CN104155451B - IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit and preparation method thereof - Google Patents
IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit and preparation method thereof Download PDFInfo
- Publication number
- CN104155451B CN104155451B CN201410410697.XA CN201410410697A CN104155451B CN 104155451 B CN104155451 B CN 104155451B CN 201410410697 A CN201410410697 A CN 201410410697A CN 104155451 B CN104155451 B CN 104155451B
- Authority
- CN
- China
- Prior art keywords
- bottle
- biotin
- nitrite ion
- antibody
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit and preparation method thereof, relates to the detection of syphilis.Kit is provided with concentrated cleaning solution bottle, yin and yang attribute reference substance bottle, biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody bottle, Horseradish peroxidase-conjugated avidin bottle, TMB nitrite ion A bottle, nitrite ion B bottle, reaction terminating liquid bottle, is coated with recombinant antigen microwell plate.To be infectious type strain Nichol strain and microspironema pallidum FFI type strain Reiter strain with microspironema pallidum, adopt proteomics methodology, by bidimensional electrophoretic separation microspironema pallidum Leaf proteins, go out to have strong immunogenic protein with the serological diagnosis of different patient or infection animal, find antigen markers.For syphilis validation test, improve diagnostic sensitivity and specificity, shorten window phase.Sensitive Detection can go out syphilis helicoid antibody and Quantitative measurement, low price, simple to operate, result is accurate.
Description
Technical field
The present invention relates to the detection of syphilis, especially relate to a kind of IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit and preparation method thereof.
Background technology
Syphilis is the sexually transmitted disease that microspironema pallidum causes, and the incidence of disease at home remains high in recent years, and the prevention and control of syphilis have become one of main task of China's public health service.
The laboratory diagnostic method of syphilis mainly contains several ([1] Lin Lirong as follows, Yang Bo, Pan Xitao, etc. the selection of potential Blood spread patient Syphilis serum test method. Chinese Journal of Nosocomiology .2010,20 (10): 1491-1494):
(1) pathogeny detection: infection by Treponema pallidum is early stage, when syphilis antibody does not also produce or content is lower, conveyor screw searched by dark-field microscope becomes laboratory diagnostic method the earliest, but be subject to the technology of drawing materials, position of drawing materials, treponemal body burden, the many factors such as local application and censored time in sample, sensitivity is lower.
(2) antibody detection test: microspironema pallidum can not carry out in vitro culture, diagnosis depends on laboratory examination, and existing Serologic detection sensitivity, specificity are not high, the equal existing defects of any one detection method, clinical fail to pinpoint a disease in diagnosis with misdiagnosis rate higher.
For serological syphilis inspection antigen mainly with recombinant antigen TPN15, TPN17, TPN37, TPN44.5, TPN47 is main ([2] Lin, L.R., Z.G.Fu, etal. " Developmentofacolloidalgold-immunochromatographyassaytod etectimmunoglobulinGantibodiestoTreponemapallidumwithTPN 17andTPN47. " Diagnosticmicrobiologyandinfectiousdisease.2010.68 (3): 193-200.), these antigens derive from microspironema pallidum adventitia mostly, but can not distinguish whether caused by Pathogenic spirochetal.
Summary of the invention
The object of the invention is to IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit and preparation method thereof.
Described IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit, is provided with:
External packing box, concentrated cleaning solution bottle, negative controls bottle, positive reference substance bottle, biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody bottle, Horseradish peroxidase-conjugated avidin bottle, TMB nitrite ion A bottle, nitrite ion B bottle, reaction terminating liquid bottle, be coated with recombinant antigen microwell plate;
Concentrated cleaning solution bottle, negative controls bottle, positive reference substance bottle, biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody bottle, Horseradish peroxidase-conjugated avidin bottle, TMB nitrite ion A bottle, nitrite ion B bottle, reaction terminating liquid bottle and be coated with recombinant antigen microwell plate and be contained in external packing box;
Concentrated cleaning solution bottle is built with concentrated cleaning solution, negative controls bottle is built with negative controls, positive reference substance bottle is built with positive reference substance, biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody bottle is built with biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody, Horseradish peroxidase-conjugated avidin bottle is built with Horseradish peroxidase-conjugated avidin, TMB nitrite ion A bottle is built with TMB nitrite ion A, nitrite ion B bottle is built with nitrite ion B, and reaction terminating liquid bottle is built with reaction terminating liquid;
Described TMB nitrite ion A is 3,3', 5,5'-tetramethyl benzidine (3,3', 5,5'-Tetramethylbenzidine, TMB) nitrite ion;
Described nitrite ion B is the superoxol of 3%.
Described concentrated cleaning solution can adopt the phosphate buffer being dissolved with Tween-20, and wherein the final concentration of Tween-20 is 0.05%, carries out 20 times of dilutions during use with distilled water.
Described negative controls is the total antibody negative controls product of microspironema pallidum, formulated by the healthy population serum of non-syphilis, uses this kit to carry out detecting its OD450nm light absorption value and is less than 0.1; Described OD450nm is predominant wavelength, and 450nm is the sample colour developing light absorption value measured.
Described positive reference substance is the total antibody positive reference substance of microspironema pallidum, formulated by the positive serum of syphilitic, carries out detecting its OD450nm light absorption value be greater than 0.5 with this kit; Described OD450nm is predominant wavelength, and 450nm is the sample colour developing light absorption value measured.
Described TMB nitrite ion A is 3,3', 5,5'-tetramethyl benzidine (3,3', 5,5'-Tetramethylbenzidine, TMB) nitrite ion, and trade name is TMB nitrite ion A, and commercially available product can purchased from Bo Sheng bio tech ltd, Xiamen.
Described nitrite ion B is the superoxol of 3%, and trade name is nitrite ion B, and commercially available product can purchased from Bo Sheng bio tech ltd, Xiamen.
Described reaction terminating liquid can adopt the sulfuric acid of volumetric molar concentration 2mmol/L.
Described bottle can adopt polyethylene bottle.
The preparation method of described IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit, comprises the following steps:
1) after extraction microspironema pallidum Nichol strain and Reiter strain outer membrane protein carry out dielectrophoresis, be transferred on nitrocellulose filter, immunoblot experiment is carried out with treponema pallidum agglutination, obtain distinctive Western blotting spot, get corresponding protein site and carry out mass spectrophotometry, find out the outer membrane protein that Nichol strain and Reiter strain have high immunogenicity respectively, and find out the peculiar and outer membrane protein of the high immunogenicity that Reiter strain lacks of Nichol strain;
2) in the whole genome sequence of microspironema pallidum, corresponding gene is found out, design primer also builds corresponding expression vector, study different inductive condition to the impact of restructuring target protein in expression in escherichia coli amount, obtain best inductive condition, a large amount of amplification target protein, collect the target protein of process LAN and carry out purifying, purity, higher than after 90%, carries out follow-up study;
3) by step 2) candidate antigens, obtain its corresponding polyclonal antibody by immune rat.Adopt enzyme-linked immunoassay method to measure antibody titer, and use western blotting method to verify the immunogenicity of these recombinant antigens.
4) adopt gene clone technology, the DNA of pcr amplification coding treponemal antigen, and insert in Escherichia coli and make it express, obtain microspironema pallidum recombinant antigen;
In step 4) in, described recombinant antigen is microspironema pallidum pathogenic strain Nichol Expression of Plant Height, and syphilis non-pathogenic strain Reiter strain is without expression; Described microspironema pallidum pathogenic strain Nichol strain is Treponemapallidumssp.pallidumstrainNichol; Described syphilis non-pathogenic strain Reiter strain is Treponemapallidumssp.pallidumstrainReiter.
5) by step 4) in microspironema pallidum recombinant antigen bag be buffered liquid and be diluted to 20ug/mL, add in microwell plate with every hole 0.1mL, 4 DEG C of bags are by 24h; Take out antigen plate, air-dry; With 1.0% skimmed milk power of 0.01mmol/LpH7.4 phosphate buffered saline, every hole 0.2mL, 4 DEG C of closed 24h, taking-up phosphate buffer washs 5 times, room temperature is air-dry, sterilization, sealing, obtain recombinant antigen bag by microwell plate;
6) with people IgM specific fragment μ chain for antigen immune Balb/c mouse, by hybridoma technology, screening obtains the hybridoma cell strain of stably excreting anti-human IgM monoclonal antibody;
7) biotin of anti-human IgM specific fragment μ chain monoclonal antibody and activation is mixed into row labels, unconjugated biotin is removed in dialysis, prepares biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody;
8) adopt Over-voltage protection to carry out horseradish peroxidase-labeled, obtain Horseradish peroxidase-conjugated avidin;
9) TMB nitrite ion A adopts commercially available product, can purchased from Bo Sheng bio tech ltd, Xiamen;
10) nitrite ion B adopts commercially available product, can purchased from Bo Sheng bio tech ltd, Xiamen;
11) preparation is dissolved with the phosphate buffer of Tween-20, and wherein the final concentration of Tween-20 is 0.05%, obtains concentrated cleaning solution, can carry out 20 times of dilutions during use with distilled water;
12) sulfuric acid of 2mmol/L is prepared as reaction terminating liquid;
13) prepare syphilis helicoid antibody negative controls by the healthy population serum of non-syphilis, use this kit to carry out detecting its OD450nm light absorption value and be less than 0.1; Described OD450nm is predominant wavelength, and 450nm is the sample colour developing light absorption value measured;
14) prepare syphilis helicoid antibody positive reference substance by the positive serum of syphilitic, carry out detecting its OD450nm light absorption value with this kit and be greater than 0.5; Described OD450nm is predominant wavelength, and 450nm is the sample colour developing light absorption value measured;
15) by anti-human for biotin labeling IgM specific fragment μ chain monoclonal antibody, Horseradish peroxidase-conjugated avidin, TMB nitrite ion A, nitrite ion B, concentrated cleaning solution, reaction terminating liquid, negative controls, positive reference substance is contained in bottle respectively, again by anti-human for biotin labeling IgM specific fragment μ chain monoclonal antibody bottle, Horseradish peroxidase-conjugated avidin bottle, TMB nitrite ion A bottle, nitrite ion B bottle, concentrated cleaning solution bottle, reaction terminating liquid bottle, negative controls bottle, positive reference substance bottle and be coated with recombinant antigen microwell plate and load in external packing box, obtain IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit.
The specificity of treponemal antigen of the present invention, it is microspironema pallidum pathogenic strain Nichol Expression of Plant Height, and syphilis non-pathogenic strain Reiter strain is without expression.
The invention provides a kind of IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit, can be used for the detection of syphilis specific antibody in clinical samples.Pathogenic syphilis helicoid antibody can be detected exactly, for Lues Assay provides a kind of efficient, easy technological means.
The present invention to be infectious type strain Nichol strain and microspironema pallidum FFI type strain Reiter strain with internationally recognized microspironema pallidum, adopt the method for protein science, by bidimensional electrophoretic separation microspironema pallidum Leaf proteins, use the serological diagnosis of different patient (or infection animal) to go out to have strong immunogenic protein again, find antigen markers.As the antigen markers of microspironema pallidum early diagnosis, set up a kind of sensitive, special IgM antibody to treponema pallidum biotin-avidin enzyme joint inspection survey technology, for syphilis validation test, improve sensitivity and the specificity of laboratory diagnosis, shorten window phase, significant to syphilis control.And product of the present invention not only Sensitive Detection can go out syphilis helicoid antibody, and can carry out accurate Quantitative measurement to it, low price, simple to operate, result is accurate, environment friendly and pollution-free, has long-range benefit.
Accompanying drawing explanation
Fig. 1 is the structure composition schematic diagram of described IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit embodiment.
In FIG, respectively be labeled as: 1, external packing box, 2, concentrated cleaning solution bottle, 3, negative controls bottle, 4, positive reference substance bottle, 5, biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody, 6, Horseradish peroxidase-conjugated avidin bottle, 7, TMB nitrite ion A bottle, 8, nitrite ion B bottle, 9, reaction terminating liquid bottle, 10, be coated with recombinant antigen microwell plate.
Embodiment
Following examples will the present invention is further illustrated by reference to the accompanying drawings.
See Fig. 1, described IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit embodiment, is provided with:
External packing box 1, concentrated cleaning solution bottle 2, negative controls bottle 3, positive reference substance bottle 4, biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody bottle 5, Horseradish peroxidase-conjugated avidin bottle 6, TMB nitrite ion A bottle 7, nitrite ion B bottle 8, reaction terminating liquid bottle 9, be coated with recombinant antigen microwell plate 10;
Concentrated cleaning solution bottle 2, negative controls bottle 3, positive reference substance bottle 4, biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody bottle 5, Horseradish peroxidase-conjugated avidin bottle 6, TMB nitrite ion A bottle 7, nitrite ion B bottle 8, reaction terminating liquid bottle 9 and be coated with recombinant antigen microwell plate 10 and be contained in external packing box 1;
Concentrated cleaning solution bottle 2 is built with concentrated cleaning solution, negative controls bottle 3 is built with negative controls, positive reference substance bottle 4 is built with positive reference substance, biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody bottle 5 is built with biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody, Horseradish peroxidase-conjugated avidin bottle 6 is built with Horseradish peroxidase-conjugated avidin, TMB nitrite ion A bottle 7 is built with TMB nitrite ion A, nitrite ion B bottle 8 is built with nitrite ion B, and reaction terminating liquid bottle 9 is built with reaction terminating liquid;
Described TMB nitrite ion A is 3,3', 5,5'-tetramethyl benzidine (3,3', 5,5'-Tetramethylbenzidine, TMB) nitrite ion;
Described nitrite ion B is the superoxol of 3%.
Described concentrated cleaning solution can adopt the phosphate buffer being dissolved with Tween-20, and wherein the final concentration of Tween-20 is 0.05%, carries out 20 times of dilutions during use with distilled water.
Described negative controls is the total antibody negative controls product of microspironema pallidum, formulated by the healthy population serum of non-syphilis, uses this kit to carry out detecting its OD450nm light absorption value and is less than 0.1; Described OD450nm is predominant wavelength, and 450nm is the sample colour developing light absorption value measured.
Described positive reference substance is the total antibody positive reference substance of microspironema pallidum, formulated by the positive serum of syphilitic, carries out detecting its OD450nm light absorption value be greater than 0.5 with this kit; Described OD450nm is predominant wavelength, and 450nm is the sample colour developing light absorption value measured.
Described TMB nitrite ion A is 3,3', 5,5'-tetramethyl benzidine (3,3', 5,5'-Tetramethylbenzidine, TMB) nitrite ion, and trade name is TMB nitrite ion A, and commercially available product can purchased from Bo Sheng bio tech ltd, Xiamen.
Described nitrite ion B is the superoxol of 3%, and trade name is nitrite ion B, and commercially available product can purchased from Bo Sheng bio tech ltd, Xiamen.
Described reaction terminating liquid can adopt the sulfuric acid of volumetric molar concentration 2mmol/L.
Described bottle can adopt polyethylene bottle.
The preparation method of described IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit, comprises the following steps:
1) after extraction microspironema pallidum Nichol strain and Reiter strain outer membrane protein carry out dielectrophoresis, be transferred on nitrocellulose filter, carry out immunoblot experiment with treponema pallidum agglutination, obtain distinctive Western blotting spot, get corresponding protein site and carry out mass spectrophotometry.Find out the outer membrane protein that Nichol strain and Reiter strain have high immunogenicity respectively, and find out the peculiar and outer membrane protein of the high immunogenicity that Reiter strain lacks of Nichol strain;
2) in the whole genome sequence of microspironema pallidum, corresponding gene is found out, design primer also builds corresponding expression vector, study different inductive condition to the impact of restructuring target protein in expression in escherichia coli amount, obtain best inductive condition, a large amount of amplification target protein, collect the target protein of process LAN and carry out purifying, purity, higher than after 90%, carries out follow-up study;
3) by step 2) candidate antigens, obtain its corresponding polyclonal antibody by immune rat.Adopt enzyme-linked immunoassay method to measure antibody titer, and use western blotting method to verify the immunogenicity of these recombinant antigens.
4) adopt gene clone technology, the DNA of pcr amplification coding treponemal antigen, and insert in Escherichia coli and make it express, obtain microspironema pallidum recombinant antigen;
5) by step 4) in microspironema pallidum recombinant antigen bag be buffered liquid and be diluted to 20ug/mL, add in microwell plate with every hole 0.1mL, 4 DEG C of bags are by 24h; Take out antigen plate, air-dry; With 1.0% skimmed milk power of 0.01mmol/LpH7.4 phosphate buffered saline, every hole 0.2mL, 4 DEG C of closed 24h, taking-up phosphate buffer washs 5 times, room temperature is air-dry, sterilization, sealing, obtain recombinant antigen bag by microwell plate;
6) with people IgM specific fragment μ chain for antigen immune Balb/c mouse, by hybridoma technology, screening obtains the hybridoma cell strain of stably excreting anti-human IgM monoclonal antibody;
7) biotin of anti-human IgM specific fragment μ chain monoclonal antibody and activation is mixed into row labels, unconjugated biotin is removed in dialysis, prepares biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody;
8) adopt Over-voltage protection to carry out horseradish peroxidase-labeled, obtain Horseradish peroxidase-conjugated avidin;
9) TMB nitrite ion A adopts commercially available product, can purchased from Bo Sheng bio tech ltd, Xiamen;
10) nitrite ion B adopts commercially available product, can purchased from Bo Sheng bio tech ltd, Xiamen;
11) preparation is dissolved with the phosphate buffer of Tween-20, and wherein the final concentration of Tween-20 is 0.05%, obtains concentrated cleaning solution, can carry out 20 times of dilutions during use with distilled water;
12) sulfuric acid of 2mmol/L is prepared as reaction terminating liquid;
13) prepare syphilis helicoid antibody negative controls by the healthy population serum of non-syphilis, use this kit to carry out detecting its OD450nm light absorption value and be less than 0.1; Described OD450nm is predominant wavelength, and 450nm is the sample colour developing light absorption value measured;
14) prepare syphilis helicoid antibody positive reference substance by the positive serum of syphilitic, carry out detecting its OD450nm light absorption value with this kit and be greater than 0.5; Described OD450nm is predominant wavelength, and 450nm is the sample colour developing light absorption value measured;
15) by anti-human for biotin labeling IgM specific fragment μ chain monoclonal antibody, Horseradish peroxidase-conjugated avidin, TMB nitrite ion A, nitrite ion B, concentrated cleaning solution, reaction terminating liquid, negative controls, positive reference substance is contained in bottle respectively, again by anti-human for biotin labeling IgM specific fragment μ chain monoclonal antibody bottle, Horseradish peroxidase-conjugated avidin bottle, TMB nitrite ion A bottle, nitrite ion B bottle, concentrated cleaning solution bottle, reaction terminating liquid bottle, negative controls bottle, positive reference substance bottle and be coated with recombinant antigen microwell plate and load in external packing box, obtain IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit.
Below provide specific embodiment.
Embodiment one
The preparation method of described IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit, comprises the following steps:
(1) the immunoproteomics research of outer membrane protein:
After extraction microspironema pallidum Nichol strain and Reiter strain outer membrane protein carry out dielectrophoresis, be transferred on nitrocellulose filter, carry out immunoblot experiment with treponema pallidum agglutination, obtain distinctive Western blotting spot, get corresponding protein site and carry out mass spectrophotometry.Find out the outer membrane protein that Nichol strain and Reiter strain have high immunogenicity respectively, and find out the peculiar and outer membrane protein of the high immunogenicity that Reiter strain lacks of Nichol strain.
(2) clone purification of candidate albumen
In the whole genome sequence of microspironema pallidum, find out corresponding gene, design primer also builds corresponding expression vector.Study different inductive condition on restructuring target protein in the impact of expression in escherichia coli amount, obtain best inductive condition, increase target protein in a large number.The target protein collecting process LAN carries out purifying.Purity, higher than after 90%, carries out follow-up study.
(3) immunogenicity of candidate albumen is detected
By step (2) candidate antigens, obtain its corresponding polyclonal antibody by immune rat.Adopt enzyme-linked immunoassay method to measure antibody titer, and use western blotting method to verify the immunogenicity of these recombinant antigens.
(4) prepare recombinant antigen: adopt gene clone technology, the DNA of pcr amplification coding treponemal antigen, and insert in Escherichia coli and make it express, obtain microspironema pallidum recombinant antigen;
(5) recombinant antigen bag is by microwell plate
Microspironema pallidum recombinant antigen bag in step (4) is buffered liquid and is diluted to 20ug/mL, add in microwell plate with every hole 0.1mL, 4 DEG C of bags are by 24h; Take out antigen plate, air-dry; With 1.0% skimmed milk power of 0.01mmol/LpH7.4 phosphate buffered saline, every hole 0.2mL, 4 DEG C of closed 24h, taking-up phosphate buffer washs 5 times, room temperature is air-dry, sterilize, seal for subsequent use.
(6) anti-human IgM specific fragment μ chain monoclonal antibody is prepared
With people IgM specific fragment μ chain for antigen immune Balb/c mouse, by hybridoma technology, screening obtains the hybridoma cell strain of the anti-human IgM monoclonal antibody of stably excreting;
(7) anti-human IgM specific fragment μ chain monoclonal antibody biotin labeling
The biotin of anti-human IgM specific fragment μ chain monoclonal antibody and activation is mixed into row labels, and unconjugated biotin is removed in dialysis, prepares biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody;
(8) Avidin horseradish peroxidase-labeled
Over-voltage protection is adopted to carry out horseradish peroxidase-labeled;
(9) TMB nitrite ion A is commercially available product, purchased from Bo Sheng bio tech ltd, Xiamen;
(10) nitrite ion B is commercially available product, purchased from Bo Sheng bio tech ltd, Xiamen;
(11) concentrated cleaning solution
Concentrated cleaning solution is the phosphate buffer being dissolved with Tween-20, and wherein the final concentration of Tween-20 is 0.05%, carries out 20 times of dilutions during use with distilled water.
(12) reaction terminating liquid
The sulfuric acid of preparation 2mmol/L is as reaction terminating liquid;
(13) reference substance
Syphilis helicoid antibody negative controls: formulated by the healthy population serum of non-syphilis, uses this kit to carry out detecting its OD450nm light absorption value and is less than 0.1;
Syphilis helicoid antibody positive reference substance: the positive serum of syphilitic is formulated, carries out detecting its OD450nm light absorption value with this kit and is greater than 0.5;
Described OD450nm is predominant wavelength, and 450nm is the sample colour developing light absorption value measured;
(14) microspironema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit is prepared
Be coated with the microspironema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit that recombinant antigen microwell plate, biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody, Horseradish peroxidase-conjugated avidin nitrite ion A, nitrite ion B, concentrated cleaning solution, reaction terminating liquid, negative controls, positive reference substance and external packing box form jointly.
Step (14) described biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody, Horseradish peroxidase-conjugated avidin nitrite ion A, nitrite ion B, concentrated cleaning solution, reaction terminating liquid, negative controls, positive reference substance are all contained in corresponding polyethylene bottle.
Embodiment two
Below provide the syphilis helicoid antibody in the clinical samples adopting IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit detection patient:
(1) sample disposal: serum: venous blood 5mL, puts 37 DEG C of water-bath 30min, the centrifugal 10min of 3000g, and supernatant is for subsequent use for detecting sample.
(2) application of sample: add the sample of 100uL and 50uL biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody in reacting hole, make blank, feminine gender and Positive control wells simultaneously.Hatch 1h for 37 DEG C.
(3) wash: button is dry afterwards to wash five times with lavation buffer solution.
(4) enzyme-added: in every hole, to add each 100 μ L of Horseradish peroxidase-conjugated avidin, put 37 DEG C and hatch 30min.
(5) wash: button is dry afterwards to wash five times with lavation buffer solution.
(6) develop the color: in every reacting hole, add each 50 μ L of nitrite ion A and nitrite ion B successively, put 37 DEG C and hatch 15min.
(7) measure: add reaction terminating liquid 50 μ L in every hole, then read the absorbance in each hole at 450nm place.
(8) result judges: reacting hole OD value/negative control hole OD value < 2.1, then this sample should be considered as feminine gender, if reacting hole OD value/negative control hole OD value all >=2.1, can be judged as the positive, be diagnosed as syphilis.
Embodiment three
Below provide the performance detecting of IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit
(1) positive sample coincidence rate
With syphilis specific antibody positive control serum 50 parts calibrating, calculate positive coincidence rate.
(2) ' negative ' specimens coincidence rate
With syphilis specific negative antibody control serum 50 parts calibrating, calculate negative match-rate.
(3) interior difference is criticized
Same batch of kit, uses characteristic Virus monitory, requires CV≤10%.
(4) differences between batches
Different batches kit, uses characteristic Virus monitory, requires CV≤12%.
(5) interference test
The interference experiment undertaken by each 50 examples of haemolysis, piarhemia and jaundice sample detects.
(6) cross reaction
Adopt this kit, carry out the detection of the autoimmune pathologies such as systemic loupus erythematosus (n=50), rheumatoid disease (n=50), autoallergic (n=50), observe cross reaction.
(7) Detection of Stability
Application Arrhenius rule, kit is placed 37 DEG C after 20 days and detect, above indices, without marked change, guarantees that finished product is preserved under drying at room temperature condition, and the term of validity is 18 months.
The cross reaction below providing IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit measures:
Adopt kit of the present invention to detect the rabbit infection experiment serum specimen of microspironema pallidum Nichol strain, result is strong positive, and the rabbit infection experiment serum specimen result of Reiter strain is negative.
Claims (7)
1. the preparation method of IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit, is characterized in that described IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit is provided with external packing box, concentrated cleaning solution bottle, negative controls bottle, positive reference substance bottle, biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody bottle, Horseradish peroxidase-conjugated avidin bottle, TMB nitrite ion A bottle, nitrite ion B bottle, reaction terminating liquid bottle, is coated with recombinant antigen microwell plate;
Concentrated cleaning solution bottle, negative controls bottle, positive reference substance bottle, biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody bottle, Horseradish peroxidase-conjugated avidin bottle, TMB nitrite ion A bottle, nitrite ion B bottle, reaction terminating liquid bottle and be coated with recombinant antigen microwell plate and be contained in external packing box;
Concentrated cleaning solution bottle is built with concentrated cleaning solution, negative controls bottle is built with negative controls, positive reference substance bottle is built with positive reference substance, biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody bottle is built with biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody, Horseradish peroxidase-conjugated avidin bottle is built with Horseradish peroxidase-conjugated avidin, TMB nitrite ion A bottle is built with TMB nitrite ion A, nitrite ion B bottle is built with nitrite ion B, and reaction terminating liquid bottle is built with reaction terminating liquid;
Described TMB nitrite ion A is 3,3', 5,5'-tetramethyl benzidine nitrite ion;
Described nitrite ion B is the superoxol of 3%;
Described preparation method, comprises the following steps:
1) after extraction microspironema pallidum Nichol strain and Reiter strain outer membrane protein carry out dielectrophoresis, be transferred on nitrocellulose filter, immunoblot experiment is carried out with treponema pallidum agglutination, obtain distinctive Western blotting spot, get corresponding protein site and carry out mass spectrophotometry, find out the outer membrane protein that Nichol strain and Reiter strain have high immunogenicity respectively, and find out the peculiar and outer membrane protein of the high immunogenicity that Reiter strain lacks of Nichol strain;
2) in the whole genome sequence of microspironema pallidum, corresponding gene is found out, design primer also builds corresponding expression vector, study different inductive condition to the impact of restructuring target protein in expression in escherichia coli amount, obtain best inductive condition, a large amount of amplification target protein, collect the target protein of process LAN and carry out purifying, purity, higher than after 90%, carries out follow-up study;
3) by step 2) candidate antigens, obtain its corresponding polyclonal antibody by immune rat, adopt enzyme-linked immunoassay method to measure antibody titer, and use western blotting method to verify the immunogenicity of these recombinant antigens;
4) adopt gene clone technology, the DNA of pcr amplification coding treponemal antigen, and insert in Escherichia coli and make it express, obtain microspironema pallidum recombinant antigen;
5) by step 4) in microspironema pallidum recombinant antigen bag be buffered liquid and be diluted to 20ug/mL, add in microwell plate with every hole 0.1mL, 4 DEG C of bags are by 24h; Take out antigen plate, air-dry; With 1.0% skimmed milk power of 0.01mmol/LpH7.4 phosphate buffered saline, every hole 0.2mL, 4 DEG C of closed 24h, taking-up phosphate buffer washs 5 times, room temperature is air-dry, sterilization, sealing, obtain recombinant antigen bag by microwell plate;
6) with people IgM specific fragment μ chain for antigen immune Balb/c mouse, by hybridoma technology, screening obtains the hybridoma cell strain of stably excreting anti-human IgM monoclonal antibody;
7) biotin of anti-human IgM specific fragment μ chain monoclonal antibody and activation is mixed into row labels, unconjugated biotin is removed in dialysis, prepares biotin labeling anti-human IgM specific fragment μ chain monoclonal antibody;
8) adopt Over-voltage protection to carry out horseradish peroxidase-labeled, obtain Horseradish peroxidase-conjugated avidin;
9) TMB nitrite ion A adopts commercially available product;
10) nitrite ion B adopts commercially available product;
11) preparation is dissolved with the phosphate buffer of Tween-20, and wherein the final concentration of Tween-20 is 0.05%, obtains concentrated cleaning solution;
12) sulfuric acid of 2mmol/L is prepared as reaction terminating liquid;
13) syphilis helicoid antibody negative controls is prepared by the healthy population serum of non-syphilis;
14) syphilis helicoid antibody positive reference substance is prepared by the positive serum of syphilitic;
15) by anti-human for biotin labeling IgM specific fragment μ chain monoclonal antibody, Horseradish peroxidase-conjugated avidin, TMB nitrite ion A, nitrite ion B, concentrated cleaning solution, reaction terminating liquid, negative controls, positive reference substance is contained in bottle respectively, again by anti-human for biotin labeling IgM specific fragment μ chain monoclonal antibody bottle, Horseradish peroxidase-conjugated avidin bottle, TMB nitrite ion A bottle, nitrite ion B bottle, concentrated cleaning solution bottle, reaction terminating liquid bottle, negative controls bottle, positive reference substance bottle and be coated with recombinant antigen microwell plate and load in external packing box, obtain IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit.
2. the preparation method of IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit as claimed in claim 1, it is characterized in that described concentrated cleaning solution adopts the phosphate buffer being dissolved with Tween-20, wherein the final concentration of Tween-20 is 0.05%.
3. the preparation method of IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit as claimed in claim 1, it is characterized in that described negative controls is the total antibody negative controls product of microspironema pallidum, formulated by the healthy population serum of non-syphilis.
4. the preparation method of IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit as claimed in claim 1, is characterized in that described positive reference substance is the total antibody positive reference substance of microspironema pallidum, formulated by the positive serum of syphilitic.
5. the preparation method of IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit as claimed in claim 1, is characterized in that described reaction terminating liquid is the sulfuric acid of volumetric molar concentration 2mmol/L.
6. the preparation method of IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit as claimed in claim 1, it is characterized in that in step 4) in, described recombinant antigen is microspironema pallidum pathogenic strain Nichol Expression of Plant Height, and syphilis non-pathogenic strain Reiter strain is without expression.
7. the preparation method of IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit as claimed in claim 6, is characterized in that described microspironema pallidum pathogenic strain Nichol strain is Treponemapallidumssp.pallidumstrainNichol; Described syphilis non-pathogenic strain Reiter strain is Treponemapallidumssp.pallidumstrainReiter.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410410697.XA CN104155451B (en) | 2014-08-20 | 2014-08-20 | IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410410697.XA CN104155451B (en) | 2014-08-20 | 2014-08-20 | IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104155451A CN104155451A (en) | 2014-11-19 |
| CN104155451B true CN104155451B (en) | 2016-01-06 |
Family
ID=51881004
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410410697.XA Active CN104155451B (en) | 2014-08-20 | 2014-08-20 | IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104155451B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002024752A2 (en) * | 2000-09-01 | 2002-03-28 | Centro De Investigacion Y De Estudios Avanzados Del Instituto Politecnico Nacional | Monoclonal antibodies against lipid structures different from the double layer of cell membranes for defining physiological states in different cell types and detecting anti lipid particle antibodies in human beings and/or animals suffering from certain antiphospholipid antibody-related diseases |
| CN201965132U (en) * | 2011-01-25 | 2011-09-07 | 厦门大学附属中山医院 | Syphilis-specific IgM antibody immune-blotting kit |
| CN103163298A (en) * | 2011-12-19 | 2013-06-19 | 天津中新科炬生物制药有限公司 | Rapid detective reagent strip of treponema pallidum immunoglobulin m (IgM) antibody and preparation method thereof |
-
2014
- 2014-08-20 CN CN201410410697.XA patent/CN104155451B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002024752A2 (en) * | 2000-09-01 | 2002-03-28 | Centro De Investigacion Y De Estudios Avanzados Del Instituto Politecnico Nacional | Monoclonal antibodies against lipid structures different from the double layer of cell membranes for defining physiological states in different cell types and detecting anti lipid particle antibodies in human beings and/or animals suffering from certain antiphospholipid antibody-related diseases |
| CN201965132U (en) * | 2011-01-25 | 2011-09-07 | 厦门大学附属中山医院 | Syphilis-specific IgM antibody immune-blotting kit |
| CN103163298A (en) * | 2011-12-19 | 2013-06-19 | 天津中新科炬生物制药有限公司 | Rapid detective reagent strip of treponema pallidum immunoglobulin m (IgM) antibody and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| Usefulness of IgM-specific enzyme immunoassays for serodiagnosis of syphilis：Comparative evaluation of three different assays;PhilippP.Bosshard;《Journal of Inferction》;20130328;第67卷;第35-42页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104155451A (en) | 2014-11-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104155450B (en) | Microspironema pallidum IgG antibody biotin-labeled pentylamine enzyme-linked immunologic detecting kit and preparation method thereof | |
| CN104237525B (en) | A kind of latex enhancing immune for measuring Procalcitonin is than turbid kit and its preparation method and application | |
| CN106459198A (en) | Method for the serological diagnosis of rheumatoid arthritis | |
| DK2569632T3 (en) | Yeast cell wall components and detection thereof | |
| CN104459159A (en) | Kit for detecting relevant autoantibody spectrum of autoimmune liver disease | |
| AU2011252840A1 (en) | Yeast cell wall components and detection thereof | |
| CN107271674B (en) | A kind of target marker GP73 and detection application method for steatohepatitis detection | |
| Major et al. | Detection of antibodies to HCV E1E2 by lectin-capture ELISA | |
| CN101315379A (en) | Reagent kit for detecting Ractopamine and application thereof | |
| CN104330562B (en) | Treponema pallidum specific antibody high throughput testing test kit and preparation method thereof | |
| CN104155451B (en) | IgM antibody to treponema pallidum biotin-labeled pentylamine enzyme-linked immunologic detecting kit and preparation method thereof | |
| CN114720691B (en) | Kit for detecting biomarkers and preparation method and application thereof | |
| CN104155439B (en) | The preparation method of microspironema pallidum total antibody biotin Avidin enzyme-linked immunologic detecting kit | |
| CN102590505A (en) | Reagent kit for detecting immunoglobulin m (IgM) AlphaLISA for resisting enterovirus71 capsid protein1 | |
| WO2011130546A1 (en) | Methods for differentiating between crohn's disease and other inflammatory bowel conditions | |
| CN111273005B (en) | Enzyme linked immunosorbent assay kit and method for detecting toxoplasma gondii IgG antibody | |
| CN113702640A (en) | Indirect ELISA method for clostridium perfringens beta 1 toxin antibody | |
| CN105652006A (en) | Dot-ELISA (dot enzyme-linked immunosorbent assay) detection method for total Treponema pallidum antibody | |
| CN107436350B (en) | A kind of separation method carrying out each ingredient in mixture using ELISA Plate and monoclonal antibody | |
| CN107870239B (en) | Application of nAChR-alpha 1-ECD protein in medical detection | |
| Nielsen et al. | Detection of immunoglobulin G antibodies to cytomegalovirus antigens by antibody capture enzyme-linked immunosorbent assay | |
| CN106841606B (en) | Detect colloidal gold immuno-chromatography test paper strip, the kit and preparation method thereof of PCT | |
| MXNL06000077A (en) | Modified antigen for detecting anti-brucella antibodies and method of use. | |
| Gurung et al. | Development of 316v antibody enzyme-linked immunosorbent assay for detection of paratuberculosis in sheep | |
| CN103614341B (en) | Anti-mannan-binding lectin monoclonal antibodies and kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |