid and whether the test is effective. However, it is not possible to effectively control the quality of the sample. For example, without adding a sample, adding a drop of saline or diluent to the reaction well can also show the quality control band, so the quality control is not very meaningful. On the contrary, in nucleic acid testing, there is an internal control to determine whether the sample is normal and whether the test process is successful; if the sample is unqualified, the internal control will not amplify, and the test can be judged to be invalid, which can rule out false negatives caused by unqualified samples.

However, colloidal gold reagents cannot do this. Sample failure cannot be reflected by the quality control line. The quality control line of samples that were not collected or were collected in insufficient quantity is still normal, which can easily lead to false negatives. In particular, during the use of self-test reagents, due to the improper operation of some users or the difficulty of sampling itself (such as nasopharyngeal swabs), false negatives may occur due to sample failure, and the sensitivity will decrease. For heterogeneous samples (such as fecal samples), large deviations may also occur due to reasons such as sample size or individual differences; therefore, it is necessary to increase the quality control of samples during immunochromatographic testing. As the most abundant protein in human plasma, HSA antibodies can be developed into an internal reference reagent for detecting HSA samples.

Therefore, it is 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-label test strips.

In view of this, the present invention is proposed.

Summary of the invention

The first purpose of the present invention is to provide an anti-HSA monoclonal antibody, which has the characteristics of high purity, high sensitivity, high specificity and the ability to recognize different HSA antigen epitopes. It can not only specifically recognize HSA in respiratory tract and nasal samples, but also recognize HSA samples in feces, thereby providing the required raw materials for immune test strip products containing trace HSA samples.

The second object of the present invention is to provide an application of an anti-HSA monoclonal antibody.

The third object of the present invention is to provide a double antibody sandwich colloidal gold test strip.

The fourth object of the present invention is to provide a HSA detection kit.

In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

The present invention provides an anti-HSA monoclonal antibody, wherein the monoclonal antibody includes monoclonal antibody anti-HSA-mab4;

and/or, monoclonal antibody anti-HSA-mab11.

Furthermore, the monoclonal antibody anti-HSA-mab4 comprises 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 its amino acid sequence is shown in SEQ ID NO.2;

The coding gene sequence of 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.

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

The CDR L1, CDR L2 and CDR L3 in the light chain variable region of the monoclonal antibody anti-HSA-mab4 are the 27th to 32nd amino acid residues, the 50th to 52nd amino acid residues and the 91st to 101st amino acid residues from the N-terminus of SEQ ID NO.1 in the sequence table, respectively.

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

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

Furthermore, the monoclonal antibody anti-HSA-mab11 comprises 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-mab11 is shown in SEQ ID NO.5, and its amino acid sequence is shown in SEQ ID NO.6;

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

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

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

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

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

The present invention provides the use of the anti-HSA monoclonal antibody in the following (A) or (B):

(A) preparing a test product for detecting HSA antigen;

(B) As a quality control preparation for immunological testing of samples containing HSA antigen;

Furthermore, the detection product includes an immunochromatographic detection reagent or kit, an ELISA detection reagent or kit, an immunomagnetic particle detection reagent or kit, an immunofluorescence detection reagent or kit, or an immunoblotting detection reagent or kit.

Further, the detection product is a colloidal gold test strip;

Preferably, the colloidal gold test strip is a double antibody sandwich colloidal gold test strip.

The present invention provides a double antibody sandwich colloidal gold test strip, which comprises a substrate, a sample pad, a labeling binding pad, an analysis membrane and a water absorption pad;

The analysis membrane is coated with monoclonal antibody anti-HSA-mab11, and the colloidal gold labeled antibody on the labeling binding pad is monoclonal antibody anti-HSA-mab4.

The present invention provides an HSA detection kit, which comprises the above-mentioned double antibody sandwich method colloidal gold test strip.

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

The anti-HSA monoclonal antibodies provided by the present invention include monoclonal antibody anti-HSA-mab4 and/or monoclonal antibody anti-HSA-mab11. The monoclonal antibodies anti-HSA-mab4 and anti-HSA-mab11 have the characteristics of high purity, high sensitivity, high specificity and the ability to recognize different HSA antigen epitopes. They can not only specifically recognize HSA in respiratory tract nasal cavity samples, but also recognize HSA samples in feces, thereby providing the required raw materials for immune test strip products containing trace HSA samples, and can be widely used in the preparation of detection products for detecting HSA antigens, such as immunoblotting, immunofluorescence and other immunological tests.

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

The double antibody sandwich colloidal gold test strip provided by the present invention comprises a substrate, a sample pad, a labeling conjugation pad, an analysis membrane and a water-absorbing pad; wherein the analysis membrane is coated with a monoclonal antibody anti-HSA-mab11, and the colloidal gold labeled antibody on the labeling conjugation pad is a monoclonal antibody anti-HSA-mab4. It has been verified that in the double antibody sandwich colloidal gold test strip, when HSA-mab11 is used as a coating antibody and HSA-mab4 is used as a labeling antibody for pairing and combination, it has high specificity for HSA, and can specifically identify not only HSA in respiratory tract nasal samples, but also HSA samples in feces; and different sample amounts can present a clear gradient.

The HSA detection kit provided by the present invention comprises the above-mentioned double antibody sandwich colloidal gold test strip, thereby being able to perform specific detection of HSA.

DETAILED DESCRIPTION

The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Generally, the nomenclature and its technology used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods as well-known in the art, and as 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 specifications, as generally achieved in the art, or as described herein. The nomenclature and its laboratory procedures and technology used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

According to one aspect of the present invention, an anti-HSA monoclonal antibody comprises monoclonal antibody anti-HSA-mab4;

and/or, monoclonal antibody anti-HSA-mab11.

The anti-HSA monoclonal antibodies provided by the present invention include monoclonal antibody anti-HSA-mab4 and/or monoclonal antibody anti-HSA-mab11. The monoclonal antibodies anti-HSA-mab4 and anti-HSA-mab11 have the characteristics of high purity, high sensitivity, high specificity and the ability to recognize different HSA antigen epitopes. They can not only specifically recognize HSA in respiratory tract nasal samples, but also recognize HSA samples in feces, thereby providing the required raw materials for immune test strip products containing trace HSA samples, and can be widely used in the preparation of detection products for detecting HSA antigens, such as immunoblotting, immunofluorescence and other immunological tests.

In a preferred embodiment of the present invention, the monoclonal antibody anti-HSA-mab4 comprises 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 its amino acid sequence is shown in SEQ ID NO.2;

The coding gene sequence of 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.

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

In the above preferred embodiment, the light chain variable region of the monoclonal antibody anti-HSA-mab4 comprises 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, respectively, the 27th to 32nd amino acid residues, the 50th to 52nd amino acid residues, and the 91st to 101st amino acid residues from the N-terminus of SEQ ID NO.1 in the sequence listing.

The heavy chain variable region of the monoclonal antibody anti-HSA-mab4 comprises 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, respectively, the 26th to 34th amino acid residues, the 52nd to 60th amino acid residues, and the 98th to 109th amino acid residues from the N-terminus of SEQ ID NO.3 in the sequence listing.

It should be noted that the "variable region" of the above-mentioned monoclonal antibody refers to the domain of the amino terminus of the heavy chain or light chain of the antibody that recognizes and binds to the antigen. The composition and arrangement of the amino acids in this segment determine the specificity of the antibody in recognizing the antigen. The heavy chain variable region can be referred to as "VH". The light chain variable region can be referred to as "VL". These domains are usually the most variable parts of the antibody and contain antigen binding sites. The variable regions of the heavy chain and light chain are each composed of three complementarity determining regions (CDR) (also known as hypervariable regions) connected by four framework regions (FR). The scope of the framework and CDR 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 a combination of methods, can identify the CDRs of the variable domain. The CDRs in each chain are held together closely by FRs to form the variable region. Generally, the variable region VL/VH of the heavy chain and light chain can be obtained by arranging and connecting the following numbered CDRs and FRs in the following combinations: FR1 CDR1 FR2 CDR2 FR3CDR3 FR4.

In a preferred embodiment of the present invention, the monoclonal antibody anti-HSA-mab11 comprises 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-mab11 is shown in SEQ ID NO.5, and its amino acid sequence is shown in SEQ ID NO.6;

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

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

In the above preferred embodiment, the light chain variable region of the monoclonal antibody anti-HSA-mab11 comprises CDRL1, CDR L2, and CDR L3;

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

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

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

According to one aspect of the present invention, a use of the above anti-HSA monoclonal antibody in the following (A) or (B):

(A) preparing a test product for detecting HSA antigen;

(B) As a quality control preparation for immunological testing of samples containing HSA antigen;

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

In a preferred embodiment of the present invention, the detection product includes an immunochromatographic detection reagent or kit, an immunomagnetic particle detection reagent or kit, an immunofluorescence detection reagent or kit, or an immunoblotting detection reagent or kit.

Preferably, the detection product is a colloidal gold test strip, and the colloidal gold test strip is a double antibody sandwich method colloidal gold test strip.

According to one aspect of the present invention, a double antibody sandwich colloidal gold test strip comprises a substrate, a sample pad, a label binding pad, an analysis membrane and a water absorbent pad;

The analysis membrane is coated with monoclonal antibody anti-HSA-mab11, and the colloidal gold labeled antibody on the labeling binding pad is monoclonal antibody anti-HSA-mab4.

The double antibody sandwich colloidal gold test strip provided by the present invention comprises a substrate, a sample pad, a labeling conjugation pad, an analysis membrane and a water-absorbing pad; wherein the analysis membrane is coated with a monoclonal antibody anti-HSA-mab11, and the colloidal gold labeled antibody on the labeling conjugation pad is a monoclonal antibody anti-HSA-mab4. It has been verified that in the double antibody sandwich colloidal gold test strip, when HSA-mab11 is used as a coating antibody and HSA-mab4 is used as a labeling antibody for pairing and combination, it has high specificity for HSA, and can specifically identify not only HSA in respiratory tract nasal samples, but also HSA samples in feces; and different sample amounts can present a clear gradient.

According to one aspect of the present invention, a HSA detection kit is provided, the kit comprising the above-mentioned double antibody sandwich colloidal gold test strip.

The HSA detection kit provided by the present invention comprises the above-mentioned double antibody sandwich colloidal gold test strip, thereby being able to perform specific detection of HSA.

The technical solution of the present invention will be further described below in conjunction with embodiments.

Example 1 Preparation of monoclonal antibodies anti-HSA-mab4&11

Healthy male New Zealand white rabbits of 6 weeks old were selected and immunized according to a pre-specified immunization scheme, including:

The 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.

The peripheral blood of rabbits that have been successfully immunized is collected, and PBMC cells are separated from the peripheral blood of rabbits using PBMC separation technology, and then antigen-specific rabbit B cells are obtained using sorting technology. After B cells are cultured in vitro, cell lines that can secrete antigen-specific antibodies are screened, and mRNA is extracted to obtain antibody sequences, which are then recombinantly expressed for a second round of screening. Positive clones are retained for sequencing verification to ensure that they are monoclonal antibodies.

(I) Experimental rabbits were immunized with rHSA antigen in stages.

The specific steps of animal immunization experiments include:

1. Select two healthy 6-week-old New Zealand white rabbits (about 2.5 kg) and let them adapt to the new living environment. After a few days of stabilization, take blood from the ear vein for the first time. Inactivate the collected blood at 37°C for 30 minutes, and then place it at 4°C overnight to coagulate and release serum. Centrifuge the coagulated blood at 3000 rpm/min for 15 minutes, and collect the upper serum as a negative control.

2. Each time two rabbits are injected with 1 mL of emulsified antigen, the antigen buffer solution must not contain chemical reagents that are harmful to rabbits. Each rabbit is first immunized with 500 ug of antigen, emulsified with Freund's complete adjuvant. Subsequent immunizations are each 250 ug of antigen, using Freund's incomplete adjuvant.

3. Each rabbit was immunized at 4 sites (the back and the thigh root were both acceptable), 250μl at each site, the needle was inserted at a 45-degree angle 1-2cm under the skin, and the injection was left for a few seconds to prevent the outflow of antigens. Immunization was performed every two weeks, and blood was collected 7-10 days after immunization, for a total of 3-5 immunizations.

(II) Detection of serum titer:

Indirect ELISA method for detecting rabbit serum or plasma titer:

1. Bottom 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°C refrigerator for 24 hours.

2. After 24 hours, take out the wells from the refrigerator and place them at 37℃ for equilibration for 30 minutes, then discard the liquid in the wells; wash the wells three times with washing solution, each time for 3 minutes.

3. Block the enzyme-labeled reaction wells: add 200 μl of blocking solution (1% BSA) to each well and block at 37°C for 120 min. After blocking, wash the wells three times with washing solution, each time for 3 min.

4. Add the sample to be tested: dilute the sample according to the required ratio, add the diluted sample to the enzyme-labeled reaction well, 100 μl per well, incubate at 37°C for 30 min; wash the well three times with washing solution, 3 min each time.

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

6. Add substrate solution: 100 μl of substrate per well, place at 37°C away from light for 15 to 30 minutes.

7. Stop reaction: Add 50 μl of stop solution to each well to stop the reaction and measure the experimental results within 20 minutes.

Two New Zealand white rabbits were immunized, and the rabbit numbers were R67 and R68. After 4-5 immunizations, the serum titer changes were detected. The test data are shown in Table 1 below.

Table 1: Serum titer test data:

(III) Screening positive cells and conducting recombination detection:

The peripheral blood of rabbits that have been successfully immunized is collected, and PBMC cells are separated from the peripheral blood of rabbits using PBMC separation technology, and then antigen-specific rabbit B cells are obtained using sorting technology. After in vitro culture of B cells, cell lines that can secrete antigen-specific antibodies are screened, and antibody sequences are extracted and plasmids are constructed for the cell lines. High-throughput recombinant expression is performed in 293 cells, and ELISA positive screening is performed on the expression supernatant. From the screened positive clones, plasmid monoclones are picked, and then paired tests are performed to screen out the best light and heavy chain plasmid pairings, and then the plasmids are amplified and transfected into 293 cells for small sample production. The antibodies are purified using Protein A antibody purification columns. The ELISA titer of the purified antibodies is >1:125000, and the purity is >90%.

The rabbits completed two rounds of screening. A total of 96 positive cell lines were screened, and recombination and subcloning were performed. Finally, 14 recombinant clones were screened. The screening standard was that the OD450 value of the expression supernatant stock solution was greater than 1.

The titer data of some recombinant clone expression supernatants are shown in Table 2:

Table 2: Recombinant clone expression supernatant titer detection data:

(IV) Production of purified antibodies in small samples

Through the above screening, 14 pairs of recombinant cloned heavy and light chain plasmids were obtained. After the plasmids were amplified, 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 test data is shown in Table 3 below:

Table 3: Purified antibody titer ELISA test data

Antibody titer testing showed that 12 monoclonal antibodies had good specific binding ability to HSA antigens. BLI detects antigen-antibody affinity and antibody sandwich pairing value.

Molecular interaction system BLI detection method:

1. Turn on the machine and prepare the reagents and consumables required for the experiment, as well as the antigens and antibodies for detection;

2. Dilute the antigen to 10-30 μg/ml, and dilute the antibody to a concentration that can reach saturation with the antigen within 3 minutes;

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

4. Select the corresponding sensor, set the file save path, and start the program;

5. After the program is finished, save and analyze the data. See Table 4 for details.

Table 4: Some data of antibody target detection:

Molecular Interaction System BLI was used to detect the affinity between antigen and antibody, and six antibodies with better affinity were screened out, including 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).

Molecular Interaction System BLI detects 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 the epitopes recognized by the two antibodies. HSA-mab1 and mab10 antibodies target the same epitope, HSA-mab5 and mab11 antibodies target the second epitope, HSA-mab4 antibodies target the third epitope, and HSA-mab8 antibodies target the 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.

Example 2 Application of monoclonal antibody anti-HSA-mab in products

Five antibodies were validated using the immunocolloidal gold platform.

The specific experimental group used the 5 antibodies obtained from the above experiment, which were labeled and coated with gold particles respectively, and then assembled into colloidal gold test strips. Four nasal swab samples were used, and two subjects (A and B) were selected to collect nasal swab samples. Two nasal swab samples were taken from each subject. Sample 1 used a sampling cotton swab to rotate along the nasal wall for 5 circles, and sample 2 used a sampling cotton swab to rotate along the nasal wall for 1 circle. The cotton swabs with collected samples were then processed and tested with ID-1 extract. The experimental data results are shown in the table below:

Table 5: HSA rabbit monoclonal antibody colloidal gold test results using nasal samples:

Note: In the above table, 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; HSA-11 is the monoclonal antibody anti-HSA-mab11 in Example 1. Samples A1 and B1 were obtained by rotating the sampling cotton swab along the nasal wall for one circle; samples A5 and B5 were obtained by rotating the sampling cotton swab along the nasal wall for five circles.

The results showed that rabbit monoclonal antibodies targeting different epitopes were screened by immunizing rabbits with HSA protein as antigen, and they can be used together on colloidal gold products and detect HSA in random nasal swab samples. Among the different combinations, HSA-mab11 as a coating antibody and HSA-mab4 as a labeling antibody had the best effect, and all four samples had high readings ≥G8. A1 and A5, B1 and B5 were different sample sizes, and the readings showed a clear gradient.

When the above combination 12 (NC membrane is HSA-11, marker pad is HSA-4) is used to detect fecal samples, the detection line reading value also meets the requirement of ≥G8. The experimental data results are shown in the following table:

Table 6: HSA rabbit monoclonal antibody colloidal gold test results using fecal samples:

In summary, the present application uses purchased HSA immunogens to immunize New Zealand white rabbits, and detects serum after immunization. When the titer is high enough to screen antibodies, the rabbit peripheral blood is extracted to separate PBMC, and then B cells are sorted to obtain B cell strains with high specificity through specific high-throughput screening. Then, the heavy and light chain sequences of the antibody are obtained through mRNA extraction, reverse transcription, and PCR, and recombined into expression vectors and transfected for expression. After multi-step screening, high-purity, high-sensitivity, high-specificity, and anti-HSA monoclonal antibodies anti-HSA-mab4&11 that recognize different antigenic epitopes are obtained.

This antibody can not only identify HSA in respiratory tract nasal samples, but also identify HSA samples in feces, providing the required raw materials for sample immune test strip products containing trace amounts of HSA. After rabbits are immunized with antigens in the present invention, monoclonal antibodies against HSA can be screened out, and the screened anti-HSA-mab 4&11 antibodies can recognize different antigen epitopes and can be used for immunological tests such as immunoblotting and immunofluorescence. The two antibodies obtained have been verified to be able to specifically recognize HSA.

Example 3 Sequence analysis of the heavy chain V region (VH) and light chain V region (VL) of monoclonal antibodies anti-HSA-mab4 and mab11.

1. Analytical methods:

1. During the recombination phase, primers for amplifying the heavy chain V region (VH) and light chain V region (VL) genes were designed.

2. Take the culture supernatant of the B cell line that is positive by Elisa test, use the magnetic bead method to extract the cell mRNA, use the mRNA as a template to reverse transcribe and synthesize the first chain of cDNA, and use the above amplification product as a template for PCR amplification of the antibody VH/VL gene.

3. Recover the heavy chain VH (about bp) and light chain VL (about bp) fragments of anti-HSA-mab4, and the heavy chain VH (about bp) and light chain VL (about bp) fragments of anti-HSA-mab11, clone and connect them to the vector, and send the constructed plasmids to the company for sequencing.

4. Then analyze the VH/VL gene sequence:

(II) Analysis results:

(1) Monoclonal antibody anti-HSA-mab4:

1. Light chain variable region:

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

GCCATCGAAATGACCCAGACTCCAGCCTCCGTGTCTGAACCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGT CAGAACATTGGTAGTTGG TTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTAT TCTGCATCC ACTCTGACATCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGCTCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGC TGCCACTTACTACTGT CAAACAGGTTATGATTTTAGTGATATTGATAGGACT TTCGGCGGAGGGACCGAGGTGGTGGTCAAA

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

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

AIEMTQTPASVSEPVGGTVTIKCQAS QNIGSW LSWYQQKPGQPPKLLIY SAS TLTSGVPSRFSGSGSGTEFTLTISDLECADAATYYC QTGYDFSDIDRT FGGGTEVVVK

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

2. Heavy chain variable region:

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

CAGGAGCAGCTGGAGGAGTCCGGGGGAGGCCTGATCAAGCCTGAGGGATCCCTGACACTCACCTGCACAGCCTCT CGTTACGACCTCATTAGCAGCTACTAC ATTTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGCAATTGAGCGCATGC ATTCGGACTGGTGGCAGTGGTTACACT TACTACGCGAGCTGGGCGAAAGGCCGATTCACCATCTCCAAAGCCTCGTCGACCGCGGTGACTCTGCAAATGACC AGGCTGACAGCCGCGGACACGGCCACCTATTTTTGT CAGGGATAT AGTCTTAGTAGTGCTGATTTCAACGTC TGGGGCCCAGGCACCCTGGTCACCGTCTCCACA

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

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

QEQLEESGGGLIKPEGSLTLTCTAS RYDLISSYY ICWVRQAPGKGLQLSAC IRTGGSGYT YYASWAKGRFTISKASSTAVTLQMTRLTAADTATYFC QGYSLSSADFNV WGPGTLVTVST

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

(2) Monoclonal antibody anti-HSA-mab11:

1. Light chain variable region:

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

GACATTGTGATGACCCAGACTCCAGCCTCCGTGTCTAAACCTGTGGGAGGCACAGTCACCATCAAGTGCCAGGCCAGT CAGAGCATTAATATCTAC TTAAACTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTAC AAGGCATCC ACTCTGGCATCTGGGGTCCCATCGCGGTTCAGCGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTG CCACTTACTACTGT CGAAGCAATTATGACAGTAGTCTTCATACTTACGGT GTCTATGCT TTCGGCGGAGGGACCGAGGTGGTGGTCAAA

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

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:

DIVMTQTPASVSKPVGGTVTIKCQAS QSINIY LNWYQQKPGQPPKRLIY KAS TLASGVPSRFSGSGSGTEFTLTISDLECADAATYYC RSNYDSSLHTYGVYA FGGGTEVVVK

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

2. Heavy chain variable region:

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

CAGGAGCAGCTGGAGGAGTCCGGGGGAGGCCTGGTCAAGCCTGAGGGATCCCTGACACTCACCTGCAAAGCCTCT CGATTCGACATCAGTGGCTACTTCTAC ATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGCATGC ATTTATGTTGGTAGTAGTGTTACAACT TACTACGCGACCTGGGCGAAAGGCCGGTTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACC AGTCTGACAGCCGCGGACACGGCCACCTATTTCTGT GTGGCCGGT TATAGTTTTGGTAATGACATT TGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA

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

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

QEQLEESGGGLVKPEGSLTLTCKAS RFDISGYFY MCWVRQAPGKGLEWIAC IYVGSSVTT YYATWAKGRFTISKTSSTTVTLQMTSLTAADTATYFC VAGYSFGNDI WGPGTLVTVSS

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

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. An anti-HSA monoclonal antibody, characterized in that the monoclonal antibody comprises monoclonal antibody anti-HSA-mab4;

And/or, monoclonal antibody anti-HSA-mab11.

2. The anti-HSA monoclonal antibody of claim 1, wherein the monoclonal antibody anti-HSA-mab4 comprises a monoclonal antibody light chain variable region and a monoclonal antibody heavy chain variable region, wherein:

The coding gene sequence of the light chain variable region of the monoclonal antibody anti-HSA-mab4 is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2;

the coding gene sequence of the heavy chain variable region of the monoclonal antibody anti-HSA-mab4 is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4.

3. The anti-HSA monoclonal antibody of claim 2, wherein the light chain variable region of the monoclonal antibody anti-HSA-mab4 comprises CDR L1, CDR L2, CDR L3;

CDR L1, CDR L2 and CDR L3 in the light chain variable region of the monoclonal antibody anti-HSA-mab4 are 27 th to 32 th amino acid residues, 50 th to 52 th amino acid residues and 91 st to 101 th amino acid residues from the N end of SEQ ID NO.1 of a sequence table in sequence;

Preferably, the heavy chain variable region of the monoclonal antibody anti-HSA-mab4 comprises CDR H1, CDR H2, CDR H3;

CDR H1, CDR H2 and CDR H3 in the heavy chain variable region of the monoclonal antibody anti-HSA-mab4 are the 26 th to 34 th amino acid residues, 52 th to 60 th amino acid residues and 98 th to 109 th amino acid residues of the SEQ ID NO.3 of the sequence table in sequence.

4. The anti-HSA monoclonal antibody of claim 1, wherein the monoclonal antibody anti-HSA-mab11 comprises a monoclonal antibody light chain variable region and a monoclonal antibody heavy chain variable region, wherein:

the coding gene sequence of the light chain variable region of the monoclonal antibody anti-HSA-mab11 is shown as SEQ ID NO.5, and the amino acid sequence is shown as SEQ ID NO. 6;

The coding gene sequence of the heavy chain variable region of the monoclonal antibody anti-HSA-mab11 is shown as SEQ ID NO.7, and the amino acid sequence is shown as SEQ ID NO. 8.

5. The anti-HSA monoclonal antibody of claim 4, wherein the light chain variable region of the monoclonal antibody anti-HSA-mab11 comprises CDR L1, CDR L2, CDR L3;

CDR L1, CDR L2 and CDR L3 in the light chain variable region of the monoclonal antibody anti-HSA-mab11 are the 27 th to 32 th amino acid residues, the 50 th to 52 th amino acid residues and the 89 th to 103 th amino acid residues from the N end of SEQ ID NO.5 of the sequence table in sequence;

Preferably, the heavy chain variable region of the monoclonal antibody anti-HSA-mab11 comprises CDR H1, CDR H2, CDR H3;

CDR H1, CDR H2 and CDR H3 in the heavy chain variable region of the monoclonal antibody anti-HSA-mab11 are the 26 th to 34 th amino acid residues, 52 th to 60 th amino acid residues and 98 th to 107 th amino acid residues from the N end of SEQ ID NO.7 of the sequence table in sequence.

6. Use of an anti-HSA monoclonal antibody according to any one of claims 1-5 in (a) or (B) as follows:

(A) Preparing a detection product for detecting the HSA antigen;

(B) As a quality control preparation for detecting products for performing immunological detection on an antigen sample containing HSA.

7. The use according to claim 6, wherein the detection product comprises an immunochromatographic detection reagent or kit, an ELISA detection reagent or kit, an immunomagnetic particle detection reagent or kit, an immunofluorescent detection reagent or kit, or an immunoimprinted detection reagent or kit.

8. The use according to claim 7, wherein the test product is a colloidal gold test strip;

preferably, the colloidal gold detection test strip is a colloidal gold test strip of a double-antibody sandwich method.

9. The colloidal gold test strip is characterized by comprising a substrate, a sample pad, a labeling binding pad, an analysis membrane and a water absorption pad;

Wherein, monoclonal antibody anti-HSA-mab11 is coated on the analysis membrane, and the colloidal gold labeled antibody on the labeled binding pad is monoclonal antibody anti-HSA-mab4.

10. An HSA detection kit, comprising the colloidal gold test strip of the double antibody sandwich method according to claim 9.