Anti-human albumin antibody 7G7, antibody composition and application thereof

Abstract

The present invention belongs to the field of immunological detection, and provides an anti-human albumin antibody 7G7, an antibody composition and its application. The anti-human albumin antibody provided by the present invention can specifically bind to human albumin and has high stability. Based on the antibody, a kit is developed that can accurately, quickly and in large quantities detect the HSA content in samples. The double antibody sandwich ELISA method for detecting human albumin has high sensitivity, specificity and accuracy, and can achieve accurate and high-throughput detection of the content of albumin in human serum or other species similar to human albumin serum, and has good application prospects in human albumin detection.

Description

Technical Field

[0001] The present invention belongs to the field of immunological detection, and in particular, relates to an anti-human albumin antibody 7G7, an antibody composition and an application thereof. Background Art

[0002] Human serum albumin (HSA) is the most abundant protein in human blood (accounting for 60% of total plasma proteins). It is synthesized only in the liver, with an average of about 12-25 g per day, accounting for about 50% of liver protein synthesis. The normal human serum albumin concentration is about 35-50 g / L, the urine albumin concentration is usually less than 30 mg / L, and the serum half-life is about 20 days. [1] . Human albumin is mainly composed of α-helices, and the overall structure resembles a heart shape. Human albumin has nine double loops spanning three homologous domains. These domains are named domains I, II, and III. Each domain has two long loops and one short loop. The first two loops of each domain are denoted as subdomain A, and the remaining loops of each domain form subdomain B. Therefore, human albumin has subdomains IA and IB in domain I, subdomains IIA and IIB in domain II, and subdomains IIIA and IIIB in domain III. Subdomains with separate helical structures mediate the binding of human albumin to various endogenous and exogenous ligands. However, although the domains have similar structures, each domain has been shown to have different ligand binding affinities and functions. [2] . HSA is essential for maintaining plasma colloidal osmotic pressure, transporting endogenous compounds, scavenging free radicals, regulating immune responses and other metabolic processes. Its medical uses are not limited to intravenous HSA as a blood substitute for the treatment of severe burns and blood loss. It can also be used as a carrier protein, as a targeted drug for inflammatory or malignant tissues or to extend the half-life of drugs. Due to its inherent properties, such as biocompatibility and biodegradability, serum albumin is widely used in clinical and biomedical research. Serum albumin has multiple roles in cell survival and tissue regeneration. Serum albumin-based composite scaffolds can recruit stem cells, adsorb nutrients, and promote cell growth and proliferation, and are therefore often used in tissue engineering and regenerative medicine. [3] .

[0003] The commonly used detection method for HSA is the bromocresol green (BCG) colorimetric method. The principle of the BCG colorimetric method is that albumin carries a positive charge in a buffer solution with a pH value of 4.2. In the presence of a non-ionic surfactant, it combines with the negatively charged dye bromocresol green to form a blue-green complex with a maximum absorption peak at a wavelength of 630nm. Its absorbance is proportional to the albumin concentration. This method can detect lower concentrations of HSA, but it is easily interfered by globulins, has poor specificity and stability, and is difficult to completely solve through equipment processing, which is not conducive to the accurate testing of HSA. [4-6] .

[0004] Other detection methods include chromatography, which provides high selectivity and resolution and can detect multiple proteins at the same time. However, it requires high equipment and maintenance costs, is time-consuming, and has a high detection limit; electrophoresis can distinguish HSA from other plasma proteins and detect very low concentrations of HSA, which can reach the mg / L level. However, the operation of this method may cause protein inactivation and aggregation, and requires special equipment and high professional requirements. [7-8] . Summary of the invention

[0005] The purpose of the present invention is to provide an anti-human albumin antibody 7G7, an antibody composition and applications thereof.

[0006] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides an anti-human albumin antibody 7G7 (monoclonal antibody) or an antigen-binding fragment thereof, wherein the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody or its antigen-binding fragment are QSILYSSSQKNY (SEQ ID NO: 9), WAS, and HQYLSSWT (SEQ ID NO: 10), respectively, and the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are GFTFSRYA (SEQ ID NO: 11), ISSGGNT (SEQ ID NO: 12), and ARGRAWSYALDY (SEQ ID NO: 13), respectively.

[0007] Furthermore, the amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NO:3, or has at least 80%, 85%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:3, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:4, or has at least 80%, 85%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:4.

[0008] In the present invention, the antibody or antigen-binding fragment thereof includes but is not limited to monoclonal antibody, Fab, Fab', F(ab')2, Fv or single-chain antibody.

[0009] In a second aspect, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof.

[0010] In a third aspect, the present invention provides biological materials containing the nucleic acid molecule, wherein the biological materials include but are not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or host cells.

[0011] In a fourth aspect, the present invention provides an antibody conjugate, wherein the antibody conjugate is obtained by conjugating the antibody or its antigen-binding fragment with a marker, wherein the marker is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, radioactive labeling, etc.

[0012] In a fifth aspect, the present invention provides an anti-human albumin antibody composition, which comprises the antibody 7G7 or an antigen-binding fragment thereof and the anti-human albumin antibody 5H11 or an antigen-binding fragment thereof.

[0013] The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody 5H11 or its antigen-binding fragment are SSVSSGY (SEQ ID NO: 14), STS, and HQYRRSPT (SEQ ID NO: 15), respectively, and the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are GYAFSRYW (SEQ ID NO: 16), VYPGDGDT (SEQID NO: 17), and ARTSRIASYYAMDY (SEQ ID NO: 18), respectively.

[0014] Furthermore, the amino acid sequence of the light chain variable region of the antibody 5H11 or its antigen-binding fragment is as shown in SEQ ID NO:7 or has at least 80%, 85%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:7, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:8 or has at least 80%, 85%, 90%, 95% or 98% similarity to the sequence shown in SEQ ID NO:8.

[0015] In a sixth aspect, the present invention provides use of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the biomaterial, the antibody conjugate, or the antibody composition in the preparation of a reagent for qualitative or quantitative detection of human albumin.

[0016] In a seventh aspect, the present invention provides any of the following uses of the antibody or antigen-binding fragment thereof, the antibody conjugate, or the antibody composition:

[0017] (1) Used for qualitative or quantitative detection of human albumin (including non-disease diagnosis and treatment purposes);

[0018] (2) Used for quality control of products containing human albumin.

[0019] In an eighth aspect, the present invention provides a human albumin double antibody sandwich ELISA kit, using the antibody 7G7 or its antigen-binding fragment as the first antibody, and the anti-human albumin antibody 5H11 or its antigen-binding fragment as the second antibody; or

[0020] The anti-human albumin antibody 5H11 or an antigen-binding fragment thereof is used as the first antibody, and the antibody 7G7 or an antigen-binding fragment thereof is used as the second antibody.

[0021] In a specific embodiment of the present invention, the antibody 7G7 is fixed on a solid phase carrier, and the solid phase carrier can be selected from ELISA plates, microspheres, nitrocellulose membranes, glass cellulose membranes or nylon membranes, etc., preferably ELISA plates.

[0022] The antibody 5H11 is labeled with an enzyme, and the enzyme labeled with the enzyme can be selected from any one of the following: horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-galactosidase, lysozyme, malate dehydrogenase, etc., preferably horseradish peroxidase.

[0023] Furthermore, the kit also includes at least one of a substrate color developing solution, a blocking solution or a stop solution.

[0024] Commonly used substrates include, but are not limited to, o-phenylenediamine (OPD), tetramethylbenzidine (3,3',5,5'-tetramethylbenzidine, TMB) and ABTS [2,2'-azino-di-(3-ethylbenziazobinesulfonate-6)], etc., preferably TMB.

[0025] The blocking solution may be 5% skim milk powder or 0.1% BSA, and the stopping solution may be 1.5-2.5 M (preferably 2 M) sulfuric acid solution.

[0026] The principle of detecting HSA content by using enzyme-linked immunosorbent assay technology based on double antibody sandwich method is as follows: anti-HSA monoclonal antibody 1 is coated on the ELISA plate; gradient dilution standard and pre-diluted sample are added respectively, and HSA in the standard and sample will fully bind to the coated antibody on the ELISA plate; after washing the plate, HRP-labeled anti-HSA antibody 2 is added, and the antibody will specifically bind to HSA in the standard and sample captured by the coated antibody on the plate; after washing the plate, the color developer substrate TMB is added, and if there are different concentrations of HSA in the sample in the reaction well, HRP will turn the colorless TMB into different shades (positively correlated) of blue substances, and the reaction well will turn yellow after adding the stop solution; finally, at λ max =450nm (OD=450nm) to measure the absorbance (OD) of the reaction well sample. The HSA concentration in the sample is proportional to the OD, and the HSA concentration in the sample can be calculated through the standard curve. This method uses an enzyme colorimetric amplification system, which has a high detection sensitivity and can detect samples with relatively low content.

[0027] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0028] (I) The anti-human albumin antibody provided by the present invention can specifically bind to human albumin and has high stability.

[0029] (II) Based on the antibody, a kit was developed that can accurately, quickly and in large quantities detect the HSA content in samples. The kit was subjected to an accelerated stability test at 37°C, and there was no significant change in the kit within 9 days, indicating high stability. A similar protein cross-experiment was conducted on the kit used, and the kit was specific and did not recognize human IgG, human IgG4, human IgA, human IL-1β, human IL-6, human IL-8, human IL-10, human IL-12, human TNF-α, human EGFR, and human MMP-2. This kit is no different from the same type of foreign kits and can replace the imported kits.

[0030] (III) The detection of human albumin by double antibody sandwich ELISA has high sensitivity, specificity and accuracy, and can achieve accurate and high-throughput detection of the content of albumin in human serum or other species similar to human albumin serum. Compared with other methods such as BCG colorimetry (g level) and electrophoresis (g level), the detection sensitivity of the kit of the present invention is high (ng level), ranging from 0.625-40ng / mL, with a sensitivity of 312.5pg / ml and R 2 =0.9996. It has good application prospects in human albumin detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1The figure is a SDS-PAGE electrophoresis detection result of the monoclonal antibodies 7G7 and 5H11 after purification in the preferred embodiment of the present invention; wherein M is a protein molecular weight marker, the unit is KDa.

[0032] Figure 2 This is a standard curve established for detecting human albumin by the double antibody sandwich ELISA method in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0033] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0034] Example 1 Preparation of anti-human HSA monoclonal antibody

[0035] 1. Animal immunization

[0036] Female BALB / c mice aged 6-8 weeks were selected and immunized with naturally extracted HSA antigen emulsified with an equal volume of Freund's adjuvant. The immunization cycle was two weeks. Blood was collected after three immunizations to measure the titer, and the immunization was boosted again three days before fusion.

[0037] 2. Cell fusion

[0038] The mice were killed by cervical dislocation, and the spleen was removed aseptically. The spleen was squeezed and ground in a plate to prepare a spleen cell suspension. The prepared syngeneic myeloma cells were mixed with mouse spleen cells in a certain proportion, and the fusion promoter polyethylene glycol (PEG) was added. Under the action of polyethylene glycol, various lymphocytes can fuse with myeloma cells to form hybridoma cells. The specific operation is as follows:

[0039] Myeloma (SP2 / 0) cell activation:

[0040] Commercial SP2 / 0 cells were thawed and revived, then resuspended in nutrient solution (RPMI 1640 medium supplemented with calf serum) and cultured in an incubator at 37°C and 5% CO2; passaged after 3-5 days;

[0041] The cells were collected and suspended in RPMI 1640 medium, and 0.5×10 6 -1×10 6 The cells were injected subcutaneously into the back of BALB / c mice and cultured for 9-10 days. When the back tumor volume increased to about 0.8 cm in diameter, the mice were killed by pulling the neck, and the tumors were removed by aseptic operation after soaking in 75% alcohol for 5 minutes.

[0042] The tumor pieces were cut and placed in a sterile homogenizer. RPMI 1640 medium was added and fully ground. 10 mL of RPMI 1640 medium was added and allowed to stand for 2 min. The upper cell suspension was aspirated and placed in another centrifuge tube. 10 mL of RPMI 1640 medium was added and the grinding was repeated twice. The cell suspension obtained above was centrifuged at 1000 r / min for 10 min to remove the supernatant and then resuspended in 30 mL of RPMI 1640 medium.

[0043] Add 15 mL of lymphocyte separation medium to another centrifuge tube, and carefully place the above cell suspension on the separation medium; then centrifuge at 1200 r / min for 15 min, pipette out the dense white cell layer at the interface, wash the cells twice with RPMI 1640 medium, resuspend them in 10 mL of RPMI 1640 medium, count and set aside.

[0044] Preparation of immune splenocytes:

[0045] Take a BALB / c mouse that has been boosted with immunity, bleed it from its orbits to kill it (collect serum, i.e., positive serum), and immerse it in 75% alcohol for 5-10 minutes for disinfection. Then fix it on a dissecting board for dissection, remove the spleen, cut it, and place it in a sterile homogenizer. The grinding and cell suspension preparation methods are the same as those described in SP2 / 0, and the cells are counted and set aside.

[0046] Preparation of feeder cells:

[0047] Take an unimmunized BALB / c mouse, bleed from the eye socket, and collect the negative serum. Inject 2~3mL RPMI 1640 culture medium into the mouse's peritoneal cavity, aspirate after blowing and place in another centrifuge tube for standby use. The liquid contains peritoneal macrophages. Prepare spleen cell suspension in the same way as above and place in the peritoneal macrophage tube. Centrifuge at 1000r / min for 10min to remove the supernatant, suspend the cells with HAT culture medium, and place in a 37℃, 5% CO2 incubator for standby use.

[0048] Fusion:

[0049] 1×10 7 -2×10 7 SP2 / 0 and 10 8Mix the immune cells in a 50mL centrifuge tube, centrifuge at 1000r / min for 8min. After discarding the supernatant, place the centrifuge tube containing the cell mixture in a 37℃ water bath, then add 0.8mL (sigma) of 50% PEG preheated to 37℃, stir and let stand for 30 s. After standing, add 10mL of RPMI 1640 medium preheated at 37℃. After mixing, centrifuge at 1000r / min for 5min, discard the supernatant and let stand at 37℃ for 5-8min. Then mix with the feeder cell suspension, seed in a 96-well culture plate, 250μL / well, and culture in a 37℃, 5% CO2 incubator. On the 4th day after fusion, change to HT medium and continue to culture. When the fused cell colony grows to 1 / 4 of the culture well and the culture medium turns slightly yellow, perform antibody detection.

[0050] 3. Screening of hybridoma positive clones and cell cloning

[0051] The purpose of selective culture is to screen fused hybridoma cells, using HAT selective culture medium. In HAT culture medium, unfused myeloma cells die because they lack hypoxanthine-guanine-phosphoribosyltransferase and cannot synthesize DNA using the salvage pathway. Although unfused lymphocytes have hypoxanthine-guanine-phosphoribosyltransferase, they cannot survive in vitro for a long time and gradually die. Only fused hybridoma cells can survive and proliferate in HAT culture medium because they have obtained hypoxanthine-guanine-phosphoribosyltransferase from spleen cells and have the characteristics of myeloma cells that can proliferate indefinitely. The specific operation is as follows:

[0052] The steps for screening positive hybridoma cells using indirect ELISA are as follows:

[0053] Coating with known antigens: dilute the purified coating antigen to 1-10 μg / mL with coating buffer; add 100 μL to each microwell, shake gently, and store in a 4°C refrigerator overnight or at 37°C for 1 hour; shake off the liquid in the wells (tap dry as much as possible); wash 3 times, 2-3 minutes each time.

[0054] Block the positions in the ELISA wells that are not coated with antigens: add 200 μL of blocking solution (5% skim milk powder or 0.1% BSA) to each well, shake gently, and incubate at 37℃ for 1h; discard the liquid in the wells; fill each well with washing buffer (containing 1×PBST, components are Na2HPO48 mM, NaCl 0.136M, KH2PO42 mM, KCl 2.6 mM, Tween-20 0.05% (V / V)), let stand for 2-3min, discard the liquid in the wells, pat dry, and use this method to wash 3 times with washing buffer. Sample addition: take 50 μL of supernatant from each well of the hybridoma to be tested and add it to the ELISA wells in order, shake gently. Incubate at 37℃ for 1h, wash, and pat dry.

[0055] Add enzyme-labeled anti-antibody: First dilute the enzyme-labeled secondary antibody to an appropriate working concentration with diluent according to the instructions, add 100μL to each well, shake gently, and place at 37℃ for 1h; then wash and pat dry. Add color development solution: Add 100μL of freshly prepared color development solution to each well, shake gently, and place at 37℃ for 10min. Stop reaction: Add 50μL of stop solution to each well.

[0056] Determination result: OD of microplate reader 450 The reading was carried out at 400 nm, and a value greater than 3 times that of the negative well was determined to be positive.

[0057] Cloning of hybridoma cells (limiting dilution method):

[0058] Prepare a mouse feeder cell layer before cloning; gently blow the hybridoma cells to be cloned from the culture well and count the number of live cells using a hemocytometer; dilute the cells to 5, 10, or 30 cells / ml with complete culture medium;

[0059] Add the three concentrations of cell suspension to the prepared 96-well culture plate of feeder cells, 100 μL / well, so that each well contains 0.5, 1 and 3 cells, respectively. Add one drop of liquid on the 4th day of culture, and carefully observe the growth of cells in each well on the 5th-6th day and record it;

[0060] Detection of specific antibodies: On the 7th to 9th day after cloning, when the cell clones cover 1 / 3-1 / 2 of the visual field, they can be detected; the cells in the positive wells can be moved to a 24-well culture plate, and when the cells in the 24-well plate grow well, they can be intraperitoneally inoculated into mice to collect ascites.

[0061] 4. Variable region sequence determination of monoclonal antibodies 7G7 and 5H11

[0062] The number of hybridoma cells collected was greater than 10 6 , and sent to Shenggong Bioengineering for subsequent construction and sequencing. The gene sequencing results were obtained: the light chain variable region sequence of the 7G7 cell line is 336bp long, encoding 112 amino acids, the DNA sequence is shown in SEQ ID NO:1, and the protein sequence is shown in SEQ ID NO:3; the heavy chain variable region sequence is 354bp long, encoding 118 amino acids, the DNA sequence is shown in SEQ ID NO:2, and the protein sequence is shown in SEQ ID NO:4. The light chain variable region sequence of the 5H11 cell line is 321bp long, encoding 107 amino acids, the DNA sequence is shown in SEQ ID NO:5, and the protein sequence is shown in SEQ ID NO:7; the heavy chain variable region sequence is 363bp long, encoding 121 amino acids, the DNA sequence is shown in SEQ ID NO:6, and the protein sequence is shown in SEQ ID NO:8.

[0063] 5. Large-scale preparation of monoclonal antibodies 7G7 and 5H11

[0064] After establishment, the hybridoma cells were injected into the peritoneal cavity of mice, and the ascites was collected after about 7 days. The antibodies were purified by Protein G affinity chromatography ( Figure 1 ).

[0065] Example 2 Establishment of a double antibody sandwich ELISA kit for HSA content

[0066] 1. Determination of relative affinity constant

[0067] HSA antigen was coated on the ELISA plate and blocked. After washing the plate with PBST, monoclonal antibodies 7G7 and 5H11 were diluted to saturation concentration and added to the ELISA plate, 100μL / well, and incubated at room temperature for 2h. After washing the plate with PBST, 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0mol / L NaSCN (sodium sulfhydrate) solution was added in sequence at 60μL / well, and incubated at room temperature for 15min. After washing the plate with PBST, HRP-labeled goat anti-mouse IgG was added, and incubated at room temperature for 45min for color development detection. The sodium thiocyanate concentration corresponding to the OD value at 450nm after elution dropped to 50% of that without elution is the relative affinity constant of the antibody, expressed in mol / L. The results showed that the relative affinity constants of monoclonal antibodies 7G7 and 5H11 were both greater than 5mol / L (Table 1), and the affinity was good.

[0068] Table 1

[0069]

[0070] 2. HRP labeling of monoclonal antibody 5H11

[0071] Add antibody 5H11 to the dialysis bag and immerse it in 0.01M CB buffer at 4℃ for overnight dialysis. Take 10mg hRP and dissolve it in 2mL water. Prepare a fresh NaIO4 solution, 0.1M, add 0.4mL 0.1M NaIO4 to 2mL hRP solution, mix well, keep it at room temperature and away from light for 45min, immerse it in 10mM NaAc buffer and dialyze it at 4℃ overnight. Take out the antibody and HRP solution that have been dialyzed overnight, add 0.1mL, 0.2M pH9.5 carbonate buffer, then immediately add the antibody that has been taken out to the HRP solution, and stir gently for 2h at room temperature and away from light. Weigh 0.04g NaBH4 and dissolve it in 10mL water, take 0.4mL and add it to the reaction solution, mix well, and place it at 4℃ for 2h. Take it out and put it in the dialysis bag, dialyze it in 0.01M PBS, change the solution once after two hours, and dialyze it at 4℃ overnight. The labeled solution dialyzed overnight was taken out, an equal volume of glycerol was added, and the solution was stored at -20°C.

[0072] 3. Preparation of monoclonal antibody 7G7 ELISA plate

[0073] Dilute monoclonal antibody 7G7 to 2 μg / mL with 0.05 M pH 9.6 carbonate coating buffer. Add 0.1 mL to the reaction wells of a 96-well polystyrene reagent plate and incubate at 4°C overnight. The next day, discard the solution in the wells and wash three times with washing buffer, 3 min each time. After the above steps, seal each well of the reaction plate with 2% BSA solution, 0.3 mL per well, for 2 h. Discard the solution in the wells, dry in a drying room, and vacuumize, and store at 4°C.

[0074] 4. Establishment of double antibody sandwich ELISA method

[0075] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 7G7, return to room temperature, wash the plate 3 times and spin dry. Add 100 μL of human HSA standard with different dilution concentrations, and the dilution concentrations of human HSA are 40ng / mL, 20ng / mL, 10ng / mL, 5ng / mL, 2.5ng / mL, 1.25ng / mL, 0.625ng / mL and set up a blank control. After sealing the plate, incubate at room temperature for 2 hours, wash the plate 4 times and spin dry. Add 100 μL of enzyme-labeled antibody HRP-5H11 working solution to the reaction well, incubate at room temperature for 60 minutes after sealing the plate, wash the plate 4 times and spin dry. Add 100 μL of color substrate TMB to the reaction well, color at room temperature in the dark for 15 minutes after sealing the plate, add 50 μL of stop solution 2M sulfuric acid solution, and immediately measure the OD value (within 5 minutes) at a wavelength of 450nm using an ELISA reader. The standard curve was drawn with different concentrations of human HSA standard as the horizontal axis and the corresponding OD value as the vertical axis, and the regression equation was established. The results showed that its detection range was 0.625-40ng / mL, R 2 is 0.9996 ( Figure 2 ).

[0076] 5. Double antibody sandwich ELISA specificity detection

[0077] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 7G7, return to room temperature, wash the plate 3 times and spin dry. Add 100 μL of human HSA standard or other components contained in serum at different dilutions, including human IgG, human IgG4, human IgA, human IL-1β, human IL-6, human IL-8, human IL-10, human IL-12, human TNF-α, human EGFR, and human MMP-2. After sealing the plate, incubate at room temperature for 2 hours, wash the plate 4 times and spin dry. Add 100 μL of enzyme-labeled antibody HRP-5H11 working solution to the reaction well, incubate at room temperature for 60 minutes after sealing the plate, wash the plate 4 times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction well, color at room temperature in the dark for 15 minutes after sealing the plate, add 50 μL of stop solution 2M sulfuric acid solution, and immediately measure the OD value at 450nm wavelength with an ELISA reader (within 5 minutes). The results showed that the antibody pair did not react with other similar proteins (Table 2), and the cross-reaction rate was less than 2%.

[0078] Table 2

[0079]

[0080] 6. Double antibody sandwich ELISA stability test

[0081] The ELISA plate coated with monoclonal antibody 7G7, HRP-labeled monoclonal antibody 5H11, and standard HSA were placed at 37°C for accelerated stability test and placed for 9 days (about 13.5 months at 4°C). Then take out the test, the test method is to take out the ELISA plate, wash the plate 3 times and spin dry. Add 100μL of HSA standard with different dilution concentrations, the HSA dilution concentrations are 40ng / mL, 20ng / mL, 10ng / mL, 5ng / mL, 2.5ng / mL, 1.25ng / mL, 0.625ng / mL and set up a blank control. After sealing the plate, incubate at room temperature for 2h, wash the plate 4 times and spin dry. Add 100μL of enzyme-labeled antibody HRP-5H11 working solution to the reaction well, seal the plate, incubate at room temperature for 60min, wash the plate 4 times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction wells, seal the plate and color for 15 minutes at room temperature in the dark, add 50 μL of stop solution 2M sulfuric acid solution, and immediately measure the OD value at 450 nm wavelength with an ELISA reader (within 5 minutes). Draw a standard curve with different concentrations of HSA standards as the horizontal axis and the corresponding OD value as the vertical axis to establish a regression equation. The results showed that the OD value was greater than 1.5 on day 9, and the gradient was good, indicating that the kit had good stability (Table 3).

[0082] Table 3

[0083]

[0084] Example 3 Comparison of test results with other foreign brands of the same type of test kit

[0085] Select foreign brand X (R&D, DY1455) for analogy testing, and conduct experiments in the most appropriate way for each brand (completely follow the instructions). Compare the performance of the two kits through sample testing.

[0086] Comparison of sample test results: 6 human serum / plasma samples were randomly selected for simultaneous testing (in mg / mL). According to literature reports, the HSA content in normal human serum ranges from 35 to 50 mg / mL (depending on age and individual content). The test results show that the test range of this kit is between 38 and 43 mg / mL. The content measured by brand X is higher, between 95 and 101 mg / mL, which is 2 to 3 times the value measured by this kit and is different from the reference range (Table 4).

[0087] Table 4

[0088]

[0089] Example 4 Linear recovery determination

[0090] 30 minutes before the experiment, take out the ELISA plate coated with monoclonal antibody 7G7, return to room temperature, wash the plate 3 times and spin dry. Select 4 sera from voluntary blood donors in the laboratory (numbered 1, 2, 3, and 4), add 100 μL of human serum samples / standards with different dilution multiples, and the sample dilution multiple is 500,000 times. On this basis, dilute 2 times, 4 times, and 8 times. The dilution concentrations of HSA standards are 40 ng / mL, 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, and 0.625 ng / mL, and set up a blank control. After sealing the plate, shake and incubate at room temperature for 2 hours, wash the plate 4 times and spin dry. Add 100 μL of enzyme-labeled antibody HRP-5H11 working solution to the reaction well, shake and incubate at room temperature for 60 minutes after sealing the plate, wash the plate 4 times and spin dry. Add 100 μL of chromogenic substrate TMB to the reaction wells, seal the plate and color for 15 min at room temperature in the dark, add 50 μL of stop solution 2M sulfuric acid solution, and immediately measure the OD value at 450 nm wavelength using an ELISA reader (within 5 minutes). Calculate the linear recovery rate, and the test results show that this method can determine that HSA has a good linear relationship within a certain range (a range of 80%-120% indicates a good result) (Table 5).

[0091] Table 5

[0092]

[0093] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

[0094] References:

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[0102] [8] Li Yaoxin. Research on serum albumin detection method based on functionalized gold nanocluster fluorescent probe[D]. Guilin University of Electronic Technology, 2023. DOI:10.27049 / d.cnki.ggldc.2023.000431.

Claims

1. Anti-human albumin antibody 7G7 or an antigen-binding fragment thereof, characterized in that: The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody or its antigen-binding fragment are QSILYSSSQKNY, WAS, and HQYLSSWT, respectively, and the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are GFTFSRYA, ISSGGNT, and ARGRAWSYALDY, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequence of the light chain variable region of the antibody or antigen-binding fragment thereof is shown in SEQ ID NO:3, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

4.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The antibody or antigen-binding fragment thereof is a monoclonal antibody, Fab, Fab', F(ab')2, Fv or a single-chain antibody.

4. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. A biological material containing the nucleic acid molecule according to claim 4, characterized in that: The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineered bacteria or host cell.

6. An antibody conjugate, characterized in that: The antibody conjugate is obtained by conjugating the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 with a label, and the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.

7. An anti-human albumin antibody composition, characterized in that: The antibody composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and the anti-human albumin antibody 5H11 or an antigen-binding fragment thereof; The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain variable region of the antibody 5H11 or its antigen-binding fragment are SSVSSGY, STS, and HQYRRSPT, respectively, and the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are GYAFSRYW, VYPGDGDT, and ARTSRIASYYAMDY, respectively.

8. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the biomaterial according to claim 5, the antibody conjugate according to claim 6, or the antibody composition according to claim 7 in the preparation of a reagent for qualitative or quantitative detection of human albumin.

9. Any of the following uses of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the antibody conjugate according to claim 6, or the antibody composition according to claim 7: (1) Qualitative or quantitative detection of human albumin for non-diagnostic and non-therapeutic purposes; (2) Used for quality control of products containing human albumin.

10. Human albumin double antibody sandwich enzyme-linked immunosorbent assay kit, characterized in that: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 is used as the first antibody, and the anti-human albumin antibody 5H11 or antigen-binding fragment thereof according to claim 7 is used as the second antibody; or The anti-human albumin antibody 5H11 or an antigen-binding fragment thereof described in claim 7 is used as the first antibody, and the antibody or an antigen-binding fragment thereof described in any one of claims 1 to 3 is used as the second antibody.

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