CN109666072B - Anti-human beta2-microglobulin antibodies and uses thereof - Google Patents

Abstract

The invention relates to a novel anti-human beta 2-microglobulin antibody and application thereof, belonging to the field of immunochemistry. The invention prepares a plurality of antibodies, and performs pairing screening to obtain an antibody combination (BM12 and BM17) with sensitivity and specificity meeting the requirements; meanwhile, the preparation method is convenient for mass production and can meet the requirement of large-scale clinical application in the future. The antibody combination is debugged and optimized, and the obtained test paper card is simple and convenient to operate, has sensitivity, specificity and related detection performance, and can meet the industrial application of the human beta 2-microglobulin colloidal gold rapid detection test paper card for human clinical sample detection.

Description

Anti-human beta2-microglobulin antibodies and uses thereof

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to two anti-human beta genes₂-microglobulin antibody, process for producing the same, and use of the antibody in human beta₂-use in the quantitative detection of microglobulin.

Background

β₂-microglobulin (β)₂-microglobulin，β₂-MG) is an endogenous small-molecule globulin produced by lymphocytes, platelets, polymorphonuclear leukocytes, with a molecular weight of 11.8 kDa; single-chain polypeptides consisting of 99 amino acids, which play an important role in immune responses; is a beta chain part of a cell surface human leukocyte antigen, contains disulfide bonds in molecules, does not contain sugar, and has a structure similar to that of immunoglobulin.

β₂MG is widely present in plasma, urine, cerebrospinal fluid, saliva and colostrum. Serum beta₂The rate of synthesis of MG and the amount released from the cell membrane are very constant, so that β is₂The MG content remains at a steady level. Normal human beta₂MG is freely filterable from the glomerulus, 99.9% is absorbed in the proximal tubule and destroyed by breakdown in the tubular epithelial cells, so that normal urine is excreted in minute amounts. Beta in blood in the case of impaired glomerular filtration function or increased filtration load₂-an increase in MG content; beta in urine₂An increase in MG content indicates an increase in tubular damage or filtration load. For renal transplant patients, beta₂The detection of MG indicates whether rejection is occurring in the body. Beta in urine₂MG also helps to identify upper and lower urinary tract infections. In summary, β₂MG is a detection index with high clinical application value.

The method for measuring beta 2-MG includes enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), immunotransmission turbidimetry, immunoscattering turbidimetry, and microparticle enzyme immunoassay. The enzyme-linked immunosorbent assay has poor repeatability and is suitable for large-scale detection. Radioimmunoassay and turbidimetric radiosity have short effective period of reagents due to half-life of nuclides; in addition, the nuclide is polluted by radiation, the construction of a nuclide laboratory needs to be supervised by an epidemic prevention department, an operator also needs to be specially trained, the nuclide laboratory is not easy to popularize, and an expensive immunoassay instrument is also needed during the measurement; the immuno-transmission turbidimetry has high specificity, simple and quick method, no radiation pollution, and stable result and repeatability which meet the clinical requirements. However, when the ratio of antigen to antibody is different, if the phenomenon of prozone or postzone occurs, the measurement result is greatly influenced. In addition, in the presence of hyperlipidemia, small particles of lipoproteins can form turbidity, giving rise to false rise in measured values, and therefore, lipemic specimens are not used as much as possible for the measurement.

Disclosure of Invention

The invention adopts the colloidal gold method, the method is rapid, simple and convenient, easy to operate and high in automation degree, has good correlation with an ELISA method, an immunoturbidimetry method and the like, has very wide clinical application, and has stronger universality compared with other method detection reagents, and the technical problem which is solved firstly is to provide a group of reagents which can effectively and specifically bind to human beta₂-antibodies to MG. More specifically:

the first purpose of the present invention is to provide two anti-human beta₂-an MG antibody.

First anti-human beta₂-MG antibody (BM12),

the heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO:1, HCDR1 as set forth in sequence SEQ ID NO:2 and HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown at 3;

and a light chain variable region sequence thereof comprising the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 4, LCDR1 shown as a sequence SEQ ID NO: 5 and LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown in fig. 6.

Preferably, the amino acid sequence of the heavy chain variable region of the BM12 antibody of the present invention is represented by SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is represented by SEQ ID NO. 8.

Second anti-human beta₂-MG antibody (BM17),

the heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 9, HCDR1 as shown in sequence SEQ ID NO: 10 and HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown in fig. 11;

and a light chain variable region sequence thereof comprising the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 12, LCDR1 as shown in sequence SEQ ID NO: 13 and LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown at 14.

Preferably, the amino acid sequence of the heavy chain variable region of the BM17 antibody of the present invention is represented by SEQ ID NO. 15, and the amino acid sequence of the light chain variable region is represented by SEQ ID NO. 16.

The second purpose of the invention is to provide two single-chain antibodies, wherein the amino acid sequence of the single-chain antibody BM12 is shown as SEQ ID NO. 17; the amino acid sequence of the antibody BM17 is shown in SEQ ID NO. 18.

The third purpose of the invention is to provide two nucleotide sequences for coding the single-chain antibody, wherein the nucleotide sequence for coding the single-chain antibody BM12 is shown as SEQ ID NO. 19, and the nucleotide sequence for coding the single-chain antibody BM17 is shown as SEQ ID NO. 20.

The fourth purpose of the invention is to provide an expression vector containing the nucleotide sequence.

The fifth object of the present invention is to provide a recombinant host bacterium containing the above expression vector.

It is a sixth object of the present invention to provide a method for producing the above single-chain antibody, comprising:

1) culturing the recombinant host bacteria under proper conditions to express the antibody;

2) then purifying and collecting the antibody from the host bacteria.

It is a seventh object of the present invention to provide the above-mentioned anti-human beta₂Detection of human beta by MG antibody₂-use in MG content.

An eighth object of the present invention is to provide a kit for pairing and detectingHuman beta₂-an antibody pair combination for MG; and the detection sensitivity is high and the specificity is good.

The ninth object of the present invention is to provide a method for producing a polypeptide using the above-mentioned antihuman beta₂MG antibody detection of beta₂-a colloidal gold immunochromatographic assay quantitative detection card for MG antibody comprising a sample absorption pad, a gold label pad, a reaction membrane and a water absorption pad; the gold label pad is sprayed with an antibody BM17 marked by colloidal gold particles, the reaction membrane is provided with a detection zone and a quality control zone, and the position of the detection zone is coated with an antibody BM 12.

The blocking solution used by the colloidal gold immunochromatographic quantitative detection card is 1-10% BSA, 0.01M PB solution, and pH is 7.4.

The gold-labeled antibody complex solution is 1-10% BSA, 5% trehalose, 0.025% tween20, 0.01M PB solution, and has a pH value of 7.4.

The antibody coating solution is 0.5-2.5% trehalose, 0.01M PB solution, pH 7.4.

The sample dilutions used were 0.1% Proclin300, 0.01M PBS, ph7.4, and may also contain 1% BSA.

The invention prepares a plurality of antibodies, and performs pairing screening to obtain a group of antibody combinations (BM12 and BM17) with sensitivity and specificity meeting the requirements; meanwhile, the preparation method is convenient for mass production and can meet the requirement of large-scale clinical application in the future. The antibody combination is debugged and optimized to obtain human beta with simple operation, sensitivity, specificity and relevant detection performance capable of meeting the requirement of human blood or urine sample detection₂-a colloidal gold immunochromatographic quantitative detection card of MG.

Drawings

FIG. 1: the specific detection effect graphs (Western Blot) of the antibodies BM12 and BM17,

wherein FIG. 1a is a Western Blot diagram of antibody BM 12; FIG. 1b is antibody BM17Western Blot. Lane 1 is standard protein (Marker); lane 2 is human beta₂-MG。

FIG. 2: the invention relates to a structure schematic diagram of a colloidal gold immunochromatography quantitative detection card. 1 is a sample pad, 2 is a reaction film, 3 is an absorption pad, 4 is a quality control line (C line), 5 is a detection line (T line), 6 is a gold label pad, and 7 is a PVC sheet.

FIG. 3: example 6. beta₂-MG detection card (12-17) detection range fitting curve

FIG. 4: example 6. beta₂-MG detection card (12-17) linear range fitting curve

FIG. 5: example 6. beta₂MG detection cards (12-17) methodological alignment of correlations

Detailed Description

Definition of

"antibody", also known as immunoglobulin, is a large Y-shaped protein secreted by B lymphocytes, and is an immunoglobulin molecule capable of specifically binding to a target antigen, such as a protein, a sugar, a polynucleotide, a lipid, a polypeptide, a small molecule compound, etc., through complementary sites (antigen-binding sites) at the two bifurcated tips of the Y.

"Single chain antibody" (scFv) refers to the variable region of the heavy chain (V) of an antibody_H) And light chain variable region (V)_L) A single-chain fusion protein is formed by connecting 15-20 amino acid short peptides (linkers), and the linkers used for connection are usually rich in glycine and serine, so that the stability and flexibility of a single-chain antibody are facilitated. The connection mode can be V_LIs connected to V_HC-terminal, or vice versa. Despite the removal of the constant region and the introduction of the linker, the single-chain antibody retains the specificity of the antibody to the antigen, and has the characteristics of small molecular weight, strong penetration, weak antigenicity and the like.

Complementary-determining regions (CDRs), also called hypervariable regions. Patterned at the amino acid end of the antibody monomer is the most critical region for binding of the target antigen to the antibody, and in immune network theory, the complementarity determining regions of each antibody are also called idiotypes or genotypes.

Example 1 anti-human beta₂Preparation of-microglobulin hybridoma cell line

1. Animal immunization

By recombination of human beta₂MG (recombinant expression in E.coli, manufactured by this company) BALB/c female mice (purchased from Calvens laboratory animals Co., Ltd., Changzhou) were immunized according to the general immunization protocol. For specific immunization, see "antibody preparation and use practiceThe test guidelines. And tracking the serum titer of the immune mice by adopting an indirect ELISA method, selecting the immune mice with the highest serum titer, and performing fusion experiments on the spleen cells and myeloma cells of the mice.

2. Cell fusion

(1) Preparation of spleen cells

Immunized mice, eyeballs are picked and blood is taken, after cervical vertebra is cut off, the immunized mice are placed in 75% (v/v) alcohol for soaking for 10 minutes, spleens of the immunized mice are taken out from a sterile operating platform, the spleens are placed in a cell screen, cells are fully ground, the cells are screened, the spleen is centrifugally washed for a plurality of times by using sterile 1640 culture medium (purchased from Gibco company), and then the cells are resuspended to prepare single cell suspension, and the single cell suspension is counted for standby.

(2) Preparation of feeder cells

Taking one female BALB/c mouse 8-10 weeks old, picking an eyeball to obtain negative serum, and immersing the negative serum in 75% (v/v) alcohol for 10 minutes after the cervical vertebra is cut off; the abdominal skin was aseptically peeled, the peritoneum was exposed, and about 10mL of 1640HT medium (purchased from SIGMA) was injected into the abdominal cavity of the mouse with a syringe, and the abdomen was gently massaged and air-blown several times. Sucking the culture medium containing the macrophages and injecting the culture medium into 20% 1640HAT culture medium for later use;

taking one female BALB/c mouse with the age of 2-3 weeks, and immersing the mouse in 75% (v/v) alcohol for 10 minutes after the mouse dies after cervical vertebra breakage; aseptically placing thymus into a cell screen, grinding, sieving to obtain thymocytes, and placing the thymocytes into the 20% 1640HAT culture medium containing macrophages for later use.

(3) Cell fusion

Mouse myeloma cell line SP2/0 was selected at the logarithmic growth phase and collected and counted. Get about 10⁸The above spleen cells were combined with 2X 10⁷Each of the above SP2/0 cell lines was mixed in a fusion tube, centrifuged at 1000rpm for 10 minutes, and the supernatant was discarded (discarded as clean as possible), and the fusion tube was gently rubbed back and forth on the palm of the hand to loosen the pellet. 1mL of preheated PEG1450 (polyethylene glycol 1450, available from SIGMA) was added slowly and quickly over 60 seconds, 30mL of 1640HT medium was added and stopped, centrifuged at 1000rpm for 10 minutes, the supernatant was removed, the precipitate was loosened by gentle rubbing, and added to 20% of 1640HAT medium obtained in step 2.

Mixing the HAT culture medium, subpackaging at 200 μ L/well into 96-well cell culture plate, standing at 37 deg.C and 5% CO₂Cultured in a cell culture box. After one week, 20% 1640HAT medium was replaced with 10% 1640HT medium, and after 3 days, the supernatant was examined.

3. Anti-human beta₂Screening of Microglobin-specific hybridoma strains

(1) Preparation of the test plate: dilution of recombinant human beta with CB-coating solution₂MG (Escherichia coli system expression, manufactured by the company) is 1 mug/mL, a 96-hole ELISA plate is coated, 100 mug/hole is coated, the temperature is 2-8 ℃ for one night, and the washing is carried out for one time and the patting is carried out; PBST buffer containing 2% bovine serum albumin was blocked (200 ul/well) for 2 hours at 37 ℃; patting dry for later use.

(2) Screening of positive clones: adding 100 μ L/well of cell culture supernatant to be detected into the detection plate, performing action at 37 deg.C for 30min, washing, drying, adding 100 μ L/well HRP-labeled goat anti-mouse IgG, performing action at 37 deg.C for 30min, washing, drying, adding 100 μ L/well TMB color development solution, performing light-shielding development at 37 deg.C for 15min, adding 50 μ L of 2M H per well₂SO₄The reaction was stopped and the value read at OD 450. Positive well determination principle: OD450 value/negative control value is not less than 2.1. Selecting positive clone strains to carry out cell cloning screening. After three to four rounds of cloning screening, the positive rate of the monoclonal cell strain is determined to be a stable cell strain with 100 percent of positive rate, and the cell strain is determined. The antibodies expressed by the hybridoma cell strains B12 and B17 both have higher titer, and then the variable region sequence of the antibody is further analyzed.

Example 2 determination of variable region sequences of antibodies of hybridoma cell lines

The sequences of the variable regions of the antibodies of the hybridoma cell lines B12 and B17 were determined.

Extraction of RNA: total RNA extraction was performed on the hybridoma cell lines B12 and B17 and reverse transcription was immediately performed with reference to the instructions of a cell total RNA extraction kit (purchased from Roche Co.);

reverse transcription of RNA into DNA: performing reverse transcription on the total RNA extracted in the previous step by referring to Thermo Scientific reversed First strand cDNA Synthesis Kit (purchased from Thermo company), preparing cDNA, and freezing and storing at-20 ℃ for later use;

c. PCR amplification and recovery of variable region sequences: performing PCR amplification on variable region sequences of a heavy chain and a light chain by using cDNA obtained in the previous step as a template and a universal primer of a mouse IgG subtype monoclonal antibody variable region sequence as a primer, and recovering a PCR product by using a DNA gel recovery kit (purchased from TIANGEN company);

d. cloning and sequencing of variable region sequences: according to the specification of cloning vector pMD18-T kit (purchased from Takara), the heavy chain and light chain variable region genes were ligated with pMD18-T vector, respectively, to transform E.coli DH 5. alpha. positive clones were picked up and submitted to Nanjing Kingsry Biotech Ltd for sequencing.

The amino acid sequence of the heavy chain variable region of the antibody of the hybridoma cell strain B12 obtained by sequencing is shown as SEQ ID NO 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown in fig. 8. The Vbase2 database analyzes the above sequences, and the amino acid sequences of the complementarity determining regions of the heavy chain variable region are: as shown in sequence SEQ ID NO:1, HCDR1 as set forth in sequence SEQ ID NO:2 and HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown at 3; the amino acid sequence of each complementarity determining region of the light chain variable region is: as shown in sequence SEQ ID NO: 4, LCDR1 shown as a sequence SEQ ID NO: 5 and LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown in fig. 6.

The amino acid sequence of the heavy chain variable region of the antibody of the hybridoma cell strain B17 obtained by sequencing is shown as SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown at 16. The Vbase2 database analyzes the above sequences, and the amino acid sequences of the complementarity determining regions of the heavy chain variable region are: as shown in sequence SEQ ID NO: 9, HCDR1 as shown in sequence SEQ ID NO: 10 and HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown in fig. 11; the amino acid sequence of each complementarity determining region of the light chain variable region is: as shown in sequence SEQ ID NO: 12, LCDR1 as shown in sequence SEQ ID NO: 13 and LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown at 14.

Example 3 recombinant expression and purification of Single chain antibodies

According to implementationAs a result of sequencing in example 2, a linker peptide (GGGGS) was added between the heavy and light chain variable regions of the antibodies of hybridoma cell lines B12 and B17₃Six histidines are introduced into the C end, and are subjected to whole gene synthesis, and a pichia pastoris expression system is used for carrying out the recombinant expression of the single-chain antibody. The expressed antibodies were designated antibody BM12 and BM17, respectively. The recombinant expression of the single-chain antibody is specifically as follows:

1. construction of expression plasmid for Single chain antibody Gene

The gene sequence of the single-chain antibody BM12 is shown as SEQ ID NO. 19, and the amino acid sequence is shown as SEQ ID NO. 17; the gene sequence of the single-chain antibody BM17 is shown in SEQ ID NO. 20, and the amino acid sequence is shown in SEQ ID NO. 18. Introducing a DNA sequence after an XhoI sequence in a pPICZ alpha A vector into a fragment synthesized by a single-chain BM12 and BM17 whole gene at the upstream, introducing a histidine tag sequence and an XbaI restriction enzyme cutting site into the downstream, and constructing into a pUC57 plasmid (purchased from Nanjing Kingsry Biotech limited) to obtain a long-term storage plasmid, wherein the plasmid is marked as pUC57-BM12-scFv and pUC57-BM 17-scFv. The PCR amplification is carried out, and the PCR amplification is carried out,

wherein the upstream primer P1 is TGT AAA ACG ACG GCC AGT;

the downstream primer P2 is CAG GAA ACA GCT ATG AC.

After a conventional PCR procedure, agarose gel electrophoresis analysis revealed that the product size was consistent with the expected size. After recovery and purification of the PCR-derived gene products, they were digested simultaneously with XhoI (# R0146S, available from New England Biolabs) and XbaI (# R0145V, available from New England Biolabs), ligated to pPICZ. alpha.A (V19520, available from Invitrogen) plasmid using T4 ligase, transformed into DH 5. alpha. competent cells, and cultured overnight at 37 ℃ in LB plates containing Zeocin (R250-01, available from Invitrogen). Screening positive clone bacteria for sequencing and comparing the sequence of the positive clone bacteria on the next day, wherein the sequence is completely consistent with an expected sequence, and expression plasmids of antibodies BM12 and BM17 are marked as pPICZ alpha-BM 12-scFv and pPICZ alpha-BM 17-scFv.

2. Construction, screening and expression of single-chain antibody gene in pichia host engineering strain

YPDS solid medium preparation: refer to the Invitrogen company EasySelectPichia Expression Kit Specification; pichia competent cells: refer to the EasySelectPichia Expression Kit Specification; preparing a BMGY culture medium: refer to the Multi-Copy Pichia Expression Kit Specification by Invitrogen; preparing a BMMY culture medium: refer to the Multi-Copy Pichia Expression Kit Specification by Invitrogen.

The pPICZ alpha-BM 12-scFv and pPICZ alpha-BM 17-scFv plasmids were linearized with SacI restriction enzyme, respectively. After ethanol precipitation, the linearized vector is electrically transformed into X-33 competent yeast cells, spread on YPDS solid medium containing Zeocin and cultured at 30 ℃ for 3-5 days, and then positive clones are generated.

The single clone obtained above was picked up and cultured to OD at 30 ℃ in 5mL of BMGY medium₆₀₀When the concentration is 2.0-6.0, 1mL of the preserved strain is taken, the residual bacterial liquid is transferred to BMMY after being resuspended, and small-amount induction expression is carried out, and methanol is supplemented every 24 hours until the final concentration is 1% (v/v).

Inoculating the obtained recombinant fusion protein engineering strain BM12, BM17 in BMGY culture medium, culturing at 30 deg.C and 220rpm until the thallus density reaches OD₆₀₀Methanol was added every 24 hours to a final concentration of 1.0% (v/v) 2.0 to 6.0. After one week, the fermentation broth was collected.

3. Single chain antibody purification

The antibodies BM12 and BM17 single-chain antibodies were purified using a histidine-tag affinity column, and HisTrap HP was selected as the pre-packed column, and the specific steps were as follows:

(1) impurity removal pretreatment of fermentation liquor: supernatant of the single-chain antibody BM12 and BM17 fusion protein fermentation liquor obtained by the expression is centrifuged and collected, and binding buffer is added to ensure that the final concentration of the supernatant is 300mM NaCl and 20mM NaH₂PO₄10mM Imidazole, pH7.5 adjusted, and filtered through a 0.45 μm filter.

(2) HisTrap HP affinity column purification: the affinity purification of the single chain antibody BM12, BM17 fusion protein fermentation broth obtained by the pretreatment was carried out using a fully automated intelligent protein purification system (AKTA avant150, available from GE healthcare Co., Ltd.) and the column was HisTrap HP (17-5248-02, available from GE healthcare Co., Ltd.). The binding buffer was 300mM NaCl, 20mM NaH₂PO₄10mM Imidazole, pH7.5, elution buffer 300mM NaCl，20mM NaH₂PO₄500mM Imidazole, pH 7.5. Linear elution was performed during elution and the individual elution peaks were collected. The purity of the purified protein reaches more than 95 percent; the collection tubes meeting the requirements were pooled, buffer changed to PBS solution and concentrated by ultrafiltration (1mg/mL), filter sterilized and stored at-20 ℃ for future use.

Example 4 evaluation of antibody Performance

1. Western blot identification of antibodies BM12 and BM17

a. Polyacrylamide gel electrophoresis: preparing 12% separation gel and 5% concentration gel, and loading standard protein and recombinant beta respectively₂Microglobulin (E.coli system expression, manufactured by this company), electrophoresed for 1 hour at constant pressure;

b. film transfer: the membrane was rotated for 1 hour under constant current (35 mA/membrane) to transfer the proteins on the polyacrylamide gel to a nitrocellulose membrane. Staining SDS-PAGE gel subjected to membrane transfer by Coomassie brilliant blue G250, and observing the residual condition of protein;

c. and (3) sealing: TBST buffer containing 5% skimmed milk was blocked (blocking solution) overnight at 4 ℃; washing with a washing solution (TBST, for details, TBST buffer of TaKaRa) once for 10 minutes after blocking;

d. antigen-antibody reaction: diluting a blocking solution (according to a volume ratio of 1: 1000), and adding a horse radish peroxidase-labeled BM12(BM12-HRP, 1mg/mL, labeled by a classic sodium periodate method in the company, the same below) and a horse radish peroxidase-labeled BM17(BM17-HRP, 1mg/mL, labeled by a classic sodium periodate method in the company, the same below) into the two cellulose nitrate membranes respectively, and reacting at room temperature for 1 hour; TBST washes 5 times for 10 minutes each;

e. and (3) color development and photographing: sucking up residual liquid on the nitrocellulose membrane, adding a mixed solution (purchased from Thermo company) of 2mL of a stable peroxidase solution (1mL) and a luminol/enhancer solution (1mL) into the nitrocellulose membrane, uniformly wetting the surface of the nitrocellulose membrane, carrying out a reaction at room temperature in a dark place for one minute, and then photographing in a gel imaging system (purchased from GE company) (figure 1) to obtain a result.

The detection result shows that the antibodies BM12 and BM17 have better specificity and can specifically detect humanβ₂-microglobulin.

2. Evaluation of single-chain antibodies BM12 and BM17 on colloidal gold detection platform

The antibodies purified in example 3 are paired and combined, and are respectively used as coating antibodies or labeled antibodies to be paired and detected for beta₂-microglobulin (β)₂-MG), the detection steps are as follows:

1) diluting BM12 or BM17 to 1.5mg/ml with antibody coating solution, and streaking on nitrocellulose membrane;

2) diluting colloidal gold-labeled BM12 or BM17 with gold-labeled antibody suspension, and spraying onto the pad;

3) pasting, slitting and clipping the film as shown in FIG. 2 (see example 5 for details of preparation)

4) Dilution of beta with sample diluent₂MG standard (manufactured by Zhonghong) to the concentration of 9MG/L and 2.25MG/L, adding 40ul of the two concentration standards and a zero concentration standard (namely sample diluent) into a colloidal gold detection card (BM 12-BM 17 mark or BM 17-BM 12 mark coated), and placing the detection card on a reading instrument for reading after 10 min. The results are shown in the following table:

from the above results, it can be seen that the double antibody sandwich method detection system composed of BM12 as a coating antibody and BM17 as a labeled antibody can be applied to a colloidal gold detection platform to perform beta-detection₂Detection of MG.

Example 5 anti-human beta₂Preparation of a colloidal gold immunoassay card for microglobulin

1. Solution preparation

1)0.01M PB buffer preparation: weighing Na₂HPO₄·12H₂O 3.22g，NaH₂PO₄·2H₂0.15g of O, 1000ml of purified water is added, the mixture is stirred by a rotor until dissolved, the pH value is measured by a ph meter and is 7.4 +/-0.1, and the mixture is filtered by a 0.45um filter membrane.

2)0.01M PBS buffer preparation: weighing 8.00g NaCl and 0.20g KCl Na₂HPO₄ 1.44g，KH₂PO₄0.24g, adding 1000ml of purified water, stirring by a rotor until the purified water is dissolved, measuring the pH value by a ph meter to be 7.4 +/-0.1, and filtering by a 0.45um filter membrane.

3) Preparing a sealing liquid: bovine Serum Albumin (BSA) 5g was weighed, and 50ml of 0.01M PB solution (pH 7.4. + -. 0.1) was added thereto, and the mixture was stirred with a rotor until dissolved.

4) Preparation of antibody coating solution: 0.2g trehalose is weighed out and added to 10ml 0.01M PB solution (pH 7.4. + -. 0.1) and stirred on a rotor for 5-10 min.

5) Preparing a gold-labeled antibody re-solution: weighing 1g bovine serum albumin and 5g trehalose, adding 100ml 0.01M PB solution (pH7.4 + -0.1), stirring with rotor until dissolved, adding 25ul Tween-20, and stirring with rotor for 5-10 min.

6) Preparation of sample diluent: weighing 10g bovine serum albumin, adding 1000ml 0.01M PBS solution (pH7.4 + -0.1), rotor stirring to dissolve, adding 1.0ml Proclin300, rotor stirring for 5-10min, and filtering with 0.45um filter membrane.

2. Human beta₂Preparation of a colloidal gold immunoassay card for microglobulin

1) Labeling of colloidal gold

Colloidal gold labeling of antibody BM17 (example labeled with 1ml of colloidal gold solution): by K₂CO₃Adjusting the pH value of the colloidal gold (5 ul of 0.2M K is added into each 1ml of the colloidal gold₂CO₃) Stirring for 5-10min, slowly adding antibody BM17 (30 ug antibody BM17 per 1ml of colloidal gold), and stirring at low speed for 30 min; adding 100ul of confining liquid, and stirring for 20 minutes; transferring the colloidal gold solution into a centrifuge tube, and centrifuging at 1500rpm and 2-8 ℃ for 15 min; transferring the supernatant into another centrifuge tube, and centrifuging at 12000rpm and 2-8 deg.C for 30 min; removing supernatant, and re-dissolving the precipitate with 200ul gold-labeled antibody re-solution to obtain colloidal gold-labeled BM17 antibody.

2) Gold label pad and reaction film preparation

Spraying BM17 gold-labeled antibody on the gold-labeled pad 6, and drying;

after diluting the antibody BM12 to 1.5mg/ml with an antibody diluent, coating the antibody BM12 on the position of a T line 5 of a reaction membrane 2 (nitrocellulose membrane); after the anti-His tag antibody is diluted to 0.1mg/ml with an antibody diluent, the antibody is coated on the position of the C line 4 of the reaction membrane 2 (nitrocellulose membrane), and the reaction membrane is dried for later use.

3) Assembling big card, cutting strip and assembling

The sample pad 1, the gold-labeled pad 6, the nitrocellulose membrane 2 coated with the antibody and the absorbent pad 3 are sequentially pasted on a PVC bottom plate from left to right (as shown in figure 2), and the T line 5 of the nitrocellulose membrane coated with the antibody is arranged on the left, and the C line 4 is arranged on the right.

Cutting the large plate into small strips with the width of 2.92 mm; placing the cut strips in the clamping grooves of the bottom card, wherein the position of a sample adding hole of the panel corresponds to the sample pad; the panel is involuted with the bottom plate and is forcibly pressed to enable the panel to be inosculated with the bottom plate; when the card is pressed, the card is vertically arranged on a conveying belt of the shell pressing machine to be pressed in sequence.

4) Kit assembly

Packing the assembled detection card and drying agent into an aluminum foil bag, sealing the aluminum foil bag by a heat sealing machine, and labeling;

subpackaging the sample diluent according to 0.7 ml/tube, filling into a self-sealing bag according to the specification of the kit, and labeling;

according to the specification of a finished product, putting a certain number of parts of inner bags, 1 self-sealing bag containing sample diluent, 1 part of specification and 1 qualified label into a packaging box, and sticking the label outside the packaging box.

3. Human beta₂Method for using-microglobulin colloidal gold detection card

1) The outer package was opened and the test card was removed from the sealed aluminum foil pouch and placed on a flat table.

2) 10 μ l of serum was pipetted and added to 490 μ l A tube sample diluent and after thorough mixing, 10ul of the mixture was pipetted therefrom and added to 490 μ l B tube sample diluent.

3) And (3) sucking 40 mu l of the treated sample from the sample diluent of the tube B, adding the sample into a sample adding hole of the detection card, and standing for 10min at room temperature.

4) The detection card is put into an immunochromatography quantitative analyzer, the detection is started by pressing a 'quick detection' key, and the detection card is automatically scanned by the analyzer.

5) The detection result is read/printed from the display screen of the immunochromatographic quantitative analyzer.

Example 6 human beta₂Evaluation of detection Effect of the microglobulin colloidal gold test card

1) Precision: detecting 2.25 and 9mg/L beta of BM12 (coating) -BM17 (marking) detection card according to the using method of the detection card₂-MG reference substance is repeatedly measured 10 times each, and the precision of the test card is calculated after outliers are removed. The experimental result shows that the coefficient of variation of the detection result is respectively 13.63% and 7.55%.

2) Detection range: BM12 (coating) -BM17 (labeling) detection card for detecting beta at different concentrations₂MG recombinant protein 1/2.25/4.5/9/18MG/L, and the fitted curve and detection range are 1-18MG/L (as shown in figure 3).

3) Linear range: preparing 5 series concentration samples from the high-value sample and the sample diluent according to a certain proportion, detecting by using a BM12 (coating) -BM17 (marking) detection card, detecting each sample for 3 times, performing regression statistics on the result and the theoretical concentration, and judging whether the concentration is linear in the concentration range. The linear range is 1-18mg/L (as shown in figure 4).

4) Accuracy: detecting 2.25 and 9mg/L beta of BM12 (coating) -BM17 (marking) detection card according to the using method of the detection card₂MG reference was repeated 3 times each, and the relative deviation of the mean value from the theoretical value was calculated, and the experimental results showed that the relative deviation of the two concentrations was 13.38% and 0.62%, respectively.

5. Accuracy-methodological comparison

Selecting Chongqing Zhongyuan biotechnology limited company beta which obtains good reputation on the market in the same kind of products₂Comparison and verification of a control product by a microglobulin detection kit (latex enhanced immunoturbidimetry). 30 clinical patient specimens were selected, numbered in the order of 1 to 30, and tested simultaneously using a control product and a colloidal gold test card of BM12 (coated) -BM17 (labeled) to be evaluated, in the sample order of 1, 2, 3. Correlation coefficient R of detection results of comparison and product to be evaluated²The results obtained by the two methods are better correlated as shown in fig. 5, which is 0.9985.

Example 7 formulation screening

In addition to the above-mentioned best preparation example 1, the applicant tried various preparation schemes, such as the following sets of test cards

Sequence listing

<110> Jiangsu Zhonghong bioengineering institute of drug creation Limited

<120> anti-human beta 2-microglobulin antibody and application thereof

<130> anti-human beta 2-microglobulin antibody and application thereof

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Gly Phe Thr Phe Gly Ser Tyr Thr

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Ile Ser Ser Gly Gly Ser Phe Thr

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Ser Arg Glu Gly Gly Pro Tyr Tyr Gly Ser His Tyr Tyr Ala Leu Asp

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Tyr

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Gln Asp Ile Thr Asn Tyr

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<213> Mus musculus

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Tyr Thr Ser

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<213> Mus musculus

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Gln Gln Gly Asn Thr Leu Pro Pro Thr

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<213> Mus musculus

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Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

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Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Ser Tyr

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Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

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Ala Ala Ile Ser Ser Gly Gly Ser Phe Thr Tyr Tyr Leu Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Lys Thr Thr Leu Tyr

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Leu Gln Met Gly Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys

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Ser Arg Glu Gly Gly Pro Tyr Tyr Gly Ser His Tyr Tyr Ala Leu Asp

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Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

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Asp Ile Gln Met Thr Gln Ser Thr Ser Ser Leu Ser Ala Ser Leu Gly

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Asp Gly Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Thr Asn Tyr

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Leu Asn Trp Tyr Gln Gln Arg Pro Asp Gly Ala Val Arg Leu Leu Ile

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Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

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Ser Gly Ser Gly Thr Asn Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

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Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Pro

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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

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Gly Phe Ser Leu Ser Thr Ser Gly Met Gly

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<213> Mus musculus

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Ile Tyr Trp Asp Asp Asp Lys

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<213> Mus musculus

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Ala Arg Val Tyr Tyr Gly Tyr Asp Gly Gly Phe Phe Asp Tyr

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<213> Mus musculus

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Ser Ser Val Ser Tyr

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<213> Mus musculus

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Ser Thr Ser

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<213> Mus musculus

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His Gln Arg Ser Ser Tyr Pro Leu Thr

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<213> Mus musculus

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Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

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Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

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Gly Met Gly Val Ser Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

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Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser

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Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val

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Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

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Cys Ala Arg Val Tyr Tyr Gly Tyr Asp Gly Gly Phe Phe Asp Tyr Trp

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Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

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<213> Mus musculus

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Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

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Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

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Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 17

<211> 246

<212> PRT

<213> Mus musculus

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Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

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Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Ser Tyr

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Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

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Ala Ala Ile Ser Ser Gly Gly Ser Phe Thr Tyr Tyr Leu Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Lys Thr Thr Leu Tyr

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Leu Gln Met Gly Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys

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Ser Arg Glu Gly Gly Pro Tyr Tyr Gly Ser His Tyr Tyr Ala Leu Asp

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Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr

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Gln Ser Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Gly Val Thr Ile

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Ser Cys Arg Ala Ser Gln Asp Ile Thr Asn Tyr Leu Asn Trp Tyr Gln

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Gln Arg Pro Asp Gly Ala Val Arg Leu Leu Ile Tyr Tyr Thr Ser Arg

180 185 190

Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asn Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr

210 215 220

Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Pro Thr Phe Gly Gly Gly

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Thr Lys Leu Glu Ile Lys

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<210> 18

<211> 243

<212> PRT

<213> Mus musculus

<400> 18

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser

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Trp Leu Ala His Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser

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Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val

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Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

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Cys Ala Arg Val Tyr Tyr Gly Tyr Asp Gly Gly Phe Phe Asp Tyr Trp

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Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

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Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser

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Glu Ile Lys

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<212> DNA

<213> Mus musculus

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gacgtgaagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60

tcctgtgcag cctctggatt cactttcggt agctacacca tgtcttgggt tcgccagact 120

ccggagaaga ggctggagtg ggtcgcagcc attagtagtg gtggtagttt cacctactat 180

ttagacagtg tgaagggccg attcaccatt tccagagaca gtgccaagac caccctgtac 240

ctgcaaatgg gcagtctgaa gtctgaggac acagccatgt attattgttc aagagaagga 300

ggtccctact acggtagtca ttactatgct ttggactact ggggtcaagg aacctcagtc 360

accgtctcct caggtggtgg tggatccgga ggtggtggtt ctggtggtgg tggttctgat 420

atccagatga cacagagtac atcctccctg tctgcctctc tgggagacgg agtcaccatc 480

agttgcaggg caagtcaaga cattaccaat tatttaaact ggtatcagca gagaccagat 540

ggagctgtta gactcctgat ctactacaca tcaagattac actcaggagt cccatcaagg 600

ttcagtggca gtgggtctgg aacaaattat tctctcacca ttagcaacct ggagcaagaa 660

gatattgcca cttacttttg ccaacaggga aatacgcttc ctccgacgtt cggtgggggc 720

accaagttgg aaatcaaa 738

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<211> 729

<212> DNA

<213> Mus musculus

<400> 20

caggttactc tgaaagagtc tggccctggg atattgcagc cctcccagac cctcagtctg 60

acttgttctt tctctgggtt ttcactgagc acttctggta tgggtgtgag ctggattcgt 120

cagccttcag gaaagggtct ggagtggctg gcacacattt actgggatga tgacaagcgc 180

tataacccat ccctgaagag ccggctcaca atctccaagg atacctccag caaccaggta 240

ttcctcaaga tcaccagtgt ggacactgca gatactgcta catactactg tgctcgggtc 300

tactatggtt acgatggggg gttctttgac tactggggcc aaggcaccac tctcacagtc 360

tcctcaggtg gtggtggatc cggaggtggt ggttctggtg gtggtggttc tcaaattgtt 420

ctcacccagt ctccagcaat catgtctgca tctccagggg agaaggtcac cataacctgc 480

agtgccagct caagtgtaag ttacatgcac tggttccagc agaagccagg cacttctccc 540

aaactctgga tttatagcac atccaacctg gcttctggag tccctgctcg cttcagtggc 600

agtggatctg ggacctctta ctctctcaca atcagccgaa tggaggctga agatgctgcc 660

acttattact gccaccaaag gagtagttac ccgctcacgt tcggtgctgg gaccaagttg 720

gaaataaaa 729

Claims (8)

1. Anti-human beta₂-a microglobulin antibody comprising:

the heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO:1, HCDR1 as set forth in sequence SEQ ID NO:2 and HCDR2 as shown in sequence SEQ ID NO: HCDR3 shown at 3;

and the light chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as the sequence SEQ ID NO: 4, LCDR1 shown as a sequence SEQ ID NO: 5 and LCDR2 as shown in sequence SEQ ID NO: LCDR3 shown in fig. 6.

2. The anti-human β according to claim 1₂The microglobulin antibody is characterized in that the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.

3. The anti-human β according to claim 2₂The microglobulin antibody is characterized in that the amino acid sequence is shown as SEQ ID NO. 17.

4. Nucleic acid encoding the antibody of claim 3, having the nucleic acid sequence shown in SEQ ID NO 19.

5. An expression vector comprising the nucleic acid sequence of claim 4.

6. A recombinant host cell comprising the expression vector of claim 5.

7. A method of producing the antibody of claim 3, comprising:

1) culturing the recombinant host cell of claim 6 under suitable conditions to express the antibody;

2) the antibody is then purified from the host cell and collected.

8. The anti-human β according to any one of claims 1 to 3₂Beta in the preparation of a test sample with a microglobulin antibody₂-use in a card for the detection of the content of microglobulin.

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