CN103323604B - A kind of tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit and preparation and application thereof - Google Patents

A kind of tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit and preparation and application thereof Download PDF

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CN103323604B
CN103323604B CN201310264724.2A CN201310264724A CN103323604B CN 103323604 B CN103323604 B CN 103323604B CN 201310264724 A CN201310264724 A CN 201310264724A CN 103323604 B CN103323604 B CN 103323604B
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CN103323604A (en
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杨致亭
王好玉
邓旭
刘树生
刘兆军
杨爱香
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Shandong Kanghua Biomedical Technology Co., Ltd
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WEIFANG KANGHUA BIOTECH CO Ltd
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Abstract

The invention discloses a kind of tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit and preparation and application thereof, comprise TB-IgG ELISA Plate, TB-IgG enzyme conjugates solution, substrate A solution, substrate B solution, the critical contrast solution of TB-IgG, TB-IgG positive control solution and stop bath, sample diluent, concentrated cleaning solution.The present invention's application indirect elisa method detects tubercle bacillus IgG antibody, to reduce the interference that false positive or false negative phenomenon are brought clinical diagnosis, and this kit is detected fast and accurately to tubercle bacillus, little with time error, detection sensitivity is high, to checkout equipment require low.

Description

A kind of tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit and preparation and application thereof
Technical field
The present invention relates to Biological Detection technical field, be specifically related to a kind of tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit and preparation and application thereof.
Background technology
Much's bacillus (M.tuberculosis) is referred to as tubercle bacillus (tuberclebacilli, TB).As far back as 1886, German bacteriologist Koch just proved that TB is pathogen lungy.This bacterium can invade each histoorgan of whole body, but the most common with pulmonary infection.Along with the development of antituberculotic and the improvement of health weather, the M & M of tuberculosis once once declined to a great extent.After the eighties in 20th century, due to the appearance of acquired immune deficiency syndrome (AIDS) and TB antibody-resistant bacterium, the application of immunodepressant, drug abuse, the factor such as poverty and movement of population, in global range, epidemic situation lungy worsens suddenly.According to WHO statistics, just have 1 people to infect TB in about every 3 people in the whole world, in some developing country adult, TB carrying rate is up to 80%, and wherein about 5% ~ 10% carrier can develop into active tuberculosis.Recent two decades is popular due to acquired immune deficiency syndrome (AIDS), has infected the TB carrier of HIV, and the immunologic function of body due to viral subversive, the possibility developing into active tuberculosis is higher than non-infected by HIV person 30 ~ 50 times, and the disease of tuberculosis is faster.In addition, in the development process of HIV, tuberculosis is a kind of opportunistic infections occurred the earliest, and tuberculosis has increased the weight of the Disease Spectrum of HIV person or aids patient, makes it more easily dead.About there are 800 ten thousand tuberculosis new cases in the current whole world, and causes about 3 million peoples dead every year.China dies from people lungy about 250,000 more than every year, is that the twice of all kinds of Death of Infectious Diseases number summation is many.Therefore, tuberculosis becomes again the global hygienic issues threatening human health, and becomes some developing countries and regions, the particularly primary cause of the death of High prevalence areas of AIDS crowd.
The tuberculosis epidemic situation of China is quite serious, and early stage, quick diagnosis lungy is for early stage chemotherapy and control to propagate and have extremely important meaning.Although tuberculosis bacteriology checking is diagnosis of tuberculosis " goldstandard ", but smear-positive rate low, cultivate length consuming time, can not meet clinical needs, especially the diagnosis of the cloudy pulmonary tuberculosis of bacterium, the outer tuberculosis of lung and childhood tuberculosis is very difficult, and immunologic test Specimen origin is convenient, easy, quick, sensitive, special, become the important auxiliary examination methods of tuberculosis at present.
Summary of the invention
Technical matters to be solved by this invention is: provide a kind of tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit and preparation and application thereof, make it to have the following advantages: application indirect elisa method detects tubercle bacillus IgG antibody, to reduce the interference that false positive or false negative phenomenon are brought clinical diagnosis, and this kit is detected fast and accurately to tubercle bacillus, little with time error, detection sensitivity is high, to checkout equipment require low.
For solving the problems of the technologies described above, technical scheme of the present invention is:
A kind of tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit: comprise TB-IgG ELISA Plate, TB-IgG enzyme conjugates solution, substrate A solution, substrate B solution, the critical contrast solution of TB-IgG, TB-IgG positive control solution and stop buffer, sample diluent, concentrated cleaning solution;
Described TB-IgG ELISA Plate is provided with the transparent reaction hole that at least 5 are coated with solid phase t B antigen;
Described TB-IgG enzyme conjugates solution is the labeled anti-human IgG antibodies of horseradish;
Containing trisodium citrate 4.16g ~ 6.24g, citric acid 7.76g ~ 11.64g, sodium acetate 7.13g ~ 10.70g, glacial acetic acid 1.52ml ~ 2.28ml, hydrogen peroxide 0.8ml ~ 1.2ml in substrate A solution described in every 1000ml;
Substrate B solution described in every 1000ml has included absolute ethyl alcohol 520ml ~ 780ml, ethylene glycol 272ml ~ 408ml, dimethyl formamide 8ml ~ 12ml, 3, and 3 ', 5,5 '-tetramethyl benzidine 0.8g ~ 1.2g;
Containing sodium chloride 6g ~ 8g, Na in the critical contrast solution of TB-IgG described in every 1000ml 2hPO 412H 2o2.2g ~ 3.4g, KH 2pO 40.16g ~ 0.24g, potassium chloride 0.16g ~ 0.24g, sucrose 40g ~ 60g, sodium azide 0.08g ~ 0.12g, sheep anti-mouse igg 100 μ l ~ 400 μ l;
Containing sodium chloride 6g ~ 8g, Na in TB-IgG positive control solution described in every 1000ml 2hPO 412H 2o2.2g ~ 3.4g, KH 2pO 40.16g ~ 0.24g, potassium chloride 0.16g ~ 0.24g, sucrose 40g ~ 60g, sodium azide 0.08g ~ 0.12g, sheep anti-mouse igg 1ml ~ 2ml;
Described stop buffer is configured by 900ml pure water and 80ml ~ 120ml sulfuric acid and forms;
Described sample diluent is be the phosphate buffered solution of the 0.01M of 7.0 containing Deporteinnized calf serum and thimerosal, pH, and wherein said Deporteinnized calf serum content is 200ml, and thiomersal is 0.4 ~ 0.6g;
The phosphate buffer of described concentrated cleaning solution to be pH=7.5 concentration be 0.1mol/L, containing the Tween-20 of volume fraction 0.05% and the thimerosal of 0.1wt% in described phosphate buffer.
Preferably, described TB antigen is gene recombinant antigens.
Preferably, described anti-human IgG antibodies is mouse-anti human IgG antibody.
The preparation method of described tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit, is characterized in that:
(1) the bag quilt of TB-IgG ELISA Plate:
1.1 preparation TB-IgG coating buffers: TB antigenic solution is diluted to the solution that concentration is 0.8 μ g/ml, pH=9.6 with sodium carbonate-bicarbonate buffer solution;
1.2 bag quilts: the TB-IgG coating buffer obtained by step 1.1 carries out bag quilt by 100 μ l/ holes to described transparent reaction hole, and 24 ~ 30 hours are left standstill at 2 ~ 8 DEG C of temperature;
1.3 cleansing solutions preparations: be the Tween-20 of 0.05% by volume content, the phosphate buffered solution of pH=7.5 and distilled water by volume 1:40 mix, obtain cleansing solution;
1.4 washings: with cleansing solution continuous washing bag by after ELISA Plate at least 1 time, pat dry;
1.5 close: in the transparent reaction hole after washing, add confining liquid by 130 μ l/ holes, and described confining liquid is containing NaHCO in every 1000ml pure water solution 32.93g, Na 2cO 31.59g, bovine serum albumin(BSA) 10g, and under 20 ~ 25 DEG C of conditions, close 2.8 ~ 3.2 hours;
1.6 throw away the confining liquid in hole, and dry 40h ~ 48h completes bag quilt;
(2) preparation of TB-IgG enzyme conjugates solution:
3 ~ 8mg horseradish peroxidase is dissolved in 1ml distilled water by 2.1, then adds the sodium periodate solution 200 μ l of 21 ~ 22mg/ml, mixes and prepare horseradish peroxidase solution under room temperature 18 DEG C ~ 25 DEG C conditions;
2.2 acetic acid-glacial acetic acid the damping fluids again horseradish peroxidase solution being added pH=4.4, dialyse 16 ~ 20 hours under 2 ~ 6 DEG C of conditions;
2.3 get the sodium carbonate-bicarbonate buffer solution that anti-human-IgG adds pH=9.6, carbonate content is 0.05mol/L, dialyse 16 ~ 20 hours under 2 ~ 6 DEG C of conditions;
2.4 by the horseradish peroxidase solution after dialysis and the mixing of anti-human-IgG solution, puts into pH=9.6, carbonate buffer solution that carbonate content is 0.05mol/L stirs dialysis 2.5 ~ 3.5 hours, then add the sodium borohydride solution of 4mg/ml;
2.5 mixed solutions that step 2.4 is obtained, by sephadex G-200 post, use the phosphate buffered solution wash-out of pH=7.1 simultaneously;
2.6, with small test tube successively subsection receiing effluent, being detected by good sheep anti mouse lath with wrapping in advance, collecting the in vitro product that invisible spectro sheep anti mouse lath shows blue look, the anti-human-IgG of obtained horseradish peroxidase-labeled;
Anti-human-the IgG of the horseradish peroxidase-labeled of collection is added the mixing of equivalent glycerine by 2.7, obtained enzyme conjugates just solution;
Sodium chloride 6g ~ 8g, Deporteinnized calf serum 140ml ~ 160ml, Sodium azide 0.8g ~ 1.2g are dissolved in pure water, and are settled to 1000ml by 2.8, obtained enzyme dilution;
The first solution of described enzyme conjugates and described enzyme dilution are pressed 1:6000 ~ 1:4000 by 2.9 to be diluted, obtained TB-IgG enzyme conjugates solution;
(3) preparation of substrate A solution
First trisodium citrate 4.16g ~ 6.24g, citric acid 7.76g ~ 11.64g and sodium acetate 7.13g ~ 10.70g pure water are dissolved completely, add the glacial acetic acid of 1.52ml ~ 2.28ml and the hydrogen peroxide of 0.8ml ~ 1.2ml again, finally be settled to 1000ml with pure water, obtained substrate A solution;
(4) preparation of substrate B solution
By 0.8g ~ 1.2g3,3 ', 5,5 '-tetramethyl benzidine is dissolved in 8ml ~ 12ml dimethyl formamide, then adds 520ml ~ 780ml absolute ethyl alcohol and 272ml ~ 408ml ethylene glycol, mixing, obtained substrate B solution;
(5) preparation of stop buffer
80 ~ 120ml sulfuric acid is added, mixing, obtained stop buffer in 900ml pure water;
(6) preparation of the critical contrast solution of TB-IgG
Sodium chloride 6g ~ 8g, Na is dissolved with 1000ml pure water 2hPO 412H 2o2.2g ~ 3.4g, KH 2pO 40.16g ~ 0.24g, potassium chloride 0.16g ~ 0.24g, sucrose 40g ~ 60g, sodium azide 0.08g ~ 0.12g, sheep anti-mouse igg 100 μ l ~ 400 μ l, the obtained critical contrast solution of TB-IgG;
(7) preparation of TB-IgG positive control solution
Sodium chloride 6g ~ 8g, Na is dissolved with 1000ml pure water 2hPO 412H 2o2.2g ~ 3.4g, KH 2pO 40.16g ~ 0.24g, potassium chloride 0.16g ~ 0.24g, sucrose 40g ~ 60g, sodium azide 0.08g ~ 0.12g, sheep anti-mouse igg 1ml ~ 2ml, obtained TB-IgG positive control solution;
(8) preparation of sample diluent
Get NaCl6g ~ 8g, Na 2hPO 412H 2o2.5g ~ 3.0g, KH 2pO 40.1g ~ 0.3g, KCl0.1g ~ 0.3g add volumetric flask, then add appropriate pure water, make it to dissolve completely, get thimerosal 0.4g ~ 0.6g, make it to dissolve completely, then get Deporteinnized calf serum 200ml and add volumetric flask, stir, be settled to 1000ml with pure water, obtained sample diluent.
(9) preparation of concentrated cleaning solution
Get NaCl149.0g ~ 151.9g, Na 2hPO 4.12H 2o92g ~ 98g, NaH 2pO 4.2H 2o28g ~ 22g adds volumetric flask; First add appropriate pure water, make it to dissolve completely; Add thimerosal 0.8g ~ 1.2g again, make it to dissolve completely; Measure 4ml ~ 6ml Tween-20 and add volumetric flask, stir, be settled to 1000ml with pure water, obtained concentrated cleaning solution.
Preferably, described enzyme conjugates is preserved under-22 ~-19 DEG C of conditions.
Preferably, described anti-human IgG antibodies is mouse-anti human IgG antibody.
Preferably, described TB antigen is gene recombinant antigens.
The using method of described tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit, is characterized in that, comprise the following steps:
(1) sample collection
Leave standstill test serum 30min, centrifugal treating is carried out 10 ~ 20 minutes to described test serum, can detect.
(2) application of sample detects
A. described TB-IgG enzyme conjugates solution bottle, substrate A solution bottle, substrate B solution bottle, the critical contrast solution bottle of TB-IgG, TB-IgG positive control solution bottle, sample diluent, concentrated cleaning solution and stop buffer solution bottle are balanced to room temperature 20 DEG C ~ 25 DEG C;
B. get in TB-IgG positive control solution joins in described ELISA Plate transparent reaction hole described at least one by 50 μ l/ holes, as Positive control wells; Get at least two described transparent reaction holes that TB-IgG critical contrast liquid joins by 50 μ l/ holes in described ELISA Plate, as critical control wells;
C. get in sample diluent joins in described ELISA Plate transparent reaction hole described at least one by 100 μ l/ holes, get serum 10 μ l/ hole to be checked and add in described transparent reaction hole, as sample well;
D. described TB-IgG ELISA Plate is sticked shrouding film, concussion mixing, is placed in 36 DEG C ~ 38 DEG C waters bath with thermostatic control and reacts 8min ~ 10min;
E. take out described TB-IgG ELISA Plate, take shrouding film off, wash 5 times with application cleansing solution, during washing, every hole fills cleansing solution, and each stop 20 seconds, pats dry;
F. in sample well, Positive control wells and critical control wells, respectively add 50 μ l enzyme conjugates solution, mixing, sticks shrouding film, is placed in 36 DEG C ~ 38 DEG C waters bath with thermostatic control and reacts 8min ~ 10min;
G. take out described TB-IgG ELISA Plate, take shrouding film off, with cleansing solution washing at least 5 times, during washing, every hole fills cleansing solution, and each stop 20 seconds, pats dry;
H. in sample well, Positive control wells and critical control wells, respectively add 50 μ l substrate solution A, 30 μ l substrate solution B, mixing, sticks shrouding film, reacts 8min ~ 10min in 36 DEG C ~ 38 DEG C waters bath with thermostatic control;
I. take shrouding film off, observations: detect the yin and yang attribute of sample if only judge, adopt visual method, the color of comparative sample hole and Positive control wells, critical control wells, can draw testing result respectively; If need read and detect numerical value accurately, after step h, in sample well, add 50 μ l stop buffers again, after mixing, use microplate reader to read result.
Preferably, if described test serum can not detect in time, need be frozen-20 DEG C ~-15 DEG C conditions.
After adopting technique scheme, the invention has the beneficial effects as follows: because the present invention adopts indirect elisa method to detect TB-IgG antibody, if containing TB-IgG antibody in test serum, the TB antigen then first wrapping quilt in lath is combined, form antigen-antibody complex, again with two anti-formation Ag-Ab-antiantibody compounds of enzyme labeling, and be combined in micro-pore wall surface, developed the color by substrate.In order to detect the TB antibody in human serum sample specifically.The present invention is highly sensitive, high specificity, experimental implementation is simple, quick, the present invention not only decreases the interference that false positive or false negative phenomenon are brought clinical diagnosis, this kit is detected fast and accurately to TB antibody, and error is little, detection sensitivity is high, require low to checkout equipment; Also owing to present invention employs the formula of described ELISA Plate and various solution, make the order of accuarcy of the testing result of kit of the present invention in the market other TB antibody reagent do not reached.
Through clinical verification, enzyme linked immunosorbent assay of the present invention (ELISA) detects lunger's serum Killing Mycobacterium Tuberculosis antibody, and its susceptibility is 99.6%, and specificity reaches 99.5%, and accuracy is 99.6%, has higher experimental specificity.Result shows that this experimental technique has reached (30min), responsive, special fast, the requirement of experiment of easy (also can estimate judgment experiment result).Be particularly suitable for basic hospital and tuberculosis control unit as routine clinical application.
Specific embodiment
Embodiment 1
A kind of tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit
Comprise TB-IgG ELISA Plate, TB-IgG enzyme conjugates solution, substrate A solution, substrate B solution, the critical contrast solution of TB-IgG, TB-IgG positive control solution and stop buffer, sample diluent, concentrated cleaning solution;
TB-IgG ELISA Plate is provided with the transparent reaction hole that 48 are coated with solid phase t 1 B gene recombinant antigen;
TB-IgG enzyme conjugates solution is the labeled mouse-anti human IgG antibody of horseradish;
Containing trisodium citrate 4.16g, citric acid 7.76g, sodium acetate 7.13g, glacial acetic acid 1.52ml, hydrogen peroxide 0.8ml in every 1000ml substrate A solution;
Every 1000ml substrate B solution has included absolute ethyl alcohol 520ml, ethylene glycol 272ml, dimethyl formamide 8ml, 3,3 ', 5,5 '-tetramethyl benzidine 0.8g;
Containing sodium chloride 6g, Na in the critical contrast solution of every 1000mlTB-IgG 2hPO 412H 2o2.2g, KH 2pO 40.16g, potassium chloride 0.16g, sucrose 40g, sodium azide 0.08g, sheep anti-mouse igg 100 μ l;
Containing sodium chloride 6g, Na in every 1000mlTB-IgG positive control solution 2hPO 412H 2o2.2g, KH 2pO 40.16g, potassium chloride 0.16g, sucrose 40g, sodium azide 0.08g, sheep anti-mouse igg 1ml;
Stop buffer is configured by 900ml pure water and 80ml sulfuric acid and forms;
Sample diluent is be the phosphate buffered solution of the 0.01M of 7.0 containing Deporteinnized calf serum and thimerosal, pH, and wherein Deporteinnized calf serum content is 200ml, and thiomersal is 0.4g;
The phosphate buffer of concentrated cleaning solution to be pH=7.5 concentration be 0.1mol/L, containing the Tween-20 of volume fraction 0.05% and the thimerosal of 0.1wt% in phosphate buffer.
Embodiment 2
A kind of tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit
Comprise TB-IgG ELISA Plate, TB-IgG enzyme conjugates solution, substrate A solution, substrate B solution, the critical contrast solution of TB-IgG, TB-IgG positive control solution and stop buffer, sample diluent, concentrated cleaning solution;
TB-IgG ELISA Plate is provided with the transparent reaction hole that 48 are coated with solid phase t 1 B gene recombinant antigen;
TB-IgG enzyme conjugates solution is the labeled mouse-anti human IgG antibody of horseradish;
Containing trisodium citrate 6.24g, citric acid 11.64g, sodium acetate 10.70g, glacial acetic acid 2.28ml, hydrogen peroxide 1.2ml in every 1000ml substrate A solution;
Every 1000ml substrate B solution has included absolute ethyl alcohol 780ml, ethylene glycol 408ml, dimethyl formamide 12ml, 3,3 ', 5,5 '-tetramethyl benzidine 1.2g;
Containing sodium chloride 8g, Na in the critical contrast solution of every 1000mlTB-IgG 2hPO 412H 2o3.4g, KH 2pO 40.24g, potassium chloride 0.24g, sucrose 60g, sodium azide 0.12g, sheep anti-mouse igg 400 μ l;
Containing sodium chloride 8g, Na in every 1000mlTB-IgG positive control solution 2hPO 412H 2o3.4g, KH 2pO 40.24g, potassium chloride 0.24g, sucrose 60g, sodium azide 0.12g, sheep anti-mouse igg 2ml;
Stop buffer is configured by 900ml pure water and 120ml sulfuric acid and forms;
Sample diluent is be the phosphate buffered solution of the 0.01M of 7.0 containing Deporteinnized calf serum and thimerosal, pH, and wherein Deporteinnized calf serum content is 200ml, and thiomersal is 0.6g;
The phosphate buffer of concentrated cleaning solution to be pH=7.5 concentration be 0.1mol/L, containing the Tween-20 of volume fraction 0.05% and the thimerosal of 0.1wt% in phosphate buffer.
Embodiment 3
A kind of tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit
Comprise TB-IgG ELISA Plate, TB-IgG enzyme conjugates solution, substrate A solution, substrate B solution, the critical contrast solution of TB-IgG, TB-IgG positive control solution and stop buffer, sample diluent, concentrated cleaning solution;
TB-IgG ELISA Plate is provided with the transparent reaction hole that 48 are coated with solid phase t 1 B gene recombinant antigen;
TB-IgG enzyme conjugates solution is the labeled mouse-anti human IgG antibody of horseradish;
Containing trisodium citrate 5.2g, citric acid 9.7g, sodium acetate 8.92g, glacial acetic acid 1.9ml, hydrogen peroxide 1ml in every 1000ml substrate A solution;
Every 1000ml substrate B solution has included absolute ethyl alcohol 650ml, ethylene glycol 340ml, dimethyl formamide 10ml, 3,3 ', 5,5 '-tetramethyl benzidine 1g;
Containing sodium chloride 7g, Na in the critical contrast solution of every 1000mlTB-IgG 2hPO 412H 2o2.8g, KH 2pO 40.2g, potassium chloride 0.2g, sucrose 50g, sodium azide 0.1g, sheep anti-mouse igg 100 μ l;
Containing sodium chloride 7g, Na in every 1000mlTB-IgG positive control solution 2hPO 412H 2o2.8g, KH 2pO 40.2g, potassium chloride 0.2g, sucrose 50g, sodium azide 0.10g, sheep anti-mouse igg 1ml;
Stop buffer is configured by 900ml pure water and 100ml sulfuric acid and forms;
Sample diluent is be the phosphate buffered solution of the 0.01M of 7.0 containing Deporteinnized calf serum and thimerosal, pH, and wherein Deporteinnized calf serum content is 200ml, and thiomersal is 0.5g;
The phosphate buffer of concentrated cleaning solution to be pH=7.5 concentration be 0.1mol/L, containing the Tween-20 of volume fraction 0.05% and the thimerosal of 0.1wt% in phosphate buffer.
Embodiment 4
Embodiment 3 tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit preparation method:
(1) the bag quilt of TB-IgG ELISA Plate:
1.1 preparation TB-IgG coating buffers: TB antigenic solution is diluted to the solution that concentration is 0.8 μ g/ml, pH=9.6 with sodium carbonate-bicarbonate buffer solution;
1.2 bag quilts: the TB-IgG coating buffer obtained by step 1.1 carries out bag quilt by 100 μ l/ holes, and 24 ~ 30 hours are left standstill at 4 DEG C of temperature;
1.3 cleansing solutions preparations: be the Tween-20 of 0.05% by volume content, the phosphate buffered solution of pH=7.5 and distilled water by volume 1:40 mix, obtain cleansing solution;
1.4 washings: with cleansing solution continuous washing bag by after ELISA Plate at least 1 time, pat dry;
1.5 close: in the transparent reaction hole after washing, add confining liquid by 130 μ l/ holes, and confining liquid is containing NaHCO in every 1000ml pure water solution 32.93g, Na 2cO 31.59g, bovine serum albumin(BSA) 10g, and under 22 DEG C of conditions, close 3 hours;
1.6 throw away the confining liquid in hole, and dry 40h ~ 48h completes bag quilt;
(2) preparation of TB-IgG enzyme conjugates solution:
5mg horseradish peroxidase is dissolved in 1ml distilled water by 2.1, then adds the sodium periodate solution 200 μ l of 21.4mg/ml, mixes and prepare horseradish peroxidase solution under room temperature 18 DEG C ~ 25 DEG C conditions;
2.2 acetic acid-glacial acetic acid the damping fluids again horseradish peroxidase solution being added pH=4.4, dialyse 16 ~ 20 hours under 2 ~ 6 DEG C of conditions;
2.3 get the sodium carbonate-bicarbonate buffer solution that anti-human-IgG adds pH=9.6, carbonate content is 0.05mol/L, dialyse 16 ~ 20 hours under 2 ~ 6 DEG C of conditions;
2.4 by the horseradish peroxidase solution after dialysis and the mixing of anti-human-IgG solution, puts into pH=9.6, carbonate buffer solution that carbonate content is 0.05mol/L stirs dialysis 2.5 ~ 3.5 hours, then add the sodium borohydride solution of 4mg/ml;
2.5 mixed solutions that step 2.4 is obtained, by sephadex G-200 post, use the phosphate buffered solution wash-out of pH=7.1 simultaneously;
2.6, with small test tube successively subsection receiing effluent, being detected by good sheep anti mouse lath with wrapping in advance, collecting the in vitro product that invisible spectro sheep anti mouse lath shows blue look, the anti-human-IgG of obtained horseradish peroxidase-labeled;
Anti-human-the IgG of the horseradish peroxidase-labeled of collection is added the mixing of equivalent glycerine by 2.7, and obtained enzyme conjugates just solution, is positioned over-20 DEG C of Refrigerator stores;
Sodium chloride 7g, Deporteinnized calf serum 150ml, Sodium azide 1.0g are dissolved in pure water, and are settled to 1000ml by 2.8, obtained enzyme dilution;
First for enzyme conjugates solution and enzyme dilution are pressed 1:5000 by 2.9 to be diluted, obtained TB-IgG enzyme conjugates solution;
(3) preparation of substrate A solution
First trisodium citrate 5.2g, citric acid 9.7g and sodium acetate 8.92g pure water are dissolved completely, then add the glacial acetic acid of 1.9ml and the hydrogen peroxide of 1ml, be finally settled to 1000ml with pure water, obtained substrate A solution;
(4) preparation of substrate B solution
By 1.0g3,3 ', 5,5 '-tetramethyl benzidine is dissolved in 10ml dimethyl formamide, then adds 650ml absolute ethyl alcohol and 340ml ethylene glycol, mixing, obtained substrate B solution;
(5) preparation of stop buffer
100ml sulfuric acid is added, mixing, obtained stop buffer in 900ml pure water;
(6) preparation of the critical contrast solution of TB-IgG
Sodium chloride 7g, Na is dissolved with 1000ml pure water 2hPO 412H 2o2.8g, KH 2pO 40.2g, potassium chloride 0.2g, sucrose 50g, sodium azide 0.1g, sheep anti-mouse igg 100 μ l, the obtained critical contrast solution of TB-IgG;
(7) preparation of TB-IgG positive control solution
Sodium chloride 7g, Na is dissolved with 1000ml pure water 2hPO 412H 2o2.8g, KH 2pO 40.2g, potassium chloride 0.2g, sucrose 50g, sodium azide 0.1g, sheep anti-mouse igg 1ml, obtained TB-IgG positive control solution;
(8) preparation of sample diluent
Get NaCl7g, Na 2hPO 412H 2o2.8g, KH 2pO 40.2g, KCl0.2g add volumetric flask, then add appropriate pure water, make it to dissolve completely, get thimerosal 0.5g, make it to dissolve completely, then get Deporteinnized calf serum 200ml and add volumetric flask, stir, are settled to 1000ml with pure water, obtained sample diluent.
(9) preparation of concentrated cleaning solution
Get NaCl150g, Na 2hPO 4.12H 2o95g, NaH 2pO 4.2H 2o26g adds volumetric flask; First add appropriate pure water, make it to dissolve completely; Add thimerosal 1.0g again, make it to dissolve completely; Measure 5ml Tween-20 and add volumetric flask, stir, be settled to 1000ml with pure water, obtained concentrated cleaning solution.
Embodiment 5
Tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit preparation method
(1) the bag quilt of TB-IgG ELISA Plate:
1.1 preparation TB-IgG coating buffers: TB antigenic solution is diluted to the solution that concentration is 0.8 μ g/ml, pH=9.6 with sodium carbonate-bicarbonate buffer solution;
1.2 bag quilts: the TB-IgG coating buffer obtained by step 1.1 carries out bag quilt by 100 μ l/ holes, and 24 hours are left standstill at 2 DEG C of temperature;
1.3 cleansing solutions preparations: be the Tween-20 of 0.05% by volume content, the phosphate buffered solution of pH=7.5 and distilled water by volume 1:40 mix, obtain cleansing solution;
1.4 washings: with cleansing solution continuous washing bag by after ELISA Plate at least 1 time, pat dry;
1.5 close: in the transparent reaction hole after washing, add confining liquid by 130 μ l/ holes, and confining liquid is containing NaHCO in every 1000ml pure water solution 32.93g, Na 2cO 31.59g, bovine serum albumin(BSA) 10g, and under 20 DEG C of conditions, close 2.8 hours;
1.6 throw away the confining liquid in hole, and dry 40h completes bag quilt;
(2) preparation of TB-IgG enzyme conjugates solution:
3 ~ 8mg horseradish peroxidase is dissolved in 1ml distilled water by 2.1, then adds the sodium periodate solution 200 μ l of 21 ~ 22mg/ml, mixes and prepare horseradish peroxidase solution under room temperature 18 DEG C of conditions;
2.2 acetic acid-glacial acetic acid the damping fluids again horseradish peroxidase solution being added pH=4.4, dialyse 16 hours under 2 ~ 6 DEG C of conditions;
2.3 get the sodium carbonate-bicarbonate buffer solution that anti-human-IgG adds pH=9.6, carbonate content is 0.05mol/L, dialyse 16 hours under 2 DEG C of conditions;
2.4 by the horseradish peroxidase solution after dialysis and the mixing of anti-human-IgG solution, puts into pH=9.6, carbonate buffer solution that carbonate content is 0.05mol/L stirs dialysis 2.5 hours, then add the sodium borohydride solution of 4mg/ml;
2.5 mixed solutions that step 2.4 is obtained, by sephadex G-200 post, use the phosphate buffered solution wash-out of pH=7.1 simultaneously;
2.6, with small test tube successively subsection receiing effluent, being detected by good sheep anti mouse lath with wrapping in advance, collecting the in vitro product that invisible spectro sheep anti mouse lath shows blue look, the anti-human-IgG of obtained horseradish peroxidase-labeled;
Anti-human-the IgG of the horseradish peroxidase-labeled of collection is added the mixing of equivalent glycerine by 2.7, obtained enzyme conjugates just solution;
Sodium chloride 6g, Deporteinnized calf serum 140ml, Sodium azide 0.8g are dissolved in pure water, and are settled to 1000ml by 2.8, obtained enzyme dilution;
First for enzyme conjugates solution and enzyme dilution are pressed 1:6000 by 2.9 to be diluted, obtained TB-IgG enzyme conjugates solution;
(3) preparation of substrate A solution
First trisodium citrate 4.16g, citric acid 7.76g and sodium acetate 7.13g pure water are dissolved completely, then add the glacial acetic acid of 1.52ml and the hydrogen peroxide of 0.8ml, be finally settled to 1000ml with pure water, obtained substrate A solution;
(4) preparation of substrate B solution
By 0.95g ~ 1.05g3,3 ', 5,5 '-tetramethyl benzidine is dissolved in 10ml dimethyl formamide, then adds 650ml absolute ethyl alcohol and 340ml ethylene glycol, mixing, obtained substrate B solution;
(5) preparation of stop buffer
100ml sulfuric acid is added, mixing, obtained stop buffer in 900ml pure water;
(6) preparation of the critical contrast solution of TB-IgG
Sodium chloride 6g, Na is dissolved with 1000ml pure water 2hPO 412H 2o2.2g, KH 2pO 40.16g, potassium chloride 0.16g, sucrose 40g, sodium azide 0.08g, sheep anti-mouse igg 100 μ l, the obtained critical contrast solution of TB-IgG;
(7) preparation of TB-IgG positive control solution
Sodium chloride 6g, Na is dissolved with 1000ml pure water 2hPO 412H 2o2.2g, KH 2pO 40.16g, potassium chloride 0.16g, sucrose 40g, sodium azide 0.08g, sheep anti-mouse igg 1ml, obtained TB-IgG positive control solution;
(8) preparation of sample diluent
Get NaCl6g, Na 2hPO 412H 2o2.5g, KH 2pO 40.1g, KCl0.1g add volumetric flask, then add appropriate pure water, make it to dissolve completely, get thimerosal 0.4g, make it to dissolve completely, then get Deporteinnized calf serum 200ml and add volumetric flask, stir, are settled to 1000ml with pure water, obtained sample diluent.
(9) preparation of concentrated cleaning solution
Get NaCl149.0g, Na 2hPO 4.12H 2o92g, NaH 2pO 4.2H 2o28g adds volumetric flask; First add appropriate pure water, make it to dissolve completely; Add thimerosal 0.8g again, make it to dissolve completely; Measure 4ml Tween-20 and add volumetric flask, stir, be settled to 1000ml with pure water, obtained concentrated cleaning solution.
Embodiment 6
Tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit preparation method
(1) the bag quilt of TB-IgG ELISA Plate:
1.1 preparation TB-IgG coating buffers: TB antigenic solution is diluted to the solution that concentration is 0.8 μ g/ml, pH=9.6 with sodium carbonate-bicarbonate buffer solution;
1.2 bag quilts: the TB-IgG coating buffer obtained by step 1.1 carries out bag quilt by 100 μ l/ holes, and 30 hours are left standstill at 8 DEG C of temperature;
1.3 cleansing solutions preparations: be the Tween-20 of 0.05% by volume content, the phosphate buffered solution of pH=7.5 and distilled water by volume 1:40 mix, obtain cleansing solution;
1.4 washings: with cleansing solution continuous washing bag by after ELISA Plate at least 1 time, pat dry;
1.5 close: in the transparent reaction hole after washing, add confining liquid by 130 μ l/ holes, and confining liquid is containing NaHCO in every 1000ml pure water solution 32.93g, Na 2cO 31.59g, bovine serum albumin(BSA) 10g, and under 25 DEG C of conditions, close 3.2 hours;
1.6 throw away the confining liquid in hole, and dry 48h completes bag quilt;
(2) preparation of TB-IgG enzyme conjugates solution:
3 ~ 8mg horseradish peroxidase is dissolved in 1ml distilled water by 2.1, then adds the sodium periodate solution 200 μ l of 22mg/ml, mixes and prepare horseradish peroxidase solution under room temperature 25 DEG C of conditions;
2.2 acetic acid-glacial acetic acid the damping fluids again horseradish peroxidase solution being added pH=4.4, dialyse 20 hours under 6 DEG C of conditions;
2.3 get the sodium carbonate-bicarbonate buffer solution that anti-human-IgG adds pH=9.6, carbonate content is 0.05mol/L, dialyse 20 hours under 6 DEG C of conditions;
2.4 by the horseradish peroxidase solution after dialysis and the mixing of anti-human-IgG solution, puts into pH=9.6, carbonate buffer solution that carbonate content is 0.05mol/L stirs dialysis 3.5 hours, then add the sodium borohydride solution of 4mg/ml;
2.5 mixed solutions that step 2.4 is obtained, by sephadex G-200 post, use the phosphate buffered solution wash-out of pH=7.1 simultaneously;
2.6, with small test tube successively subsection receiing effluent, being detected by good sheep anti mouse lath with wrapping in advance, collecting the in vitro product that invisible spectro sheep anti mouse lath shows blue look, the anti-human-IgG of obtained horseradish peroxidase-labeled;
Anti-human-the IgG of the horseradish peroxidase-labeled of collection is added the mixing of equivalent glycerine by 2.7, obtained enzyme conjugates just solution;
Sodium chloride 8g, Deporteinnized calf serum 160ml, Sodium azide 1.2g are dissolved in pure water, and are settled to 1000ml by 2.8, obtained enzyme dilution;
First for enzyme conjugates solution and enzyme dilution are pressed 1:4000 by 2.9 to be diluted, obtained TB-IgG enzyme conjugates solution;
(3) preparation of substrate A solution
First trisodium citrate 6.24g, citric acid 11.64g and sodium acetate 10.70g pure water are dissolved completely, then add the glacial acetic acid of 2.28ml and the hydrogen peroxide of 1.2ml, be finally settled to 1000ml with pure water, obtained substrate A solution;
(4) preparation of substrate B solution
By 1.05g3,3 ', 5,5 '-tetramethyl benzidine is dissolved in 10ml dimethyl formamide, then adds 650ml absolute ethyl alcohol and 340ml ethylene glycol, mixing, obtained substrate B solution;
(5) preparation of stop buffer
100ml sulfuric acid is added, mixing, obtained stop buffer in 900ml pure water;
(6) preparation of the critical contrast solution of TB-IgG
Sodium chloride 8g, Na is dissolved with 1000ml pure water 2hPO 412H 2o3.4g, KH 2pO 40.24g, potassium chloride 0.24g, sucrose 60g, sodium azide 0.12g, sheep anti-mouse igg 400 μ l, the obtained critical contrast solution of TB-IgG;
(7) preparation of TB-IgG positive control solution
Sodium chloride 8g, Na is dissolved with 1000ml pure water 2hPO 412H 2o3.4g, KH 2pO 40.24g, potassium chloride 0.24g, sucrose 60g, sodium azide 0.12g, sheep anti-mouse igg 2ml, obtained TB-IgG positive control solution;
(8) preparation of sample diluent
Get NaCl8g, Na 2hPO 412H 2o3.0g, KH 2pO 40.3g, KCl0.3g add volumetric flask, then add appropriate pure water, make it to dissolve completely, get thimerosal 0.6g, make it to dissolve completely, then get Deporteinnized calf serum 200ml and add volumetric flask, stir, are settled to 1000ml with pure water, obtained sample diluent.
(9) preparation of concentrated cleaning solution
Get NaCl151.9g, Na 2hPO 4.12H 2o98g, NaH 2pO 4.2H 2o22g adds volumetric flask; First add appropriate pure water, make it to dissolve completely; Add thimerosal 1.2g again, make it to dissolve completely; Measure 6ml Tween-20 and add volumetric flask, stir, be settled to 1000ml with pure water, obtained concentrated cleaning solution.
Embodiment 7
Tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit using method
The using method of the tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit described in embodiment 3:
(1) sample collection
Leave standstill test serum 30min, centrifugal treating is carried out 10 ~ 20 minutes to described test serum, detects.
(2) application of sample detects
A. described TB-IgG enzyme conjugates solution bottle, substrate A solution bottle, substrate B solution bottle, the critical contrast solution bottle of TB-IgG, TB-IgG positive control solution bottle, concentrated cleaning solution, sample diluent and the stop buffer solution bottle preserved in cold storage environment are balanced to room temperature 20 DEG C ~ 25 DEG C;
B. get at least one transparent reaction hole that TB-IgG positive control solution joins by 50 μ l/ holes in described ELISA Plate, as Positive control wells; Get at least two transparent reaction holes that TB-IgG critical contrast liquid joins by 50 μ l/ holes in described ELISA Plate, as critical control wells;
C. get at least one transparent reaction hole that sample diluent joins by 100 μ l/ holes in described ELISA Plate, get serum 10 μ l/ hole to be checked and add in transparent reaction hole, as sample well;
D. described TB-IgG ELISA Plate is sticked shrouding film, concussion mixing, is placed in 36 DEG C ~ 38 DEG C waters bath with thermostatic control and reacts 8min ~ 10min;
E. take out described TB-IgG ELISA Plate, take shrouding film off, wash 5 times with application cleansing solution, during washing, every hole fills cleansing solution, and each stop 20 seconds, pats dry;
F. in sample well, Positive control wells and critical control wells, respectively add 50 μ l enzyme conjugates solution, mixing, sticks shrouding film, is placed in 36 DEG C ~ 38 DEG C waters bath with thermostatic control and reacts 8min ~ 10min;
G. take out described TB-IgG ELISA Plate, take shrouding film off, wash 5 times with application cleansing solution, during washing, every hole fills cleansing solution, and each stop 20 seconds, pats dry.
H. in sample well, Positive control wells and critical control wells, respectively add 50 μ l substrate solution A successively, 30 μ l substrate solution B, mixing, sticks shrouding film, reacts 8min ~ 10min in 36 DEG C ~ 38 DEG C waters bath with thermostatic control;
I. take shrouding film off, observations: detect the yin and yang attribute of sample if only judge, adopt visual method, the color of comparative sample hole and Positive control wells, critical control wells, can draw testing result respectively; If need read and detect numerical value accurately, after step g, in sample well, add 50 μ l stop buffers again, after mixing, use microplate reader to read result.
(3) Analysis of test results
Under 450nm wavelength, read the absorbance in each hole with blank well zeroing, and by following computing method, draw testing result:
Colourimetry:
If sample well absorbance >=critical value, testing result is positive;
If sample well absorbance < critical value, testing result is negative;
The testing performance index of tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit of the present invention:
(1) negative reference product coincidence rate: detect with 10 parts of these reagent negative reference product, testing result all becomes negative.
(2) positive reference material coincidence rate: with the positive reference materials of 10 parts of these reagent detect (comprise strong, in, weak) detect, testing result all becomes positive.
(3) accuracy: do 10 tests by 1 part of accuracy reference material, CV value is not higher than 10%.
(4) clinical trial results: detect clinical sample 1000 example (wherein positive 50 examples of TB-IgG) made a definite diagnosis with this kit, testing result shows, negative match-rate 100%, positive coincidence rate 100%, total coincidence rate 100%.
Detection example: detect clinical serum sample 1000 example (wherein positive 50 examples of TB-IgG) altogether, use the inventive method to detect, its specificity and susceptibility are 100%.
Table 1 TB-IgG kit of the present invention testing result compares with clinical diagnoses
The present invention is not limited to above-mentioned concrete embodiment, and those of ordinary skill in the art is from above-mentioned design, and without performing creative labour, done all conversion, all drop within protection scope of the present invention.

Claims (4)

1. a preparation method for tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit, is characterized in that:
(1) the bag quilt of TB-IgG ELISA Plate:
1.1 preparation TB-IgG coating buffers: TB antigenic solution is diluted to the solution that concentration is 0.8 μ g/mlpH=9.6 with sodium carbonate-bicarbonate buffer solution;
1.2 bag quilts: the TB-IgG coating buffer obtained by step 1.1 carries out bag quilt by 100 μ l/ holes to transparent reaction hole, and 24 ~ 30 hours are left standstill at 2 ~ 8 DEG C of temperature;
1.3 cleansing solutions preparations: be the Tween-20 of 0.05% by volume content, the phosphate buffered solution of pH=7.5 and distilled water by volume 1:40 mix, obtain cleansing solution;
1.4 washings: with cleansing solution continuous washing bag by after ELISA Plate at least 1 time, pat dry;
1.5 close: in the transparent reaction hole after washing, add confining liquid by 130 μ l/ holes, and described confining liquid is containing NaHCO in every 1000ml pure water solution 32.93g, Na 2cO 31.59g, bovine serum albumin(BSA) 10g, and under 20 ~ 25 DEG C of conditions, close 2.8 ~ 3.2 hours;
1.6 throw away the confining liquid in hole, and dry 40h ~ 48h completes bag quilt;
(2) preparation of TB-IgG enzyme conjugates solution:
3 ~ 8mg horseradish peroxidase is dissolved in 1ml distilled water by 2.1, then adds the sodium periodate solution 200 μ l of 21 ~ 22mg/ml, mixes and prepare horseradish peroxidase solution under room temperature 18 DEG C ~ 25 DEG C conditions;
2.2 acetic acid-glacial acetic acid the damping fluids again horseradish peroxidase solution being added pH=4.4, dialyse 16 ~ 20 hours under 2 ~ 6 DEG C of conditions;
2.3 get the sodium carbonate-bicarbonate buffer solution that anti-human-IgG adds pH=9.6, carbonate content is 0.05mol/L, dialyse 16 ~ 20 hours under 2 ~ 6 DEG C of conditions;
2.4 by the horseradish peroxidase solution after dialysis and the mixing of anti-human-IgG solution, puts into pH=9.6, carbonate buffer solution that carbonate content is 0.05mol/L stirs dialysis 2.5 ~ 3.5 hours, then add the sodium borohydride solution of 4mg/ml;
2.5 mixed solutions that step 2.4 is obtained, by sephadex G-200 post, use the phosphate buffered solution wash-out of pH=7.1 simultaneously;
2.6, with small test tube successively subsection receiing effluent, being detected by good sheep anti mouse lath with wrapping in advance, collecting the in vitro product that invisible spectro sheep anti mouse lath shows blue look, the anti-human-IgG of obtained horseradish peroxidase-labeled;
Anti-human-the IgG of the described horseradish peroxidase-labeled of collecting is added the mixing of equivalent glycerine by 2.7, obtained enzyme conjugates just solution;
Sodium chloride 6g ~ 8g, bSA 140ml ~ 160ml, Sodium azide 0.8g ~ 1.2g are dissolved in pure water, and are settled to 1000ml by 2.8, obtained enzyme dilution;
The first solution of described enzyme conjugates and described enzyme dilution are pressed 1:6000 ~ 1:4000 by 2.9 to be diluted, obtained TB-IgG enzyme conjugates solution;
(3) preparation of substrate A solution
First trisodium citrate 4.16g ~ 6.24g, citric acid 7.76g ~ 11.64g and sodium acetate 7.13g ~ 10.70g pure water are dissolved completely, add the glacial acetic acid of 1.52ml ~ 2.28ml and the hydrogen peroxide of 0.8ml ~ 1.2ml again, finally be settled to 1000ml with pure water, obtained substrate A solution;
(4) preparation of substrate B solution
By 0.8g ~ 1.2g3,3', 5,5'-tetramethyl benzidine is dissolved in 8ml ~ 12ml dimethyl formamide, then adds 520ml ~ 780ml absolute ethyl alcohol and 272ml ~ 408ml ethylene glycol, mixing, obtained substrate B solution;
(5) preparation of stop buffer
80 ~ 120ml sulfuric acid is added, mixing, obtained stop buffer in 900ml pure water;
(6) preparation of the critical contrast solution of TB-IgG
Sodium chloride 6g ~ 8g, Na is dissolved with 1000ml pure water 2hPO 412H 2o2.2g ~ 3.4g, KH 2pO 40.16g ~ 0.24g, potassium chloride 0.16g ~ 0.24g, sucrose 40g ~ 60g, sodium azide 0.08g ~ 0.12g, sheep anti-mouse igg 100 μ l ~ 400 μ l, the obtained critical contrast solution of TB-IgG;
(7) preparation of TB-IgG positive control solution
Sodium chloride 6g ~ 8g, Na is dissolved with 1000ml pure water 2hPO 412H 2o2.2g ~ 3.4g, KH 2pO 40.16g ~ 0.24g, potassium chloride 0.16g ~ 0.24g, sucrose 40g ~ 60g, sodium azide 0.08g ~ 0.12g, sheep anti-mouse igg 1ml ~ 2ml, obtained TB-IgG positive control solution;
(8) preparation of sample diluent
Get NaCl6g ~ 8g, Na 2hPO 412H 2o2.5g ~ 3.0g, KH 2pO 40.1g ~ 0.3g, KCl0.1g ~ 0.3g add volumetric flask, then add appropriate pure water, make it to dissolve completely, get thimerosal 0.4g ~ 0.6g, make it to dissolve completely, then get bSA 200ml and add volumetric flask, stir, be settled to 1000ml with pure water, obtained sample diluent;
(9) preparation of concentrated cleaning solution
Get NaCl149.0g ~ 151.9g, Na 2hPO 4.12H 2o92g ~ 98g, NaH 2pO 4.2H 2o28g ~ 22g adds volumetric flask; First add appropriate pure water, make it to dissolve completely; Add thimerosal 0.8g ~ 1.2g again, make it to dissolve completely; Measure 4ml ~ 6ml Tween-20 and add volumetric flask, stir, be settled to 1000ml with pure water, obtained concentrated cleaning solution.
2. the preparation method of tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit as claimed in claim 1, is characterized in that: by described enzyme conjugates just solution preserve under-22 ~-19 DEG C of conditions.
3. the preparation method of tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit as claimed in claim 1, is characterized in that: described anti-human IgG antibodies is mouse-anti human IgG antibody.
4. the preparation method of tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit as claimed in claim 1, is characterized in that: described TB antigen is gene recombinant antigens.
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