CN113621059B - Beta-form 2 Microglobulin detection kit and clinical application thereof - Google Patents

Beta-form 2 Microglobulin detection kit and clinical application thereof Download PDF

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CN113621059B
CN113621059B CN202010185527.1A CN202010185527A CN113621059B CN 113621059 B CN113621059 B CN 113621059B CN 202010185527 A CN202010185527 A CN 202010185527A CN 113621059 B CN113621059 B CN 113621059B
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马永
杨芸
赵利利
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ZONHON BIOPHARMA INSTITUTE Inc
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Abstract

The invention relates to five anti-human beta 2 Microglobulin (beta) 2‑ MG) antibodies and their use in detecting human beta in blood samples 2 -the use of a kit for the detection of the content of microglobulin. In particular, the invention provides 5 kinds of mouse anti-human beta 2‑ MG monoclonal antibody and application thereof in latex enhanced turbidimetric immunoassay kit. The invention prepares a plurality of antibodies, and performs pairing screening to obtain antibody combinations with sensitivity and specificity meeting requirements; meanwhile, the invention adopts recombinant human beta 2‑ MG is used as an antigen calibrator, is convenient and stable, has controllable quality and can meet the requirement of large-scale clinical application in the future. The latex immunoturbidimetric quantitative detection reagent which is easy and convenient to operate, and has the sensitivity, the specificity and the related detection performance can meet the requirements of human clinical sample detection is obtained by carrying out debugging and optimizing operation of a detection system on the antibody combination.

Description

Beta-form 2 Microglobulin detection kit and clinical application thereof
Technical Field
The invention belongs to the field of medical immune in-vitro diagnosis, in particular to a recombinant human beta 2 Microglobulin (beta) 2 -microglobulin，β 2 -MG) and its use in quantitative detection of human serum beta 2 -application in a kit for detecting MG content.
Background
β 2 Microglobulin (beta) 2 -microglobulin，β 2 MG) is a protein with a molecular weight of 11.8kD, which is present in all nucleated cells (except erythrocytes, placental trophoblasts), especially in lymphocytes and monocytes. The same individual produced a constant amount of beta 2-MG daily and secreted into various body fluids. Determination of beta in blood, urine, etc 2 MG is of importance for diagnosing diseases, in which case beta in the blood is caused 2 -an increase in MG content: (1) renal function decline: acute and chronic nephritis, renal failure, renal tumor, etc., (2) malignant tumor: primary liver cancer, lung cancer, etc., (3) autoimmune diseases: systemic lupus erythematosus, AIDS disease, etc., (4) organ transplant rejection.
Beta pair 2 The detection method of the-MG mainly comprises an enzyme-linked immunosorbent assay and a radioimmunoassay, and the methods are immunological based and utilize the specific combination of antigen and antibody to quantitatively detect beta in serum 2 -MG content. However, the methods have disadvantages of complicated operation, long period or high cost, which prevent the methods from being widely used clinicallyIs used. The full-automatic biochemical analyzer is almost fully used clinically (from a secondary hospital to a tertiary hospital), and the biochemical turbidimetry has the advantages of short reaction time, good precision, easiness in automation and the like, and becomes a main flow detection method clinically at home and abroad.
The invention adopts latex enhanced immunoturbidimetry, and the particle size of the antigen-antibody polymer is obviously increased by means of a plurality of specific murine monoclonal antibody coated 'latex' media, thereby improving the detection sensitivity and precision.
Disclosure of Invention
The invention provides a recombinant human beta 2 Microglobulin (hereinafter referred to as beta) 2- MG) having an amino acid sequence as set forth in SEQ ID NO:1, the codon sequence after optimization is shown as SEQ ID NO: 2.
The invention also provides 5 kinds of mouse anti-human beta 2- MG monoclonal antibody, first anti-human beta 2- MG monoclonal antibody (BM 15) whose heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:3, HCDR1 as set forth in SEQ ID NO:4 and/or HCDR2 as set forth in SEQ ID NO: HCDR3 as shown in 5;
and light chain variable region sequences thereof comprise the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:6, LCDR1 as set forth in SEQ ID NO:7 and/or LCDR2 as set forth in SEQ ID NO: LCDR3 as shown in fig. 8.
Preferably, the amino acid sequence of the heavy chain variable region of the BM15 antibody is shown as SEQ ID NO. 9, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 10.
The amino acid sequence of the preferred BM15 monoclonal antibody is shown as SEQ ID NO. 11; the nucleotide sequence of the coding monoclonal antibody is shown as SEQ ID NO. 12.
Second anti-human beta 2- MG monoclonal antibody (BM 18) whose heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:13, HCDR1 as set forth in SEQ ID NO:14 and/or HCDR2 as set forth in SEQ ID NO: HCDR3 as shown at 15;
and light chain variable region sequences thereof comprise the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:16, LCDR1 as set forth in SEQ ID NO:17 and/or LCDR2 as set forth in SEQ ID NO:18, LCDR3.
Preferably, the amino acid sequence of the heavy chain variable region of the BM18 antibody of the present invention is shown as SEQ ID NO. 19, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 20.
The amino acid sequence of the preferred BM18 monoclonal antibody is shown as SEQ ID NO. 21; the nucleotide sequence of the coding monoclonal antibody is shown as SEQ ID NO. 22.
Third anti-human beta 2- MG monoclonal antibody (BM 20) whose heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:23, HCDR1 as set forth in SEQ ID NO:24 and/or HCDR2 as set forth in SEQ ID NO:25, HCDR3;
and light chain variable region sequences thereof comprise the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:26, LCDR1 as set forth in SEQ ID NO:27 and/or LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 28.
Preferably, the amino acid sequence of the heavy chain variable region of the BM20 antibody of the present invention is shown as SEQ ID NO. 29, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 30.
The amino acid sequence of the preferred BM20 monoclonal antibody is shown as SEQ ID NO. 31; the nucleotide sequence of the coding monoclonal antibody is shown as SEQ ID NO. 32.
Fourth antihuman beta 2- MG monoclonal antibody (BM 22) whose heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:33, HCDR1 as set forth in SEQ ID NO:34 and/or HCDR2 as set forth in SEQ ID NO: HCDR3 as shown at 35;
and light chain variable region sequences thereof comprise the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:36, LCDR1 as set forth in SEQ ID NO:37 and/or LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 38.
Preferably, the amino acid sequence of the heavy chain variable region of the BM22 antibody of the present invention is shown as SEQ ID NO. 39, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 40.
The amino acid sequence of the preferred BM22 monoclonal antibody is shown as SEQ ID NO. 41; the nucleotide sequence of the coding monoclonal antibody is shown as SEQ ID NO. 42.
Fifth anti-human beta 2- MG monoclonal antibody (BM 23) whose heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:43, HCDR1 as set forth in SEQ ID NO:44 and/or HCDR2 as set forth in SEQ ID NO: HCDR3 as shown at 45;
and light chain variable region sequences thereof comprise the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:46, LCDR1 as set forth in SEQ ID NO:47 and/or LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 48. Preferably, the amino acid sequence of the heavy chain variable region of the BM23 antibody of the present invention is shown as SEQ ID NO. 49, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 50.
The amino acid sequence of the preferred BM23 monoclonal antibody is shown as SEQ ID NO. 51; the nucleotide sequence of the coding monoclonal antibody is shown as SEQ ID NO. 52.
The invention also provides the recombinant mouse anti-human beta 2 Latex enhanced turbidimetric immunoassay kit prepared by MG monoclonal antibody. The invention discloses a beta 2-microglobulin immune latex enhanced turbidimetric detection kit, which comprises an R1 reagent, an R2 reagent and a standard substance, wherein the R1 reagent comprises: buffer solution 1, stabilizer, coagulant, protective agent, preservative and chelating agent; the R2 reagent includes: buffer solution 2, preservative, stabilizer and protective agent; the standard comprises: buffer solution 3, protective agent and stabilizer.
Preferably, in the R1 reagent: buffer 1 is phosphate buffer with pH of 7.0-7.4 and 0.01-0.05M; the stabilizer is 40g/L-80g/L NaCl; the coagulant enhancer is PEG6000 of 10-20g/L, the protectant is BSA of 0-40 g/L, the preservative is Proclin300 of 0.05-0.1g/L, and the chelating agent is EDTA of 2-10 g/L.
Preferably, in the R2 reagent: buffer 2 is phosphate buffer with pH of 7.0-7.4 and 0.01-0.05M; the preservative is Proclin300 with the concentration of 0.05-0.1 g/L; the stabilizer is 0g/L-20The protective agent comprises NaCl of g/L and sucrose of 50-100g/L, and BSA of 5-20 g/L; in addition, five recombinant mouse anti-human beta markers are marked 2 Latex microspheres with a MG protein antibody mass concentration of 0.2% -0.5%.
Preferably, the standard comprises a quantity of recombinant human beta packaged with different concentrations 2 -a solution of MG, wherein buffer 3 is a phosphate buffer of 0.01M-0.02M, ph 7.0-7.4; the protecting agent is BSA of 1g/L-10g/L and NaCl of 9-13 g/L.
Preferably, the recombinant murine anti-human beta is labeled 2 The preparation method of the latex microsphere of the MG protein antibody comprises the following steps:
5 recombinant murine anti-human beta 2 Diluting the mixture with equal mass of the MG protein monoclonal antibody to 0.5-2MG/mL by using a phosphate buffer solution to prepare an antibody working solution; diluting latex microsphere (polystyrene latex, available from JSR company, particle size 80-200 nm) with MES buffer solution to 1% (mass concentration), adding EDC 1% -4% (mass ratio of EDC/latex microsphere), stirring at 30deg.C for 30min, centrifuging to remove unreacted EDC after reaction, and adding recombinant mouse anti-human beta 0.001-0.2 (mass ratio of antibody/latex microsphere) 2 The working solution of the mixture with equal mass of the monoclonal antibody of the MG protein is stirred and reacts for 30min to 120min at the temperature of 16 ℃ to 37 ℃, unreacted antibody is removed through centrifugation after the reaction is finished, then stop reaction is carried out by adding into stop reaction solution, centrifugation is carried out, finally buffer solution composed of buffer solution 2, preservative (such as Proclin 300), stabilizer (such as NaCl and sucrose) and protective agent (such as BSA) is added, and the mixture is stirred and mixed uniformly, thus obtaining R2 reagent (wherein recombinant mouse anti-human beta is marked 2 The mass concentration of the latex microsphere of the MG protein antibody is 0.2% -0.5%).
The invention adopts the escherichia coli as an expression system to prepare the recombinant human beta 2 The preparation method of the MG protein has the advantages of short period, large expression quantity and low cost. Beta of the preparation 2 The MG protein can be used as a calibrator raw material of a detection kit, can be used as an effective immunogen for preparing a mouse monoclonal antibody, and can be subjected to recombinant expression on the basis of obtaining a mouse anti-sequence, and the obtained single-chain antibody is applied to beta 2 -coated antibodies of R2 reagent in the microglobulin quantitative detection kit. The invention performs pairing screening on the prepared multiple single-chain antibodies to obtain the antibody combination with sensitivity and specificity meeting the requirements; meanwhile, the method is convenient for mass production, and can meet the requirement of large-scale clinical application in the future. The antibody combination is subjected to debugging and optimizing operation of a detection system, so that beta which is easy and convenient to operate, and can meet the requirements of detection of human clinical samples in terms of sensitivity, specificity and related detection performance is obtained 2 Quantitative detection kit for microglobulin (latex immunoturbidimetry).
Drawings
The standard curve of the kit of FIG. 1 is plotted with beta on the abscissa 2 MG calibrator concentration, absorbance Δa on the ordinate.
FIG. 2 compares test performance evaluation results.
Detailed Description
The present invention will be described in further detail by way of examples, but the present invention is not limited to these examples.
EXAMPLE 1 preparation of beta 2-MG mouse monoclonal antibody
1. Immunization of animals BALB/c female mice (purchased from Kwangsi laboratory animal Co., ltd.) were immunized with recombinant human beta 2-MG protein (amino acid sequence shown as SEQ ID NO:1, optimized codon sequence shown as SEQ ID NO: 2) according to the general immunization program. Specific immunization conditions are described in the guidelines for antibody preparation and use. The serum titer of the immunized mice is tracked by adopting an indirect ELISA method, and the immunized mice with the highest serum titer are selected to perform fusion experiments on spleen cells and myeloma cells of the mice.
2. Cell fusion
(1) Preparation of spleen cells
Mice were immunized, blood was collected from the eyeballs, sacrificed by cervical spine breakage, immersed in 75% (v/v) alcohol for 10 minutes, the spleens were removed in a sterile operating table, placed in a cell screen, the cells were ground thoroughly, the screen was washed by centrifugation with sterile 1640 medium (available from Gibco company) for several times, and the cells were resuspended to make a single cell suspension and counted for use.
(2) Preparation of feeder cells
Taking one female BALB/c mouse of 8-10 weeks old, picking eyeball to obtain negative serum, killing by cervical vertebra and soaking in 75% (v/v) alcohol for 10 min; the abdominal skin was aseptically uncovered, the peritoneum was exposed, and about 10ml of 1640ht medium (purchased from SIGMA) was injected into the abdominal cavity of the mice using a syringe, the abdomen was gently massaged and blown several times. Sucking the medium containing the macrophages and injecting the medium into 20%1640HAT medium for later use;
taking one female BALB/c mouse with 2-3 weeks of age, killing the female BALB/c mouse by cervical vertebra fracture, and soaking the female BALB/c mouse in 75% (v/v) alcohol for 10 minutes; taking thymus in a cell screen in a sterile manner, grinding, screening to obtain thymus cells, and placing the thymus cells in the 20%1640HAT medium containing macrophages for later use.
(3) Cell fusion
Mouse myeloma cell line SP2/0 in log phase was selected, collected and counted. Get about 10 8 Spleen cells as described above and 2X 10 7 Each of the above SP2/0 cell lines was added to the fusion tube and mixed, the supernatant was discarded (discarded as much as possible) after centrifugation at 1000rpm, and the fusion tube was gently rubbed back and forth over the palm to loosen the pellet. 1mL of preheated PEG1450 (polyethylene glycol 1450, available from SIGMA company) was added slowly followed by 30mL of 1640HT medium to terminate, centrifuged at 1000rpm for 10 min, the supernatant removed, gently rubbed to loosen the pellet, and added to 20% of 1640HAT medium obtained in step 2.
Mixing the HAT culture medium, sub-packaging into 96-well cell culture plate at 200 μl/well, and placing at 37deg.C and 5% CO 2 Is cultured in a cell culture incubator. After one week, 20%1640hat medium was replaced with 10%1640ht medium, and the supernatant was taken after 3 days for detection.
3. Screening of anti-human beta 2-MG specific hybridoma strains
(1) Preparation of the detection plate: diluting recombinant human beta 2-MG protein to 1 mug/mL by using CB coating liquid, coating a 96-hole ELISA plate, coating 100 mug/hole, coating overnight at 2-8 ℃, washing and beating to dryness once; blocking (200 ul/well) with 2% bovine serum albumin in PBST buffer, blocking for 2 hours at 37 ℃; and (5) drying the mixture for standby.
(2) Screening of positive clones: will beAdding 100 mu L/well of cell culture supernatant to be detected into the detection plate, washing and drying after 30 minutes of action at 37 ℃, adding 100 mu L/well of HRP-labeled goat anti-mouse IgG, washing and drying after 30 minutes of action at 37 ℃, adding 100 mu L/well of TMB color development liquid, developing for 15 minutes at 37 ℃ in a dark place, and adding 50 mu L of 2M H to each well 2 SO 4 The reaction was terminated and the values were read at OD 450. Positive well determination principle: OD450 value/negative control value is more than or equal to 2.1. And selecting positive clone strains for cell cloning screening. After three to four rounds of cloning and screening, determining that the positive rate of the monoclonal cell strain is 100 percent as a stable cell strain, and determining the cell strain. The hybridoma cell lines BMG15, BMG18, BMG20, BMG22 and BMG23 all have higher titers, and then the antibody variable region sequence of the hybridoma cell lines is further analyzed in sequence.
EXAMPLE 2 recombinant expression and purification of Single chain antibodies
According to the sequencing results in example 2, the hybridoma cell lines BM15, BM18, BM20, BM22 and BM23 were introduced with a connecting peptide (GGGGS) 3 between their heavy and light chain variable regions, six histidines were introduced, and their whole genes were directly fused and recombinant expression of single chain antibodies was performed using a Pichia pastoris expression system. The expressed antibodies were designated as antibodies BM15, BM18, BM20, BM22 and BM23, respectively. The recombinant expression of the single chain antibody is specifically as follows:
a) Construction of single-chain antibody gene expression vector
The gene sequence of the single-chain antibody BM15 is shown as SEQ ID NO. 12, and the amino acid sequence is shown as SEQ ID NO. 11; the gene sequence of the single-chain antibody BM18 is shown as SEQ ID NO. 22, and the amino acid sequence is shown as SEQ ID NO. 21; the gene sequence of the single-chain antibody BM20 is shown as SEQ ID NO. 32, and the amino acid sequence is shown as SEQ ID NO. 31; the gene sequence of the single-chain antibody BM22 is shown as SEQ ID NO. 42, and the amino acid sequence is shown as SEQ ID NO. 41; the gene sequence of the single-chain antibody BM23 is shown as SEQ ID NO. 52, and the amino acid sequence is shown as SEQ ID NO. 51. The single chain antibodies BM15, BM18, BM20, BM22 and BM23 were introduced with XhoI cleavage site upstream and XhoI sequence in pPICZ alpha A vector, histidine tag and XbaI cleavage site downstream, and were subjected to total gene synthesis and constructed into pUC57 plasmid (available from Nanjing Jinsrui Biotechnology Co., ltd.) to obtain a long-term storage plasmid designated pUC57-BM15-scFv, pUC57-BM18-scFv, pUC57-BM20-scFv, pUC57-BM22-scFv and pUC57-BM23-scFv. Performing PCR amplification in which
The upstream primer P1 is TGT AAA ACG ACG GCC AGT;
the downstream primer P2 was CAG GAA ACA GCT ATG AC.
After conventional PCR procedures, agarose gel electrophoresis analysis showed that the product size was consistent with the expected size. After the PCR-derived gene products were recovered and purified, they were digested with XhoI (#R0146S, available from New England Biolabs) and XbaI (#R0145V, available from New England Biolabs), ligated into pPICZαA (V19520, available from Invitrogen) plasmid with T4 ligase, transformed into DH5 α competent cells, and cultured overnight at 37℃in LB plates containing Zeocin (R250-01, available from Invitrogen). The next day of screening positive clone bacteria, sequencing, comparing and completely conforming to the expected sequence to obtain the expression vector of the single-chain antibody, namely pPICZ alpha-BM 15-scFv, pPICZ alpha-BM 18-scFv, pPICZ alpha-BM 20-scFv, pPICZ alpha-BM 22-scFv and pPICZ alpha-BM 23-scFv.
b) Construction, screening and expression of single-chain antibody gene in pichia pastoris host engineering strain
Preparation of YPDS solid medium: refer to Invitrogen company EasySelect Pichia Expression Kit specification; pichia pastoris competent cells: reference is made to EasySelect Pichia Expression Kit description; BMGY medium preparation: refer to Invitrogen company Multi-Copy Pichia Expression Kit Specification; preparing BMMY culture medium: refer to Invitrogen company Multi-Copy Pichia Expression Kit Specification.
pPICZ alpha-BM 15-scFv, pPICZ alpha-BM 18-scFv, pPICZ alpha-BM 20-scFv, pPICZ alpha-BM 22-scFv, and pPICZ alpha-BM 23-scFv were linearized with SacI restriction enzymes, respectively. After ethanol precipitation, the linearized vector was electrotransformed into X-33 competent yeast cells, plated onto Zeocin-containing YPDS solid medium, and cultured at 30℃for 3-5 days, and positive clones were generated.
Genetically engineering bacteria of the recombinant single-chain antibody obtained by the methodInoculating the strain into BMGY culture medium, culturing at 30deg.C and 220rpm until the bacterial density reaches OD 600 =2.0 to 6.0, methanol was added every 24 hours to a final concentration of 1.0% (v/v). After one week, the fermentation broth was collected.
c) Single chain antibody purification
The single chain antibodies BM15, BM18, BM20, BM22 and BM23 were purified using histidine-tagged affinity columns, and the pre-loaded columns were selected as HisTrap HP, comprising the following steps:
(1) Impurity removal pretreatment of fermentation liquor: the single-chain antibodies BM15, BM18, BM20, BM22 and BM23 supernatant obtained by the above expression were centrifuged, the supernatant was collected and a binding buffer was added thereto so that the final concentration of the supernatant was 300mM NaCl,20mM NaH2PO4, 10mM Imidazole, pH7.5 was adjusted, and filtration was performed with a 0.45 μm filter.
(2) Purification by HisTrap HP affinity column: the single chain antibodies BM15, BM18, BM20, BM22 and BM23 fermentation broth obtained from the pretreatment were affinity purified using a fully automatic intelligent protein purification system (AKTA avant150, available from GE healthcare Co.), as a column HisTrap HP (17-5248-02, available from GE healthcare Co.). The binding buffer was 300mM NaCl,20mM NaH 2 PO 4 10mM Imidazole,pH7.5 elution buffer 300mM NaCl,20mM NaH 2 PO 4 500mM Imidazole,pH7.5. The elution was performed linearly and the individual elution peaks were collected. The purity of the purified protein reaches more than 95 percent; combining collecting pipes meeting the requirements, changing buffer solution into PBS solution, ultrafiltering and concentrating (1 mg/ml), filtering and sterilizing, and preserving at-20deg.C for use.
Example 3 preparation and use of detection kit
The detection kit of the invention is a liquid double reagent, wherein the liquid double reagent comprises an R1 reagent, an R2 reagent and a calibrator, and the specific steps are as follows:
1. reagent R1: adding a stabilizer (such as NaCl or KCl), a coagulant (such as PEG6000 or PEG 8000), a protective agent (such as BSA or casein), a preservative (such as Proclin 300) and a chelating agent (such as EDTA) into the buffer solution 1, and stirring and mixing uniformly to obtain the R1 reagent.
2. R2 reagent: 5 recombinant antihuman beta 2 -MG protein sheetDiluting the cloned antibody to 1.0mg/mL by using phosphate buffer solution to prepare antibody working solution; diluting latex microspheres (polystyrene latex, available from JSR corporation, particle size 80-200 nm) with MES buffer to 1% (mass concentration), adding 4% (mass ratio of EDC/latex microsphere) EDC, stirring at 30deg.C for 30min, centrifuging to remove unreacted EDC after the reaction, and adding 0.1 (mass ratio of antibody/latex microsphere) recombinant anti-human beta 2 The MG protein monoclonal antibody working solution is stirred and reacted for 30min-120min at 30 ℃, unreacted antibody is removed by centrifugation after the reaction is finished, then the stop reaction solution is added to stop the reaction, the centrifugation is carried out, finally the buffer solution composed of buffer solution 2, preservative (such as Proclin 300), stabilizer (such as NaCl and sucrose) and protective agent (such as BSA) is added to be stirred and mixed uniformly, and the R2 reagent (wherein recombinant mouse anti-human beta is marked) 2 The mass concentration of latex microspheres of the mixture of the MG protein monoclonal antibodies is 0.2% -0.5%).
3. Calibration material: different volumes of recombinant human beta are added to a buffer solution composed of a buffer solution 3, a protective agent (such as BSA), a stabilizer (such as NaCl) and a preservative (such as Proclin 300) 2 -MG protein, to prepare a set of calibrator with final concentration of 22, 12, 5, 2.5, 0 MG/L.
Preferably, in order to improve the accuracy and detection range of the kit, different R1, R2 and calibrator reagents are prepared for performance detection and comparison of accuracy (recovery rate) and analysis sensitivity, and specific evaluation results are shown in the following table 1. The kit prepared by selecting various buffers, stabilizers, protective agents, preservatives, chelating agents and the like has better detection performance.
Analytical sensitivity: at a known beta 2 Microglobulin (or simply beta) 2 -MG) content of (2.00+/-0.50) MG/L, and recording the absorbance difference of the kit under the specified parameters. Converted into 2.00mg/L beta 2 -difference in absorbance caused by MG.
Accuracy (addition recovery): adding a certain volume of calibrator (beta) into a human serum sample (short for routine sample) with proper concentration 2 Theoretical concentration of MG ofC Theory of ) Preparing a recovered sample, adding deionized water with the same volume into a conventional sample to prepare a basic sample, wherein the volume of the added calibrator is not more than 1/10 of the total volume, and the measured concentrations of the recovered sample and the basic sample are C 1 And C 0 . According to the calculation formula: recovery = (C 1 -C 0 )/C Theory of * The respective recovery rates were calculated at 100%.
Table 1 preparation of the kit and comparison results
Figure BDA0002414045080000081
Example 4 Performance test of detection kit
Control kit: the recombinant antibody used in the reagent R2 is replaced by commercial rabbit anti-human beta of A manufacturer 2 The preparation of the MG polyclonal antibody, other reagents and experimental protocols were as described in example 3;
the kit comprises: specifically, see preparation example 3 for beta 2 -an MG detection kit;
commercial kit: clinical beta purchased from well-known company B 2 -MG latex enhanced turbidimetric kit;
the key performances of the above kits were compared.
1. Linear range
Determination of 5 beta-concentrations using Beckmann full-automatic Biochemical Analyzer AU480 2 Absorbance at 600nm of MG standard, working curve of calibrator was plotted (see fig. 1). As can be seen from the results, the detection range of the kit provided by the invention has a wider linear range than that of a control kit, is more beneficial to directly detecting a high-concentration pathological sample, does not need to dilute the sample, and is easy to operate.
The linear range of the control kit cannot meet the clinical application requirements, and further research is not performed. The following performance evaluations were only developed for the kits of the present invention and commercial kits.
2. Precision of
Repeatability: samples of physiological and pathological concentrations were measured separately, the test was repeated 10 times, and Coefficient of Variation (CV) was calculated separately. The detection result shows that the repeatability of the kit and the commercialized kit completely reaches the industry standard that CV is less than or equal to 10 percent, and the CV value of the kit is lower and better than that of the commercialized kit (see Table 2).
Batch-to-batch difference: samples of both physiological and pathological concentrations were measured with 3 different lot numbers of the detection reagent of the present invention, each lot number was repeated 3 times, and the inter-lot difference R of the reagent (kit) was calculated. The detection result shows that the batch-to-batch difference of the kit can completely meet the industry standard R which is less than or equal to 10 percent (see Table 2).
3. Accuracy of
And (3) adding and recycling: adding a certain volume of calibrator (beta) into a human serum sample (short for routine sample) with proper concentration 2 Theoretical concentration of-MG of C Theory of ) Preparing a recovered sample, adding deionized water with the same volume into a conventional sample to prepare a basic sample, wherein the added calibrator is not more than 1/10 of the total volume, detecting each recovered sample and the basic sample for 3 times, and determining that the average concentration corresponds to C 1 And C 0 . According to the calculation formula: recovery = (C 1 -C 0 )/C Theory of * The respective recovery rates were calculated at 100%. The results show that the recovery rate of the kit can meet the requirements of 80% -120%, and the recovery rate of a high-concentration sample of the commercial kit is significantly higher than 120%, which shows that the kit has better specificity and accuracy (see table 2).
TABLE 2 evaluation results of accuracy and precision Properties
Figure BDA0002414045080000091
4. Comparison test
57 parts of human serum are detected by the kit and the commercial kit of the like products respectively, and are measured simultaneously by a Beckmann AU480 biochemical analyzer, the parameter setting of the kit is shown in example 3, and the commercial kit is carried out by referring to the instruction book. Measuring the measured value with a commercial kitThe correlation analysis of the values is carried out, and FIG. 2 shows that the kit of the invention has good correlation (R) with the commercial kit (i.e.the control kit) 2 =0.997), the clinical criteria can be met.
Example 5 applicability of the test kit
The detection kit of the invention is applied to a full-automatic biochemical analyzer, but is not limited to the full-automatic biochemical analyzer, wherein the main parameter setting and detection method is as follows (for example, beckmann full-automatic biochemical analyzer AU 5800):
detection wavelength: the main wavelength is 600nm, and the auxiliary wavelength is absent; detecting the temperature: 37 ℃;
the detection method comprises the following steps: mixing 110 mu L of reagent R1 with 1.5 mu L of sample, incubating for 5min, adding 40 mu L of reagent R2, immediately reading absorbance A1, incubating for 5min, reading absorbance A2, and calculating absorbance change DeltaA=A2-A1. Working curve of calibrator is drawn by using multipoint nonlinear calibration, and beta is in sample 2 The MG content can be calculated on the operating curve from the Δa variation.
The performance of the kit of the invention is evaluated on a Beckmann AU5800 biochemical analyzer for precision, accuracy and the like (see in particular example 4, wherein the accuracy adopts a relative deviation method), and the results are shown in Table 3. The results show that the precision and the accuracy of the kit meet the requirements, and the kit can be applied to other models of Beckmann biochemical analyzers and has wider applicability.
TABLE 3 Performance of the inventive kits on AU5800
Figure BDA0002414045080000101
/>
Sequence listing
<110> Jiangsu Suzhong Red bioengineering medicine laboratory Co., ltd
<120> beta 2-microglobulin detection kit and clinical application thereof
<130> beta 2-microglobulin detection kit and clinical application thereof
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<170> SIPOSequenceListing 1.0
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Met Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala
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Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His
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Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu
35 40 45
Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr
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Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala
65 70 75 80
Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp
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Asp Arg Asp Met
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atgattcagc gtaccccgaa aatccaagtt tattcacgcc atccggcgga aaacggcaaa 60
tcgaactttc tgaattgcta cgtcagtggt ttccacccgt ccgatattga agtggacctg 120
ctgaaaaatg gcgaacgtat cgaaaaagtc gaacatagcg atctgagctt ttctaaagac 180
tggtctttct atctgctgta ttacaccgaa tttaccccga cggaaaaaga tgaatacgcc 240
tgtcgtgtga accatgttac gctgtcccag ccgaaaattg tgaaatggga tcgcgacatg 300
taa 303
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<213> Mus musculus
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Gly Tyr Thr Phe Thr Ser Tyr Asp
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<213> Mus musculus
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Ile Tyr Pro Gly His Gly Ser Thr
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<213> Mus musculus
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Ala Arg Ser Asp Gly Asn His His Phe Asp Tyr
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<213> Mus musculus
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Gln Ser Leu Leu Tyr Leu Asn Asn Gln Lys Asn Tyr
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<213> Mus musculus
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Trp Ala Ser
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<210> 8
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<213> Mus musculus
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Gln Gln Tyr Tyr Ser Tyr Pro Arg Thr
1 5
<210> 9
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<213> Mus musculus
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Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Leu Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
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Gly Trp Ile Tyr Pro Gly His Gly Ser Thr Lys Phe Asn Glu Lys Phe
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Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
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Met Gln Leu Ser Ser Leu Thr Ser Glu Asn Ser Ala Val Tyr Phe Cys
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Ala Arg Ser Asp Gly Asn His His Phe Asp Tyr Trp Gly Gln Gly Thr
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Thr Leu Thr Val Ser Ser
115
<210> 10
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<213> Mus musculus
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Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Leu
20 25 30
Asn Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
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Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
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Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
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Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
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Tyr Tyr Ser Tyr Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
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Lys
<210> 11
<211> 246
<212> PRT
<213> Mus musculus
<400> 11
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Leu Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Tyr Pro Gly His Gly Ser Thr Lys Phe Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asn Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Ser Asp Gly Asn His His Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu
130 135 140
Ala Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln
145 150 155 160
Ser Leu Leu Tyr Leu Asn Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr
180 185 190
Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val
210 215 220
Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Arg Thr Phe Gly Gly Gly
225 230 235 240
Thr Lys Leu Glu Ile Lys
245
<210> 12
<211> 741
<212> DNA
<213> Mus musculus
<400> 12
caggttcagc tgcaacagtc tggacctgag ctggtgaagc ctggggcttt agtgaagata 60
tcctgcaagg cttctggtta caccttcaca agctacgata taaactgggt gaagcagagg 120
cctggacagg gacttgagtg gattggatgg atttatcctg gacatggtag tactaagttc 180
aatgagaaat tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcctac 240
atgcagctca gcagcctgac ttctgagaac tctgcagtct atttctgtgc aagatcggat 300
ggtaaccacc actttgacta ctggggccaa ggcaccactc tcacagtctc ctcaggtggt 360
ggtggatccg gaggtggtgg ttctggtggt ggtggttctg acattgtgat gtcacagtct 420
ccatcctccc tagctgtgtc agttggagag aaggttacta tgagctgcaa gtccagtcag 480
agccttttat atcttaacaa tcaaaagaac tacttggcct ggtaccagca gaaaccaggg 540
cagtctccta aactgctgat ttactgggca tccactaggg aatctggggt ccctgatcgc 600
ttcacaggca gtggatctgg gacagatttc actctcacca tcagcagtgt gaaggctgaa 660
gacctggcag tttattactg tcagcaatat tatagctatc ctcggacgtt cggtggaggc 720
accaagctgg agatcaaata a 741
<210> 13
<211> 8
<212> PRT
<213> Mus musculus
<400> 13
Gly Phe Ser Leu Pro Ser Tyr Gly
1 5
<210> 14
<211> 7
<212> PRT
<213> Mus musculus
<400> 14
Ile Trp Arg Gly Gly Ser Thr
1 5
<210> 15
<211> 10
<212> PRT
<213> Mus musculus
<400> 15
Ala Lys Asn Gly Arg Tyr Ala Leu Asp Tyr
1 5 10
<210> 16
<211> 6
<212> PRT
<213> Mus musculus
<400> 16
Gln Asp Ile Gly Asn Ser
1 5
<210> 17
<211> 3
<212> PRT
<213> Mus musculus
<400> 17
Ala Thr Ser
1
<210> 18
<211> 9
<212> PRT
<213> Mus musculus
<400> 18
Leu Gln Tyr Ala Ser Ser Pro Tyr Thr
1 5
<210> 19
<211> 116
<212> PRT
<213> Mus musculus
<400> 19
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Pro Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Met
50 55 60
Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Lys Asn Gly Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val
100 105 110
Thr Val Ser Ser
115
<210> 20
<211> 107
<212> PRT
<213> Mus musculus
<400> 20
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Asn Ser
20 25 30
Leu Asn Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile
35 40 45
Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser
65 70 75 80
Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 21
<211> 238
<212> PRT
<213> Mus musculus
<400> 21
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15
Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Pro Ser Tyr
20 25 30
Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Met
50 55 60
Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80
Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Lys Asn Gly Arg Tyr Ala Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val
100 105 110
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
130 135 140
Ser Leu Gly Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile
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Gly Asn Ser Leu Asn Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Lys
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Arg Leu Ile Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Lys Arg
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Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser
195 200 205
Leu Glu Ser Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser
210 215 220
Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
225 230 235
<210> 22
<211> 717
<212> DNA
<213> Mus musculus
<400> 22
caggtgcagc tgaagcagtc aggacctggc ctggtgcagc cctcacagag cctgtccata 60
acctgcacag tctctggttt ctcattacct agctatggtg tacactgggt tcgccagtct 120
ccaggaaagg gtctggagtg gctgggagtg atatggagag gtggaagcac agactacaat 180
gcagctttca tgtccagact gagcatcacc aaggacaact ccaagagcca agttttcttt 240
aaaatgaaca gtctgcaagc tgatgacact gccatatact actgtgccaa aaatggacgg 300
tatgctttgg actactgggg tcaaggaacc tcagtcaccg tctcctcagg tggtggtgga 360
tccggaggtg gtggttctgg tggtggtggt tctgacatcc agatgaccca gtctccatcc 420
tccttatctg cctctctggg agaaagagtc agtctcactt gtcgggcaag tcaggacatt 480
ggtaatagct taaactggct tcagcaggaa ccagatggaa ctattaaacg cctgatctac 540
gccacatcca gtttagattc tggtgtcccc aaaaggttca gtggcagtag gtctgggtca 600
gattattctc tcaccatcag cagccttgag tctgaagatt ttgtagacta ttactgtcta 660
caatatgcta gttctccgta cacgttcgga ggggggacca agctggaaat aaaataa 717
<210> 23
<211> 8
<212> PRT
<213> Mus musculus
<400> 23
Gly Tyr Ser Phe Thr Gly Tyr Tyr
1 5
<210> 24
<211> 8
<212> PRT
<213> Mus musculus
<400> 24
Val Asn Pro Asn Asn Gly Gly Thr
1 5
<210> 25
<211> 16
<212> PRT
<213> Mus musculus
<400> 25
Ala Arg Val Pro Gly Ile Asn Gly Asn Tyr Ala His Ser Met Asp Tyr
1 5 10 15
<210> 26
<211> 6
<212> PRT
<213> Mus musculus
<400> 26
Gln Thr Ile Gly Thr Trp
1 5
<210> 27
<211> 3
<212> PRT
<213> Mus musculus
<400> 27
Ala Ala Thr
1
<210> 28
<211> 9
<212> PRT
<213> Mus musculus
<400> 28
Gln Gln Leu Tyr Ser Ala Pro Leu Thr
1 5
<210> 29
<211> 123
<212> PRT
<213> Mus musculus
<400> 29
Glu Val Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Tyr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Arg Val Asn Pro Asn Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Pro Gly Ile Asn Gly Asn Tyr Ala His Ser Met Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 30
<211> 107
<212> PRT
<213> Mus musculus
<400> 30
Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Gln Ser Ala Ser Leu Gly
1 5 10 15
Glu Ser Val Thr Ile Thr Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile
35 40 45
Tyr Ala Ala Thr Ser Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Lys Phe Ser Phe Lys Ile Ser Ser Leu Gln Ala
65 70 75 80
Glu Asp Phe Val Ser Tyr Tyr Cys Gln Gln Leu Tyr Ser Ala Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 31
<211> 245
<212> PRT
<213> Mus musculus
<400> 31
Glu Val Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Tyr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Arg Val Asn Pro Asn Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Pro Gly Ile Asn Gly Asn Tyr Ala His Ser Met Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln
130 135 140
Ser Pro Ala Ser Gln Ser Ala Ser Leu Gly Glu Ser Val Thr Ile Thr
145 150 155 160
Cys Leu Ala Ser Gln Thr Ile Gly Thr Trp Leu Ala Trp Tyr Gln Gln
165 170 175
Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile Tyr Ala Ala Thr Ser Leu
180 185 190
Ala Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Lys
195 200 205
Phe Ser Phe Lys Ile Ser Ser Leu Gln Ala Glu Asp Phe Val Ser Tyr
210 215 220
Tyr Cys Gln Gln Leu Tyr Ser Ala Pro Leu Thr Phe Gly Gly Gly Thr
225 230 235 240
Lys Leu Glu Ile Lys
245
<210> 32
<211> 738
<212> DNA
<213> Mus musculus
<400> 32
gaggtccagc tgcagcagtc tgggcctgac ctggtgaagc ctggggcttc agtgaagatt 60
tcctgcaagg cttctggtta ctcattcact ggctactaca tgcactgggt gaagcagagc 120
catggaaaga gccttgagtg gattggacgt gttaatccta acaatggtgg tactagctac 180
aaccagaagt tcaagggcaa ggccatatta actgtagaca agtcatccag cacagcctac 240
atggagctcc gcagcctgac atctgaggac tctgcggtct attactgtgc aagagtaccg 300
ggtataaatg gtaactacgc ccattctatg gactactggg gtcaaggaac ctcagtcacc 360
gtctcctcag gtggtggtgg atccggaggt ggtggttctg gtggtggtgg ttctgacatt 420
cagatgaccc agtctcctgc ctcccagtct gcatctctgg gagaaagtgt caccatcaca 480
tgcctggcaa gtcagaccat tggtacatgg ttagcatggt atcagcagaa accagggaaa 540
tctcctcagc tcctgattta tgctgcaacc agcttggcag atggggtccc atcaaggttc 600
agtggtagtg gatctggcac aaaattttct ttcaagatca gcagccttca ggctgaagat 660
tttgtaagtt attactgtca acaactttac agtgctcctc tgacgttcgg tggaggcacc 720
aagctggaaa tcaaataa 738
<210> 33
<211> 8
<212> PRT
<213> Mus musculus
<400> 33
Gly Tyr Thr Phe Thr Ser Tyr Asp
1 5
<210> 34
<211> 8
<212> PRT
<213> Mus musculus
<400> 34
Ile Tyr Pro Gly His Gly Ser Thr
1 5
<210> 35
<211> 11
<212> PRT
<213> Mus musculus
<400> 35
Ala Arg Ser Asp Gly Asn His His Phe Asp Tyr
1 5 10
<210> 36
<211> 12
<212> PRT
<213> Mus musculus
<400> 36
Gln Ser Leu Leu Tyr Ser Asn Asn Gln Lys Asn Tyr
1 5 10
<210> 37
<211> 3
<212> PRT
<213> Mus musculus
<400> 37
Trp Ala Ser
1
<210> 38
<211> 9
<212> PRT
<213> Mus musculus
<400> 38
Gln Gln Tyr Tyr Ser Tyr Pro Arg Thr
1 5
<210> 39
<211> 118
<212> PRT
<213> Mus musculus
<400> 39
Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Gly Ala
1 5 10 15
Leu Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Tyr Pro Gly His Gly Ser Thr Arg Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asn Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Ser Asp Gly Asn His His Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Leu Thr Val Ser Ser
115
<210> 40
<211> 113
<212> PRT
<213> Mus musculus
<400> 40
Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Asn Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Tyr Ser Tyr Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 41
<211> 246
<212> PRT
<213> Mus musculus
<400> 41
Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Gly Ala
1 5 10 15
Leu Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Tyr Pro Gly His Gly Ser Thr Arg Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asn Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Ser Asp Gly Asn His His Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu
130 135 140
Ala Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln
145 150 155 160
Ser Leu Leu Tyr Ser Asn Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln
165 170 175
Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr
180 185 190
Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr
195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val
210 215 220
Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Arg Thr Phe Gly Gly Gly
225 230 235 240
Thr Lys Leu Glu Ile Lys
245
<210> 42
<211> 741
<212> DNA
<213> Mus musculus
<400> 42
caggttcagc tgcagcagtc tggacctggg ctggtgaagc ctggggcttt agtgaagata 60
tcctgcaagg cttctggtta caccttcaca agctacgata taaactgggt gaagcagagg 120
cctggacagg gacttgagtg gattggatgg atttatcctg gacatggtag tactaggtac 180
aatgagaaat tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcctac 240
atgcagctca gcagcctgac ttctgagaac tctgcagtct atttctgtgc aagatcggat 300
ggtaaccacc actttgacta ctggggccaa ggcaccactc tcacagtctc ctcaggtggt 360
ggtggatccg gaggtggtgg ttctggtggt ggtggttctg acattgtgat gtcacagtct 420
ccatcctccc tagctgtgtc agttggagag aaggttacta tgagctgcaa gtccagtcag 480
agccttttat atagtaacaa tcaaaagaac tacttggcct ggtaccagca gaaaccaggg 540
cagtctccta aactgctgat ttactgggca tccactaggg aatctggggt ccctgatcgc 600
ttcacaggca gtggatctgg gacagatttc actctcacca tcagcagtgt gaaggctgaa 660
gacctggcag tttattactg tcagcaatat tatagctatc ctcggacgtt cggtggaggc 720
accaagctgg agatcaaata a 741
<210> 43
<211> 8
<212> PRT
<213> Mus musculus
<400> 43
Gly Tyr Thr Phe Thr Ser Tyr Asp
1 5
<210> 44
<211> 8
<212> PRT
<213> Mus musculus
<400> 44
Ile Tyr Pro Gly His Gly Ser Thr
1 5
<210> 45
<211> 11
<212> PRT
<213> Mus musculus
<400> 45
Ala Arg Ser Asp Gly Asn His His Phe Asp Tyr
1 5 10
<210> 46
<211> 6
<212> PRT
<213> Mus musculus
<400> 46
Gln Asp Ile Ser Asn Tyr
1 5
<210> 47
<211> 3
<212> PRT
<213> Mus musculus
<400> 47
Tyr Thr Ser
1
<210> 48
<211> 9
<212> PRT
<213> Mus musculus
<400> 48
Gln Gln Gly Tyr Thr Leu Pro Pro Thr
1 5
<210> 49
<211> 118
<212> PRT
<213> Mus musculus
<400> 49
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Leu Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Tyr Pro Gly His Gly Ser Thr Lys Phe Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asn Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Ser Asp Gly Asn His His Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Leu Thr Val Ser Ser
115
<210> 50
<211> 107
<212> PRT
<213> Mus musculus
<400> 50
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Asn Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Arg Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Pro
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 51
<211> 240
<212> PRT
<213> Mus musculus
<400> 51
Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Leu Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Tyr Pro Gly His Gly Ser Thr Lys Phe Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asn Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Ser Asp Gly Asn His His Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu
130 135 140
Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Asn Cys Arg Ala Ser Gln
145 150 155 160
Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Arg Pro Asp Gly Thr
165 170 175
Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro
180 185 190
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile
195 200 205
Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly
210 215 220
Tyr Thr Leu Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
225 230 235 240
<210> 52
<211> 723
<212> DNA
<213> Mus musculus
<400> 52
caggttcagc tgcaacagtc tggacctgag ctggtgaagc ctggggcttt agtgaagata 60
tcctgcaagg cttctggtta caccttcaca agctacgata taaactgggt gaagcagagg 120
cctggacagg gacttgagtg gattggatgg atttatcctg gacatggtag tactaagttc 180
aatgagaaat tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcctac 240
atgcagctca gcagcctgac ttctgagaac tctgcagtct atttctgtgc aagatcggat 300
ggtaaccacc actttgacta ctggggccaa ggcaccactc tcacagtctc ctcaggtggt 360
ggtggatccg gaggtggtgg ttctggtggt ggtggttctg atatccagat gacacagact 420
acatcctccc tgtctgcctc tctgggagac agagtcacca tcaattgcag ggcaagtcag 480
gacattagca attatttaaa ctggtatcag cagagaccag atggaactgt taaactcctg 540
atctactaca catcaagatt acactcaggg gtcccatcaa ggttcagtgg cagtgggtct 600
ggaacagatt attctctcac cattagcaac ctggagcaag aagatattgc cacttacttt 660
tgccaacagg gttatacgct tcctccgacg ttcggtggag gcaccaagct ggagatcaaa 720
taa 723

Claims (12)

1. Anti-human beta 2- MG monoclonal antibody, heavy chain thereofThe variable region contains the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:3, HCDR1 as set forth in SEQ ID NO:4 and HCDR2 as set forth in SEQ ID NO: HCDR3 as shown in 5;
and light chain variable region sequences thereof comprise the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:6, LCDR1 as set forth in SEQ ID NO:7 and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown in fig. 8.
2. The anti-human beta of claim 1 2- The amino acid sequence of the MG monoclonal antibody is shown as SEQ ID NO. 11.
3. Anti-human beta 2- An MG monoclonal antibody whose heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:13, HCDR1 as set forth in SEQ ID NO:14 and HCDR2 as set forth in SEQ ID NO: HCDR3 as shown at 15;
and light chain variable region sequences thereof comprise the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:16, LCDR1 as set forth in SEQ ID NO:17 and LCDR2 as set forth in SEQ ID NO:18, LCDR3.
4. The anti-human beta of claim 1 2- The amino acid sequence of the MG monoclonal antibody is shown as SEQ ID NO. 21.
5. Anti-human beta 2- An MG monoclonal antibody whose heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:23, HCDR1 as set forth in SEQ ID NO:24 and HCDR2 as set forth in SEQ ID NO:25, HCDR3;
and light chain variable region sequences thereof comprise the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:26, LCDR1 as set forth in SEQ ID NO:27 and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 28.
6. The anti-human beta of claim 5 2- MG monoclonal antibodies, amino acid sequences such asSEQ ID NO. 31.
7. Anti-human beta 2- An MG monoclonal antibody whose heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:33, HCDR1 as set forth in SEQ ID NO:34 and HCDR2 as set forth in SEQ ID NO: HCDR3 as shown at 35;
and light chain variable region sequences thereof comprise the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:36, LCDR1 as set forth in SEQ ID NO:37 and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 38.
8. The anti-human beta of claim 7 2- The amino acid sequence of the MG monoclonal antibody is shown as SEQ ID NO. 41.
9. Anti-human beta 2- An MG monoclonal antibody whose heavy chain variable region comprises the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:43, HCDR1 as set forth in SEQ ID NO:44 and HCDR2 as set forth in SEQ ID NO: HCDR3 as shown at 45;
and light chain variable region sequences thereof comprise the following complementarity determining regions: the amino acid sequence is shown as SEQ ID NO:46, LCDR1 as set forth in SEQ ID NO:47 and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown at 48.
10. Beta as claimed in claim 9 2- The amino acid sequence of the MG monoclonal antibody is shown as SEQ ID NO. 51.
11. Beta-form 2 -microglobulin latex-enhanced turbidimetric immunoassay kit comprising an R1 reagent, an R2 reagent and a standard substance, wherein the R2 reagent contains the anti-human beta simultaneously marked with the reagents of claims 1, 3, 5, 7 and 9 2- Latex microspheres of MG monoclonal antibodies.
12. Beta as claimed in claim 11 2 -a microglobulin latex-enhanced turbidimetric immunoassay kit characterized in that: the R1 reagent comprises the following components: buffer solutionPhosphate buffer of 0.01M-0.05M, pH 7.0-7.4; the stabilizer is 40g/L-80g/L NaCl; the coagulant enhancer is PEG6000 of 10-20g/L, the protectant is BSA of 0-40 g/L, the preservative is Proclin300 of 0.05-0.1g/L, and the chelating agent is EDTA of 2-10 g/L; the R2 reagent comprises the following components: the buffer solution is phosphate buffer solution with the concentration of 0.01M-0.05M and the pH value of 7.0-7.4; the preservative is Proclin300 with the concentration of 0.05-0.1 g/L; the stabilizer is NaCl of 0g/L-20g/L and sucrose of 50 g/L-100 g/L, and the protecting agent is BSA of 5g/L-20 g/L; in addition, five recombinant mouse anti-human beta markers are marked 2 -latex microspheres with a MG protein antibody mass concentration of 0.2% -0.5%; the standard comprises 5 recombinant human beta with different concentrations 2 -a solution of MG, wherein the buffer is a phosphate buffer of 0.01M-0.02M, ph 7.0-7.4; the protecting agent is BSA of 1g/L-10g/L and NaCl of 9-13 g/L.
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CN101951953A (en) * 2007-02-27 2011-01-19 株式会社未来创药研究所 Contain the pharmaceutical composition of anti-GRP78 antibody as effective ingredient
CN107759699A (en) * 2017-10-18 2018-03-06 银丰生物工程集团有限公司 Target transgenic T cells of CD30 antigens and preparation method and application
CN108318695A (en) * 2018-01-30 2018-07-24 江苏晶红生物医药科技股份有限公司 People RBP colloidal gold immunochromatographimethod quantitative testing test paper cards and its clinical application
CN109575133A (en) * 2018-12-28 2019-04-05 江苏众红生物工程创药研究院有限公司 Anti-human β2- MG antibody and its application
CN109666072A (en) * 2018-12-28 2019-04-23 江苏众红生物工程创药研究院有限公司 Anti-human β2Microglobulin antibody and its application
CN109725159A (en) * 2018-12-28 2019-05-07 江苏众红生物工程创药研究院有限公司 People β2The quantitative testing test paper card of microglobulin and clinical application
CN110642949A (en) * 2019-10-11 2020-01-03 上海钹乐诗生物技术有限公司 Preparation method of beta 2-microglobulin monoclonal antibody

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101951953A (en) * 2007-02-27 2011-01-19 株式会社未来创药研究所 Contain the pharmaceutical composition of anti-GRP78 antibody as effective ingredient
CN107759699A (en) * 2017-10-18 2018-03-06 银丰生物工程集团有限公司 Target transgenic T cells of CD30 antigens and preparation method and application
CN108318695A (en) * 2018-01-30 2018-07-24 江苏晶红生物医药科技股份有限公司 People RBP colloidal gold immunochromatographimethod quantitative testing test paper cards and its clinical application
CN109575133A (en) * 2018-12-28 2019-04-05 江苏众红生物工程创药研究院有限公司 Anti-human β2- MG antibody and its application
CN109666072A (en) * 2018-12-28 2019-04-23 江苏众红生物工程创药研究院有限公司 Anti-human β2Microglobulin antibody and its application
CN109725159A (en) * 2018-12-28 2019-05-07 江苏众红生物工程创药研究院有限公司 People β2The quantitative testing test paper card of microglobulin and clinical application
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