CN103323604A - Mycobacterium tuberculosis IgG antibody enzyme-linked immunosorbent assay (ELISA) kit and preparation and application methods thereof - Google Patents
Mycobacterium tuberculosis IgG antibody enzyme-linked immunosorbent assay (ELISA) kit and preparation and application methods thereof Download PDFInfo
- Publication number
- CN103323604A CN103323604A CN2013102647242A CN201310264724A CN103323604A CN 103323604 A CN103323604 A CN 103323604A CN 2013102647242 A CN2013102647242 A CN 2013102647242A CN 201310264724 A CN201310264724 A CN 201310264724A CN 103323604 A CN103323604 A CN 103323604A
- Authority
- CN
- China
- Prior art keywords
- solution
- igg
- preparation
- pure water
- substrate
- 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.)
- Granted
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a mycobacterium tuberculosis IgG antibody enzyme-linked immunosorbent assay (ELISA) kit and preparation and application methods thereof. The mycobacterium tuberculosis IgG antibody ELISA kit comprises a TB-IgG enzyme label plate, a TB-IgG enzyme conjugate solution, a substrate solution A, a substrate solution B, a TB-IgG critical control solution, a TB-IgG positive control solution, a stop solution, a sample diluent and a concentrated detergent. The kit applies an indirect ELISA method for detecting a mycobacterium tuberculosis IgG antibody so as to reduce the interference of a false positive or false negative phenomenon in clinical diagnosis, the kit can be used for rapidly and accurately detecting mycobacterium tuberculosis, and meanwhile the kit is small in error, high in detection sensitivity and low in requirements on detection equipment.
Description
Technical field
The present invention relates to the Biological Detection technical field, be specifically related to a kind of tubercle bacillus IgG antibody ELISA immunity detection reagent and preparation and application thereof.
Background technology
Much's bacillus (M.tuberculosis) is referred to as tubercle bacillus (tubercle bacilli, TB).As far back as 1886, German bacteriologist Koch just proved that TB is pathogen lungy.This bacterium can be invaded 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, because acquired immune deficiency syndrome (AIDS) and the appearance of TB antibody-resistant bacterium, the factors such as application, drug abuse, poverty and movement of population of immunodepressant, epidemic situation lungy worsens suddenly in the global range.According to the WHO statistics, just there is 1 people to infect TB among approximately per 3 people in the whole world, the TB carrying rate is up to 80% in some adult of developing country, and wherein approximately 5%～10% carrier can develop into active tuberculosis.Recent two decades has infected the TB carrier of HIV because acquired immune deficiency syndrome (AIDS) popular and since virus damage the immunologic function of body, develop into the possibility of active tuberculosis than infected by HIV person is not high 30～50 times, and the disease of tuberculosis is faster.In addition, in the development process that HIV infects, tuberculosis is a kind of opportunistic infections that occur the earliest, and tuberculosis has increased the weight of the disease burden of HIV the infected or aids patient, makes its easier death.The whole world 800 ten thousand tuberculosis new cases approximately occur every year at present, and causes approximately 3 million peoples death.China dies from people lungy approximately 250,000 more than every year, is that the twice of all kinds of Death of Infectious Diseases number summations is many.Therefore, tuberculosis becomes again the global hygienic issues that threatens human health, and becomes some developing countries and regions, particularly High prevalence areas of AIDS crowd's the primary cause of the death.
The tuberculosis epidemic situation of China is quite serious, and early stage, quick diagnosis lungy is propagated for early stage chemotherapy and control extremely important meaning.Although the tuberculosis bacteriology checking is diagnosis of tuberculosis " goldstandard ", yet the smear-positive rate is 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 the immunologic test Specimen origin is convenient, easy, quick, sensitive, special, has become the important auxiliary examination means of tuberculosis at present.
Summary of the invention
Technical matters to be solved by this invention is: a kind of tubercle bacillus IgG antibody ELISA immunity detection reagent and preparation and application thereof are provided, make it to have the following advantages: use indirect elisa method and detect tubercle bacillus IgG antibody, the interference that clinical diagnosis is brought to reduce false positive or false negative phenomenon, and this kit can be detected fast and accurately to tubercle bacillus, little with time error, detection sensitivity is high, it is low that checkout equipment is required.
For solving the problems of the technologies described above, technical scheme of the present invention is:
A kind of tubercle bacillus IgG antibody ELISA immunity detection reagent: 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 at least 5 transparent reaction holes that are coated with solid phase t B antigen;
Described TB-IgG enzyme conjugates solution is the anti-human IgG antibody that the horseradish mark is crossed;
The described substrate A solution of every 1000ml contains 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;
The described substrate B solution of every 1000ml contains absolute ethyl alcohol 520ml～780ml, ethylene glycol 272ml～408ml, dimethyl formamide 8ml～12ml, 3,3 ', 5,5 '-tetramethyl benzidine 0.8g～1.2g;
The critical contrast solution of the described TB-IgG of every 1000ml contains sodium chloride 6g～8g, Na
2HPO
412H
2O 2.2g～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 described TB-IgG positive control of every 1000ml solution contains sodium chloride 6g～8g, Na
2HPO
412H
2O 2.2g～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 formed by 900ml pure water and 80ml～120ml sulfuric acid configuration;
Described sample diluent is to contain the phosphate buffered solution that Deporteinnized calf serum and thimerosal, pH are 7.0 0.01M, and wherein said Deporteinnized calf serum content is 200ml, and thimerosal content is 0.4～0.6g;
Described concentrated cleaning solution is that pH=7.5 concentration is the phosphate buffer of 0.1mol/L, contains the Tween-20 of volume fraction 0.05% and the thimerosal of 0.1wt% in the described phosphate buffer.
Preferably, described TB antigen is gene recombinant antigens.
Preferably, described anti-human IgG antibody is the mouse-anti human IgG antibody.
The preparation method of described tubercle bacillus IgG antibody ELISA immunity detection reagent is characterized in that:
(1) the TB-IgG ELISA Plate is coated:
1.1 preparation TB-IgG coating buffer: the TB antigenic solution is diluted to the solution that concentration is 0.8 μ g/ml, pH=9.6 with sodium carbonate-sodium bicarbonate buffer solution;
1.2 coated: the TB-IgG coating buffer that makes with step 1.1 is coated with described transparent reaction hole by 100 μ l/ holes, and leaves standstill 24～30 hours under 2～8 ℃ of temperature;
1.3 cleansing solution preparation: with volume content be the phosphate buffered solution of 0.05% Tween-20, pH=7.5 and distilled water by volume 1:40 mix, make cleansing solution;
1.4 washing: the ELISA Plate after coated with the cleansing solution continuous washing at least 1 time pats dry;
1.5 sealing: add confining liquid by 130 μ l/ holes in the transparent reaction hole after the washing, described confining liquid is to contain NaHCO in every 1000ml pure water solution
32.93g, Na
2CO
31.59g, bovine serum albumin(BSA) 10g, and under 20～25 ℃ of conditions, sealed 2.8～3.2 hours;
1.6 throw away the confining liquid in the hole, dry 40h～48h finishes coated;
(2) preparation of TB-IgG enzyme conjugates solution:
2.1 3～8mg horseradish peroxidase is dissolved in the 1ml distilled water, add again the sodium periodate solution 200 μ l of 21～22mg/ml, under 18 ℃～25 ℃ conditions of room temperature, mix and prepare horseradish peroxidase solution;
2.2 again horseradish peroxidase solution is added the acetic acid of pH=4.4-glacial acetic acid damping fluid, under 2～6 ℃ of conditions, dialysed 16～20 hours;
Anti-human-IgG adds pH=9.6, carbonate content is sodium carbonate-sodium bicarbonate buffer solution of 0.05mol/L 2.3 get, and dialyses 16～20 hours under 2～6 ℃ of conditions;
2.4 the horseradish peroxidase solution after will dialysing and anti-human-IgG solution mixing, the carbonate buffer solution of putting into pH=9.6, carbonate content and be 0.05mol/L stirs dialysis 2.5～3.5 hours, adds the sodium borohydride solution of 4mg/ml again;
2.5 the mixed solution that step 2.4 is obtained passes through sephadex G-200 post, uses simultaneously the phosphate buffered solution wash-out of pH=7.1;
2.6 receive effluent with small test tube successively segmentation, detect with prior coated good sheep anti mouse lath, collect the in vitro product of the aobvious blue look of invisible spectro sheep anti mouse lath, make horseradish peroxidase-labeled anti-human-IgG;
2.7 with the horseradish peroxidase-labeled of collecting anti-human-IgG adds equivalent glycerine mixing, makes just solution of enzyme conjugates;
2.8 sodium chloride 6g～8g, Deporteinnized calf serum 140ml～160ml, Sodium azide 0.8g～1.2g are dissolved in the pure water, and are settled to 1000ml, make the enzyme dilution;
2.9 the first solution of described enzyme conjugates and described enzyme dilution are pressed 1:6000～1:4000 dilution, make 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 are dissolved fully with pure water, add again the glacial acetic acid of 1.52ml～2.28ml and the hydrogen peroxide of 0.8ml～1.2ml, be settled to 1000ml with pure water at last, make substrate A solution;
(4) preparation of substrate B solution
With 0.8g～1.2g3,3 ', 5,5 '-tetramethyl benzidine is dissolved in 8ml～12ml dimethyl formamide, adds 520ml～780ml absolute ethyl alcohol and 272ml～408ml ethylene glycol again, and mixing makes substrate B solution;
(5) preparation of stop buffer
Add 80～120ml sulfuric acid in the 900ml pure water, mixing makes stop buffer;
(6) preparation of the critical contrast solution of TB-IgG
With 1000ml pure water dissolving sodium chloride 6g～8g, Na
2HPO
412H
2O 2.2g～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 make the critical contrast solution of TB-IgG;
(7) preparation of TB-IgG positive control solution
With 1000ml pure water dissolving sodium chloride 6g～8g, Na
2HPO
412H
2O 2.2g～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 makes TB-IgG positive control solution;
(8) preparation of sample diluent
Get NaCl 6g～8g, Na
2HPO
412H
2O 2.5g～3.0g, KH
2PO
40.1g～0.3g, KCl 0.1g～0.3g adds volumetric flask, adds an amount of pure water again, makes it dissolve complete, get thimerosal 0.4g～0.6g, make it dissolve complete, get again Deporteinnized calf serum 200ml and add volumetric flask, stir, be settled to 1000ml with pure water, make sample diluent.
(9) preparation of concentrated cleaning solution
Get NaCl 149.0g～151.9g, Na
2HPO
4.12H
2O 92g～98g, NaH
2PO
4.2H
2O 28g～22g adds volumetric flask; Add first an amount of pure water, make it dissolve complete; Add again thimerosal 0.8g～1.2g, make it dissolve complete; Measure 4ml～6ml Tween-20 and add volumetric flask, stir, be settled to 1000ml with pure water, make concentrated cleaning solution.
Preferably, described enzyme conjugates is preserved under-22～-19 ℃ of conditions.
Preferably, described anti-human IgG antibody is the mouse-anti human IgG antibody.
Preferably, described TB antigen is gene recombinant antigens.
The using method of described tubercle bacillus IgG antibody ELISA immunity detection reagent is characterized in that, may further comprise the steps:
(1) sample collection
Leave standstill test serum 30min, described test serum was carried out centrifugal treating 10～20 minutes, can detect.
(2) application of sample detects
A. with 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 balance to 20 ℃～25 ℃ of room temperatures;
B. getting TB-IgG positive control solution joins at least one described transparent reaction hole on the described ELISA Plate, as the positive control hole by 50 μ l/ holes; Getting the critical contrast liquid of TB-IgG joins in two described transparent reaction holes on the described ELISA Plate, as critical control wells by 50 μ l/ holes at least;
C. this dilution of label taking joins at least one described transparent reaction hole on the described ELISA Plate by 100 μ l/ holes, gets serum to be checked 10 μ l/ holes and adds in the described transparent reaction hole, as sample well;
D. described TB-IgG ELISA Plate is sticked the shrouding film, the concussion mixing places 36 ℃～38 ℃ waters bath with thermostatic control to react 8min～10min;
E. take out described TB-IgG ELISA Plate, take the shrouding film off, with using cleansing solution washing 5 times, cleansing solution is filled with in every hole during washing, stops 20 seconds at every turn, pats dry;
F. respectively add 50 μ l enzyme conjugates solution in sample well, positive control hole and critical control wells, mixing sticks the shrouding film, places 36 ℃～38 ℃ waters bath with thermostatic control to react 8min～10min;
G. take out described TB-IgG ELISA Plate, take the shrouding film off, with cleansing solution washing at least 5 times, cleansing solution is filled with in every hole during washing, stops 20 seconds at every turn, pats dry;
H. in sample well, positive control hole and critical control wells, respectively add 50 μ l substrate solution A, 30 μ l substrate solution B, mixing sticks the shrouding film, reacts 8min～10min in 36 ℃～38 ℃ waters bath with thermostatic control;
I. take the shrouding film off, observations: if only judge the yin and yang attribute that detects sample, adopt visual method, the color of comparative sample hole and positive control hole, critical control wells can draw testing result respectively; If need read and detect accurately numerical value, behind step h, in sample well, add again 50 μ l stop buffers, behind the mixing, use the microplate reader reading result.
Preferably, if described test serum can not in time detect, need frozen-20 ℃～-15 ℃ 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 contain TB-IgG antibody in the test serum, the TB antigen that then is coated with in lath first is combined, form antigen-antibody complex, again with two anti-formation Ag-Ab-antiantibody compounds of enzyme labeling, and be combined in the micro-pore wall surface, develop the color by substrate.In order to detect specifically the TB antibody among the human serum sample.The present invention is highly sensitive, high specificity, experimental implementation is simple, quick, the present invention has not only reduced the interference that false positive or false negative phenomenon are brought clinical diagnosis, this kit can be detected fast and accurately to TB antibody, and error is little, detection sensitivity is high, require low to checkout equipment; Also because the present invention has adopted the prescription of described ELISA Plate and various solution, so that the order of accuarcy of the testing result of kit of the present invention is that other TB antibody reagent institute is inaccessible in the market.
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 experiment specificity.The result shows that this experimental technique has reached fast (30min), sensitivity, special, the requirement of experiment of easy (also can estimate the 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 ELISA immunity detection reagent
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;
The TB-IgG ELISA Plate is provided with 48 transparent reaction holes that are coated with solid phase t B gene recombinant antigens;
TB-IgG enzyme conjugates solution is the mouse-anti human IgG antibody that the horseradish mark is crossed;
Every 1000ml substrate A solution contains trisodium citrate 4.16g, citric acid 7.76g, sodium acetate 7.13g, glacial acetic acid 1.52ml, hydrogen peroxide 0.8ml;
Every 1000ml substrate B solution contains absolute ethyl alcohol 520ml, ethylene glycol 272ml, dimethyl formamide 8ml, 3,3 ', 5,5 '-tetramethyl benzidine 0.8g;
The critical contrast solution of every 1000mlTB-IgG contains sodium chloride 6g, Na
2HPO
412H
2O 2.2g, KH
2PO
40.16g, potassium chloride 0.16g, sucrose 40g, sodium azide 0.08g, sheep anti-mouse igg 100 μ l;
Every 1000mlTB-IgG positive control solution contains sodium chloride 6g, Na
2HPO
412H
2O 2.2g, KH
2PO
40.16g, potassium chloride 0.16g, sucrose 40g, sodium azide 0.08g, sheep anti-mouse igg 1ml;
Stop buffer is formed by 900ml pure water and the configuration of 80ml sulfuric acid;
Sample diluent is to contain the phosphate buffered solution that Deporteinnized calf serum and thimerosal, pH are 7.0 0.01M, and wherein Deporteinnized calf serum content is 200ml, and thimerosal content is 0.4g;
Concentrated cleaning solution is that pH=7.5 concentration is the phosphate buffer of 0.1mol/L, contains the Tween-20 of volume fraction 0.05% and the thimerosal of 0.1wt% in the phosphate buffer.
Embodiment 2
A kind of tubercle bacillus IgG antibody ELISA immunity detection reagent
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;
The TB-IgG ELISA Plate is provided with 48 transparent reaction holes that are coated with solid phase t B gene recombinant antigens;
TB-IgG enzyme conjugates solution is the mouse-anti human IgG antibody that the horseradish mark is crossed;
Every 1000ml substrate A solution contains trisodium citrate 6.24g, citric acid 11.64g, sodium acetate 10.70g, glacial acetic acid 2.28ml, hydrogen peroxide 1.2ml;
Every 1000ml substrate B solution contains absolute ethyl alcohol 780ml, ethylene glycol 408ml, dimethyl formamide 12ml, 3,3 ', 5,5 '-tetramethyl benzidine 1.2g;
The critical contrast solution of every 1000mlTB-IgG contains sodium chloride 8g, Na
2HPO
412H
2O 3.4g, KH
2PO
40.24g, potassium chloride 0.24g, sucrose 60g, sodium azide 0.12g, sheep anti-mouse igg 400 μ l;
Every 1000mlTB-IgG positive control solution contains sodium chloride 8g, Na
2HPO
412H
2O 3.4g, KH
2PO
40.24g, potassium chloride 0.24g, sucrose 60g, sodium azide 0.12g, sheep anti-mouse igg 2ml;
Stop buffer is formed by 900ml pure water and the configuration of 120ml sulfuric acid;
Sample diluent is to contain the phosphate buffered solution that Deporteinnized calf serum and thimerosal, pH are 7.0 0.01M, and wherein Deporteinnized calf serum content is 200ml, and thimerosal content is 0.6g;
Concentrated cleaning solution is that pH=7.5 concentration is the phosphate buffer of 0.1mol/L, contains the Tween-20 of volume fraction 0.05% and the thimerosal of 0.1wt% in the phosphate buffer.
Embodiment 3
A kind of tubercle bacillus IgG antibody ELISA immunity detection reagent
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;
The TB-IgG ELISA Plate is provided with 48 transparent reaction holes that are coated with solid phase t B gene recombinant antigens;
TB-IgG enzyme conjugates solution is the mouse-anti human IgG antibody that the horseradish mark is crossed;
Every 1000ml substrate A solution contains trisodium citrate 5.2g, citric acid 9.7g, sodium acetate 8.92g, glacial acetic acid 1.9ml, hydrogen peroxide 1ml;
Every 1000ml substrate B solution contains absolute ethyl alcohol 650ml, ethylene glycol 340ml, dimethyl formamide 10ml, 3,3 ', 5,5 '-tetramethyl benzidine 1g;
The critical contrast solution of every 1000mlTB-IgG contains sodium chloride 7g, Na
2HPO
412H
2O 2.8g, KH
2PO
40.2g, potassium chloride 0.2g, sucrose 50g, sodium azide 0.1g, sheep anti-mouse igg 100 μ l;
Every 1000mlTB-IgG positive control solution contains sodium chloride 7g, Na
2HPO
412H
2O 2.8g, KH
2PO
40.2g, potassium chloride 0.2g, sucrose 50g, sodium azide 0.10g, sheep anti-mouse igg 1ml;
Stop buffer is formed by 900ml pure water and the configuration of 100ml sulfuric acid;
Sample diluent is to contain the phosphate buffered solution that Deporteinnized calf serum and thimerosal, pH are 7.0 0.01M, and wherein Deporteinnized calf serum content is 200ml, and thimerosal content is 0.5g;
Concentrated cleaning solution is that pH=7.5 concentration is the phosphate buffer of 0.1mol/L, contains the Tween-20 of volume fraction 0.05% and the thimerosal of 0.1wt% in the phosphate buffer.
Embodiment 4
Embodiment 3 tubercle bacillus IgG antibody ELISA immunity detection reagent preparation methods:
(1) the TB-IgG ELISA Plate is coated:
1.1 preparation TB-IgG coating buffer: the TB antigenic solution is diluted to the solution that concentration is 0.8 μ g/ml, pH=9.6 with sodium carbonate-sodium bicarbonate buffer solution;
1.2 coated: the TB-IgG coating buffer that makes with step 1.1 is coated with by 100 μ l/ holes, and leaves standstill 24～30 hours under 4 ℃ of temperature;
1.3 cleansing solution preparation: with volume content be the phosphate buffered solution of 0.05% Tween-20, pH=7.5 and distilled water by volume 1:40 mix, make cleansing solution;
1.4 washing: the ELISA Plate after coated with the cleansing solution continuous washing at least 1 time pats dry;
1.5 sealing: add confining liquid by 130 μ l/ holes in the transparent reaction hole after the washing, confining liquid is to contain NaHCO in every 1000ml pure water solution
32.93g, Na
2CO
31.59g, bovine serum albumin(BSA) 10g, and under 22 ℃ of conditions, sealed 3 hours;
1.6 throw away the confining liquid in the hole, dry 40h～48h finishes coated;
(2) preparation of TB-IgG enzyme conjugates solution:
2.1 the 5mg horseradish peroxidase is dissolved in the 1ml distilled water, add again the sodium periodate solution 200 μ l of 21.4mg/ml, under 18 ℃～25 ℃ conditions of room temperature, mix and prepare horseradish peroxidase solution;
2.2 again horseradish peroxidase solution is added the acetic acid of pH=4.4-glacial acetic acid damping fluid, under 2～6 ℃ of conditions, dialysed 16～20 hours;
Anti-human-IgG adds pH=9.6, carbonate content is sodium carbonate-sodium bicarbonate buffer solution of 0.05mol/L 2.3 get, and dialyses 16～20 hours under 2～6 ℃ of conditions;
2.4 the horseradish peroxidase solution after will dialysing and anti-human-IgG solution mixing, the carbonate buffer solution of putting into pH=9.6, carbonate content and be 0.05mol/L stirs dialysis 2.5～3.5 hours, adds the sodium borohydride solution of 4mg/ml again;
2.5 the mixed solution that step 2.4 is obtained passes through sephadex G-200 post, uses simultaneously the phosphate buffered solution wash-out of pH=7.1;
2.6 receive effluent with small test tube successively segmentation, detect with prior coated good sheep anti mouse lath, collect the in vitro product of the aobvious blue look of invisible spectro sheep anti mouse lath, make horseradish peroxidase-labeled anti-human-IgG;
2.7 with the horseradish peroxidase-labeled of collecting anti-human-IgG adds equivalent glycerine mixing, makes just solution of enzyme conjugates, is positioned over-20 ℃ of Refrigerator stores;
2.8 sodium chloride 7g, Deporteinnized calf serum 150ml, Sodium azide 1.0g are dissolved in the pure water, and are settled to 1000ml, make the enzyme dilution;
2.9 the first solution of enzyme conjugates and enzyme dilution are pressed the 1:5000 dilution, make 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 are dissolved fully with pure water, add again the glacial acetic acid of 1.9ml and the hydrogen peroxide of 1ml, be settled to 1000ml with pure water at last, make substrate A solution;
(4) preparation of substrate B solution
With 1.0g3,3 ', 5,5 '-tetramethyl benzidine is dissolved in the 10ml dimethyl formamide, adds 650ml absolute ethyl alcohol and 340ml ethylene glycol again, and mixing makes substrate B solution;
(5) preparation of stop buffer
Add 100ml sulfuric acid in the 900ml pure water, mixing makes stop buffer;
(6) preparation of the critical contrast solution of TB-IgG
With 1000ml pure water dissolving sodium chloride 7g, Na
2HPO
412H
2O 2.8g, KH
2PO
40.2g, potassium chloride 0.2g, sucrose 50g, sodium azide 0.1g, sheep anti-mouse igg 100 μ l make the critical contrast solution of TB-IgG;
(7) preparation of TB-IgG positive control solution
With 1000ml pure water dissolving sodium chloride 7g, Na
2HPO
412H
2O 2.8g, KH
2PO
40.2g, potassium chloride 0.2g, sucrose 50g, sodium azide 0.1g, sheep anti-mouse igg 1ml makes TB-IgG positive control solution;
(8) preparation of sample diluent
Get NaCl 7g, Na
2HPO
412H
2O 2.8g, KH
2PO
40.2g, KCl 0.2g adds volumetric flask, adds an amount of pure water again, makes it dissolve complete, gets thimerosal 0.5g, makes it dissolve complete, gets Deporteinnized calf serum 200ml again and adds volumetric flask, stirs, and is settled to 1000ml with pure water, makes sample diluent.
(9) preparation of concentrated cleaning solution
Get NaCl 150g, Na
2HPO
4.12H
2O 95g, NaH
2PO
4.2H
2O 26g adds volumetric flask; Add first an amount of pure water, make it dissolve complete; Add again thimerosal 1.0g, make it dissolve complete; Measure the 5ml Tween-20 and add volumetric flask, stir, be settled to 1000ml with pure water, make concentrated cleaning solution.
Embodiment 5
Tubercle bacillus IgG antibody ELISA immunity detection reagent preparation method
(1) the TB-IgG ELISA Plate is coated:
1.1 preparation TB-IgG coating buffer: the TB antigenic solution is diluted to the solution that concentration is 0.8 μ g/ml, pH=9.6 with sodium carbonate-sodium bicarbonate buffer solution;
1.2 coated: the TB-IgG coating buffer that makes with step 1.1 is coated with by 100 μ l/ holes, and leaves standstill 24 hours under 2 ℃ of temperature;
1.3 cleansing solution preparation: with volume content be the phosphate buffered solution of 0.05% Tween-20, pH=7.5 and distilled water by volume 1:40 mix, make cleansing solution;
1.4 washing: the ELISA Plate after coated with the cleansing solution continuous washing at least 1 time pats dry;
1.5 sealing: add confining liquid by 130 μ l/ holes in the transparent reaction hole after the washing, confining liquid is to contain NaHCO in every 1000ml pure water solution
32.93g, Na
2CO
31.59g, bovine serum albumin(BSA) 10g, and under 20 ℃ of conditions, sealed 2.8 hours;
1.6 throw away the confining liquid in the hole, dry 40h finishes coated;
(2) preparation of TB-IgG enzyme conjugates solution:
2.1 3～8mg horseradish peroxidase is dissolved in the 1ml distilled water, add again the sodium periodate solution 200 μ l of 21～22mg/ml, under 18 ℃ of conditions of room temperature, mix and prepare horseradish peroxidase solution;
2.2 horseradish peroxidase solution is added the acetic acid of pH=4.4-glacial acetic acid damping fluid, dialysis is 16 hours under 2～6 ℃ of conditions again;
Anti-human-IgG adds pH=9.6, carbonate content is sodium carbonate-sodium bicarbonate buffer solution of 0.05mol/L 2.3 get, and dialysis is 16 hours under 2 ℃ of conditions;
2.4 the horseradish peroxidase solution after will dialysing and anti-human-IgG solution mixing, the carbonate buffer solution of putting into pH=9.6, carbonate content and be 0.05mol/L stirs dialysis 2.5 hours, adds the sodium borohydride solution of 4mg/ml again;
2.5 the mixed solution that step 2.4 is obtained passes through sephadex G-200 post, uses simultaneously the phosphate buffered solution wash-out of pH=7.1;
2.6 receive effluent with small test tube successively segmentation, detect with prior coated good sheep anti mouse lath, collect the in vitro product of the aobvious blue look of invisible spectro sheep anti mouse lath, make horseradish peroxidase-labeled anti-human-IgG;
2.7 with the horseradish peroxidase-labeled of collecting anti-human-IgG adds equivalent glycerine mixing, makes just solution of enzyme conjugates;
2.8 sodium chloride 6g, Deporteinnized calf serum 140ml, Sodium azide 0.8g are dissolved in the pure water, and are settled to 1000ml, make the enzyme dilution;
2.9 the first solution of enzyme conjugates and enzyme dilution are pressed the 1:6000 dilution, make 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 are dissolved fully with pure water, add again the glacial acetic acid of 1.52ml and the hydrogen peroxide of 0.8ml, be settled to 1000ml with pure water at last, make substrate A solution;
(4) preparation of substrate B solution
With 0.95g～1.05g3,3 ', 5,5 '-tetramethyl benzidine is dissolved in the 10ml dimethyl formamide, adds 650ml absolute ethyl alcohol and 340ml ethylene glycol again, and mixing makes substrate B solution;
(5) preparation of stop buffer
Add 100ml sulfuric acid in the 900ml pure water, mixing makes stop buffer;
(6) preparation of the critical contrast solution of TB-IgG
With 1000ml pure water dissolving sodium chloride 6g, Na
2HPO
412H
2O 2.2g, KH
2PO
40.16g, potassium chloride 0.16g, sucrose 40g, sodium azide 0.08g, sheep anti-mouse igg 100 μ l make the critical contrast solution of TB-IgG;
(7) preparation of TB-IgG positive control solution
With 1000ml pure water dissolving sodium chloride 6g, Na
2HPO
412H
2O 2.2g, KH
2PO
40.16g, potassium chloride 0.16g, sucrose 40g, sodium azide 0.08g, sheep anti-mouse igg 1ml makes TB-IgG positive control solution;
(8) preparation of sample diluent
Get NaCl 6g, Na
2HPO
412H
2O 2.5g, KH
2PO
40.1g, KCl 0.1g adds volumetric flask, adds an amount of pure water again, makes it dissolve complete, gets thimerosal 0.4g, makes it dissolve complete, gets Deporteinnized calf serum 200ml again and adds volumetric flask, stirs, and is settled to 1000ml with pure water, makes sample diluent.
(9) preparation of concentrated cleaning solution
Get NaCl 149.0g, Na
2HPO
4.12H
2O 92g, NaH
2PO
4.2H
2O 28g adds volumetric flask; Add first an amount of pure water, make it dissolve complete; Add again thimerosal 0.8g, make it dissolve complete; Measure the 4ml Tween-20 and add volumetric flask, stir, be settled to 1000ml with pure water, make concentrated cleaning solution.
Embodiment 6
Tubercle bacillus IgG antibody ELISA immunity detection reagent preparation method
(1) the TB-IgG ELISA Plate is coated:
1.1 preparation TB-IgG coating buffer: the TB antigenic solution is diluted to the solution that concentration is 0.8 μ g/ml, pH=9.6 with sodium carbonate-sodium bicarbonate buffer solution;
1.2 coated: the TB-IgG coating buffer that makes with step 1.1 is coated with by 100 μ l/ holes, and leaves standstill 30 hours under 8 ℃ of temperature;
1.3 cleansing solution preparation: with volume content be the phosphate buffered solution of 0.05% Tween-20, pH=7.5 and distilled water by volume 1:40 mix, make cleansing solution;
1.4 washing: the ELISA Plate after coated with the cleansing solution continuous washing at least 1 time pats dry;
1.5 sealing: add confining liquid by 130 μ l/ holes in the transparent reaction hole after the washing, confining liquid is to contain NaHCO in every 1000ml pure water solution
32.93g, Na
2CO
31.59g, bovine serum albumin(BSA) 10g, and under 25 ℃ of conditions, sealed 3.2 hours;
1.6 throw away the confining liquid in the hole, dry 48h finishes coated;
(2) preparation of TB-IgG enzyme conjugates solution:
2.1 3～8mg horseradish peroxidase is dissolved in the 1ml distilled water, add again the sodium periodate solution 200 μ l of 22mg/ml, under 25 ℃ of conditions of room temperature, mix and prepare horseradish peroxidase solution;
2.2 horseradish peroxidase solution is added the acetic acid of pH=4.4-glacial acetic acid damping fluid, dialysis is 20 hours under 6 ℃ of conditions again;
Anti-human-IgG adds pH=9.6, carbonate content is sodium carbonate-sodium bicarbonate buffer solution of 0.05mol/L 2.3 get, and dialysis is 20 hours under 6 ℃ of conditions;
2.4 the horseradish peroxidase solution after will dialysing and anti-human-IgG solution mixing, the carbonate buffer solution of putting into pH=9.6, carbonate content and be 0.05mol/L stirs dialysis 3.5 hours, adds the sodium borohydride solution of 4mg/ml again;
2.5 the mixed solution that step 2.4 is obtained passes through sephadex G-200 post, uses simultaneously the phosphate buffered solution wash-out of pH=7.1;
2.6 receive effluent with small test tube successively segmentation, detect with prior coated good sheep anti mouse lath, collect the in vitro product of the aobvious blue look of invisible spectro sheep anti mouse lath, make horseradish peroxidase-labeled anti-human-IgG;
2.7 with the horseradish peroxidase-labeled of collecting anti-human-IgG adds equivalent glycerine mixing, makes just solution of enzyme conjugates;
2.8 sodium chloride 8g, Deporteinnized calf serum 160ml, Sodium azide 1.2g are dissolved in the pure water, and are settled to 1000ml, make the enzyme dilution;
2.9 the first solution of enzyme conjugates and enzyme dilution are pressed the 1:4000 dilution, make 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 are dissolved fully with pure water, add again the glacial acetic acid of 2.28ml and the hydrogen peroxide of 1.2ml, be settled to 1000ml with pure water at last, make substrate A solution;
(4) preparation of substrate B solution
With 1.05g3,3 ', 5,5 '-tetramethyl benzidine is dissolved in the 10ml dimethyl formamide, adds 650ml absolute ethyl alcohol and 340ml ethylene glycol again, and mixing makes substrate B solution;
(5) preparation of stop buffer
Add 100ml sulfuric acid in the 900ml pure water, mixing makes stop buffer;
(6) preparation of the critical contrast solution of TB-IgG
With 1000ml pure water dissolving sodium chloride 8g, Na
2HPO
412H
2O 3.4g, KH
2PO
40.24g, potassium chloride 0.24g, sucrose 60g, sodium azide 0.12g, sheep anti-mouse igg 400 μ l make the critical contrast solution of TB-IgG;
(7) preparation of TB-IgG positive control solution
With 1000ml pure water dissolving sodium chloride 8g, Na
2HPO
412H
2O 3.4g, KH
2PO
40.24g, potassium chloride 0.24g, sucrose 60g, sodium azide 0.12g, sheep anti-mouse igg 2ml makes TB-IgG positive control solution;
(8) preparation of sample diluent
Get NaCl 8g, Na
2HPO
412H
2O 3.0g, KH
2PO
40.3g, KCl 0.3g adds volumetric flask, adds an amount of pure water again, makes it dissolve complete, gets thimerosal 0.6g, makes it dissolve complete, gets Deporteinnized calf serum 200ml again and adds volumetric flask, stirs, and is settled to 1000ml with pure water, makes sample diluent.
(9) preparation of concentrated cleaning solution
Get NaCl 151.9g, Na
2HPO
4.12H
2O 98g, NaH
2PO
4.2H
2O 22g adds volumetric flask; Add first an amount of pure water, make it dissolve complete; Add again thimerosal 1.2g, make it dissolve complete; Measure the 6ml Tween-20 and add volumetric flask, stir, be settled to 1000ml with pure water, make concentrated cleaning solution.
Embodiment 7
Tubercle bacillus IgG antibody ELISA immunity detection reagent using method
The using method of embodiment 3 described tubercle bacillus IgG antibody ELISA immunity detection reagents:
(1) sample collection
Leave standstill test serum 30min, described test serum was carried out centrifugal treating 10～20 minutes, detect.
(2) application of sample detects
A. the described TB-IgG enzyme conjugates solution bottle that will preserve in cold storage environment, 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 stop buffer solution bottle balance are to 20 ℃～25 ℃ of room temperatures;
B. getting TB-IgG positive control solution joins at least one transparent reaction hole on the described ELISA Plate, as the positive control hole by 50 μ l/ holes; Getting the critical contrast liquid of TB-IgG joins in two transparent reaction holes on the described ELISA Plate, as critical control wells by 50 μ l/ holes at least;
C. this dilution of label taking joins at least one transparent reaction hole on the described ELISA Plate by 100 μ l/ holes, gets serum to be checked 10 μ l/ holes and adds in the transparent reaction hole, as sample well;
D. described TB-IgG ELISA Plate is sticked the shrouding film, the concussion mixing places 36 ℃～38 ℃ waters bath with thermostatic control to react 8min～10min;
E. take out described TB-IgG ELISA Plate, take the shrouding film off, with using cleansing solution washing 5 times, cleansing solution is filled with in every hole during washing, stops 20 seconds at every turn, pats dry;
F. respectively add 50 μ l enzyme conjugates solution in sample well, positive control hole and critical control wells, mixing sticks the shrouding film, places 36 ℃～38 ℃ waters bath with thermostatic control to react 8min～10min;
G. take out described TB-IgG ELISA Plate, take the shrouding film off, with using cleansing solution washing 5 times, cleansing solution is filled with in every hole during washing, stops 20 seconds at every turn, pats dry.
H. in sample well, positive control hole and critical control wells, respectively add 50 μ l substrate solution A successively, 30 μ l substrate solution B, mixing sticks the shrouding film, reacts 8min～10min in 36 ℃～38 ℃ waters bath with thermostatic control;
I. take the shrouding film off, observations: if only judge the yin and yang attribute that detects sample, adopt visual method, the color of comparative sample hole and positive control hole, critical control wells can draw testing result respectively; Detect accurately numerical value if need read, after step g, in sample well, add again 50 μ l stop buffers, behind the mixing, use the microplate reader reading result.
(3) Analysis of test results
Under the 450nm wavelength, read the absorbance in each hole with the 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 ELISA immunity detection reagent of the present invention:
(1) negative reference material coincidence rate: detect with the negative reference material of 10 parts of these reagent, it is negative that testing result all becomes.
(2) positive reference material coincidence rate: with the positive reference materials of 10 parts of these reagent detect (comprise strong, in, weak) detect, it is positive that testing result all becomes.
(3) accuracy: do 10 tests with 1 part of accuracy reference material, the CV value is not higher than 10%.
(4) clinical trial result: detect clinical sample 1000 examples (wherein positive 50 examples of TB-IgG) of having 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 altogether clinical serum sample 1000 examples (wherein positive 50 examples of TB-IgG), use the inventive method to detect, its specificity and susceptibility are 100%.
Table 1 TB-IgG kit of the present invention testing result and clinical diagnosis result are relatively
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, all conversion of having done all drop within protection scope of the present invention.
Claims (9)
1. tubercle bacillus IgG antibody ELISA immunity detection reagent is characterized in that:
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 at least 5 transparent reaction holes that are coated with solid phase t B antigen;
Described TB-IgG enzyme conjugates solution is the anti-human IgG antibody that the horseradish mark is crossed;
The described substrate A solution of every 1000ml contains 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;
The described substrate B solution of every 1000ml contains absolute ethyl alcohol 520ml～780ml, ethylene glycol 272ml～408ml, dimethyl formamide 8ml～12ml, 3,3 ', 5,5 '-tetramethyl benzidine 0.8g～1.2g;
The critical contrast solution of the described TB-IgG of every 1000ml contains sodium chloride 6g～8g, Na
2HPO
412H
2O 2.2g～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 described TB-IgG positive control of every 1000ml solution contains sodium chloride 6g～8g, Na
2HPO
412H
2O 2.2g～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 formed by 900ml pure water and 80ml～120ml sulfuric acid configuration;
Described sample diluent is to contain the phosphate buffered solution that Deporteinnized calf serum and thimerosal, pH are 7.0 0.01M, and wherein said Deporteinnized calf serum content is 200ml, and thimerosal content is 0.4～0.6g;
Described concentrated cleaning solution is that PH=7.5 concentration is the phosphate buffer of 0.1mol/L, contains the Tween-20 of volume fraction 0.05% and the thimerosal of 0.1wt% in the described phosphate buffer.
2. a kind of tubercle bacillus IgG antibody ELISA immunity detection reagent as claimed in claim 1, it is characterized in that: described TB antigen is the TB gene recombinant antigens.
3. a kind of tubercle bacillus IgG antibody ELISA immunity detection reagent as claimed in claim 1, it is characterized in that: described anti-human IgG antibody is the mouse-anti human IgG antibody.
4. the preparation method of the described tubercle bacillus IgG of claim 1 antibody ELISA immunity detection reagent is characterized in that:
(1) the TB-IgG ELISA Plate is coated:
1.1 preparation TB-IgG coating buffer: the TB antigenic solution is diluted to the solution that concentration is 0.8 μ g/mlpH=9.6 with sodium carbonate-sodium bicarbonate buffer solution;
1.2 coated: the TB-IgG coating buffer that makes with step 1.1 is coated with described transparent reaction hole by 100 μ l/ holes, and leaves standstill 24～30 hours under 2～8 ℃ of temperature;
1.3 cleansing solution preparation: with volume content be the phosphate buffered solution of 0.05% Tween-20, pH=7.5 and distilled water by volume 1:40 mix, make cleansing solution;
1.4 washing: the ELISA Plate after coated with the cleansing solution continuous washing at least 1 time pats dry;
1.5 sealing: add confining liquid by 130 μ l/ holes in the transparent reaction hole after the washing, described confining liquid is to contain NaHCO in every 1000ml pure water solution
32.93g, Na
2CO
31.59g, bovine serum albumin(BSA) 10g, and under 20～25 ℃ of conditions, sealed 2.8～3.2 hours;
1.6 throw away the confining liquid in the hole, dry 40h～48h finishes coated;
(2) preparation of TB-IgG enzyme conjugates solution:
2.1 3～8mg horseradish peroxidase is dissolved in the 1ml distilled water, add again the sodium periodate solution 200 μ l of 21～22mg/ml, under 18 ℃～25 ℃ conditions of room temperature, mix and prepare horseradish peroxidase solution;
2.2 again horseradish peroxidase solution is added the acetic acid of pH=4.4-glacial acetic acid damping fluid, under 2～6 ℃ of conditions, dialysed 16～20 hours;
Anti-human-IgG adds pH=9.6, carbonate content is sodium carbonate-sodium bicarbonate buffer solution of 0.05mol/L 2.3 get, and dialyses 16～20 hours under 2～6 ℃ of conditions;
2.4 the horseradish peroxidase solution after will dialysing and anti-human-IgG solution mixing, the carbonate buffer solution of putting into pH=9.6, carbonate content and be 0.05mol/L stirs dialysis 2.5～3.5 hours, adds the sodium borohydride solution of 4mg/ml again;
2.5 the mixed solution that step 2.4 is obtained passes through sephadex G-200 post, uses simultaneously the phosphate buffered solution wash-out of pH=7.1;
2.6 receive effluent with small test tube successively segmentation, detect with prior coated good sheep anti mouse lath, collect the in vitro product of the aobvious blue look of invisible spectro sheep anti mouse lath, make horseradish peroxidase-labeled anti-human-IgG;
2.7 the described horseradish peroxidase-labeled that will collect is anti-human-and IgG adds equivalent glycerine mixing, makes just solution of enzyme conjugates;
2.8 sodium chloride 6g～8g, Deporteinnized calf serum 140ml～160ml, Sodium azide 0.8g～1.2g are dissolved in the pure water, and are settled to 1000ml, make the enzyme dilution;
2.9 the first solution of described enzyme conjugates and described enzyme dilution are pressed 1:6000～1:4000 dilution, make 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 are dissolved fully with pure water, add again the glacial acetic acid of 1.52ml～2.28ml and the hydrogen peroxide of 0.8ml～1.2ml, be settled to 1000ml with pure water at last, make substrate A solution;
(4) preparation of substrate B solution
With 0.8g～1.2g3,3 ', 5,5 '-tetramethyl benzidine is dissolved in 8ml～12ml dimethyl formamide, adds 520ml～780ml absolute ethyl alcohol and 272ml～408ml ethylene glycol again, and mixing makes substrate B solution;
(5) preparation of stop buffer
Add 80～120ml sulfuric acid in the 900ml pure water, mixing makes stop buffer;
(6) preparation of the critical contrast solution of TB-IgG
With 1000ml pure water dissolving sodium chloride 6g～8g, Na
2HPO
412H
2O 2.2g～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 make the critical contrast solution of TB-IgG;
(7) preparation of TB-IgG positive control solution
With 1000ml pure water dissolving sodium chloride 6g～8g, Na
2HPO
412H
2O 2.2g～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 makes TB-IgG positive control solution;
(8) preparation of sample diluent
Get NaCl 6g～8g, Na
2HPO
412H
2O 2.5g～3.0g, KH
2PO
40.1g～0.3g, KCl 0.1g～0.3g adds volumetric flask, adds an amount of pure water again, makes it dissolve complete, get thimerosal 0.4g～0.6g, make it dissolve complete, get again Deporteinnized calf serum 200ml and add volumetric flask, stir, be settled to 1000ml with pure water, make sample diluent;
(9) preparation of concentrated cleaning solution
Get NaCl 149.0g～151.9g, Na
2HPO
4.12H
2O 92g～98g, NaH
2PO
4.2H
2O 28g～22g adds volumetric flask; Add first an amount of pure water, make it dissolve complete; Add again thimerosal 0.8g～1.2g, make it dissolve complete; Measure 4ml～6ml Tween-20 and add volumetric flask, stir, be settled to 1000ml with pure water, make concentrated cleaning solution.
5. the preparation method of tubercle bacillus IgG antibody ELISA immunity detection reagent as claimed in claim 4 is characterized in that: with described enzyme conjugates just solution under-22～-19 ℃ of conditions, preserve.
6. the preparation method of tubercle bacillus IgG antibody ELISA immunity detection reagent as claimed in claim 4, it is characterized in that: described anti-human IgG antibody is the mouse-anti human IgG antibody.
7. the preparation method of tubercle bacillus IgG antibody ELISA immunity detection reagent as claimed in claim 4, it is characterized in that: described TB antigen is gene recombinant antigens.
8. the using method of tubercle bacillus IgG antibody ELISA immunity detection reagent as claimed in claim 1 is characterized in that, may further comprise the steps:
(1) sample collection
Leave standstill test serum 30min, described test serum was carried out centrifugal treating 10～20 minutes, can detect;
(2) application of sample detects
A. with 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 balance to 20 ℃～25 ℃ of room temperatures;
B. getting TB-IgG positive control solution joins at least one described transparent reaction hole on the described ELISA Plate, as the positive control hole by 50 μ l/ holes; Getting the critical contrast liquid of TB-IgG joins in two described transparent reaction holes on the described ELISA Plate, as critical control wells by 50 μ l/ holes at least;
C. this dilution of label taking joins at least one described transparent reaction hole on the described ELISA Plate by 100 μ l/ holes, gets serum to be checked 10 μ l/ holes and adds in the described transparent reaction hole, as sample well;
D. described TB-IgG ELISA Plate is sticked the shrouding film, the concussion mixing places 36 ℃～38 ℃ waters bath with thermostatic control to react 8min～10min;
E. take out described TB-IgG ELISA Plate, take the shrouding film off, with using cleansing solution washing 5 times, cleansing solution is filled with in every hole during washing, stops 20 seconds at every turn, pats dry;
F. respectively add 50 μ l enzyme conjugates solution in sample well, positive control hole and critical control wells, mixing sticks the shrouding film, places 36 ℃～38 ℃ waters bath with thermostatic control to react 8min～10min;
G. take out described TB-IgG ELISA Plate, take the shrouding film off, with cleansing solution washing at least 5 times, cleansing solution is filled with in every hole during washing, stops 20 seconds at every turn, pats dry;
H. in sample well, positive control hole and critical control wells, respectively add 50 μ l substrate solution A, 30 μ l substrate solution B, mixing sticks the shrouding film, reacts 8min～10min in 36 ℃～38 ℃ waters bath with thermostatic control;
I. take the shrouding film off, observations: if only judge the yin and yang attribute that detects sample, adopt visual method, the color of comparative sample hole and positive control hole, critical control wells can draw testing result respectively; If need read and detect accurately numerical value, behind step h, in sample well, add again 50 μ l stop buffers, behind the mixing, use the microplate reader reading result.
9. the using method of tubercle bacillus IgG antibody ELISA immunity detection reagent as claimed in claim 8 is characterized in that: if test serum can not in time detect, need described test serum is frozen-20 ℃～-15 ℃ conditions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310264724.2A CN103323604B (en) | 2013-06-27 | 2013-06-27 | A kind of tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit and preparation and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310264724.2A CN103323604B (en) | 2013-06-27 | 2013-06-27 | A kind of tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit and preparation and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103323604A true CN103323604A (en) | 2013-09-25 |
| CN103323604B CN103323604B (en) | 2015-12-23 |
Family
ID=49192481
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310264724.2A Active CN103323604B (en) | 2013-06-27 | 2013-06-27 | A kind of tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit and preparation and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103323604B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114966055A (en) * | 2022-05-26 | 2022-08-30 | 北京美联泰科生物技术有限公司 | Application of buffer solution in detection kit for acidic protein in glial fibers |
| CN115097138A (en) * | 2022-05-26 | 2022-09-23 | 北京美联泰科生物技术有限公司 | Application of buffer solution in PGP9.5 detection kit |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6245331B1 (en) * | 1997-01-02 | 2001-06-12 | New York Univ. Medical Center | Early detection of mycobacterial disease |
| CN1908667A (en) * | 2005-08-05 | 2007-02-07 | 珠海经济特区海泰生物制药有限公司 | Toxoplasmosis IgM antigen testing reagent and its application |
| CN102253225A (en) * | 2011-04-20 | 2011-11-23 | 珠海经济特区海泰生物制药有限公司 | Kit for detecting Southeast Asia type alpha-thalassemia |
| CN102520155A (en) * | 2011-12-13 | 2012-06-27 | 潍坊市康华生物技术有限公司 | Clenbuterol hydrochloride assay kit and its preparation method and use method |
| CN102660559A (en) * | 2012-04-28 | 2012-09-12 | 吉林大学 | Mycobacterium tuberculosis (TB) recombinant protein and preparation method thereof |
| CN102707052A (en) * | 2012-05-11 | 2012-10-03 | 中国农业科学院北京畜牧兽医研究所 | Bovine tuberculosis detection reagent containing recombinant protein mixture |
| CN102866254A (en) * | 2012-09-21 | 2013-01-09 | 潍坊市康华生物技术有限公司 | High-sensitivity antenatal screening kit of Down's Syndrome in pregnant women at second trimester as well as preparation and detection methods of kit |
| CN103063841A (en) * | 2012-12-24 | 2013-04-24 | 潍坊市康华生物技术有限公司 | Epidemic hemorrhagic fever (EHF) virus immunoglobulin M (I g M) antibody enzyme-linked immunoassay detection kit and preparation and use method thereof |
-
2013
- 2013-06-27 CN CN201310264724.2A patent/CN103323604B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6245331B1 (en) * | 1997-01-02 | 2001-06-12 | New York Univ. Medical Center | Early detection of mycobacterial disease |
| CN1908667A (en) * | 2005-08-05 | 2007-02-07 | 珠海经济特区海泰生物制药有限公司 | Toxoplasmosis IgM antigen testing reagent and its application |
| CN102253225A (en) * | 2011-04-20 | 2011-11-23 | 珠海经济特区海泰生物制药有限公司 | Kit for detecting Southeast Asia type alpha-thalassemia |
| CN102520155A (en) * | 2011-12-13 | 2012-06-27 | 潍坊市康华生物技术有限公司 | Clenbuterol hydrochloride assay kit and its preparation method and use method |
| CN102660559A (en) * | 2012-04-28 | 2012-09-12 | 吉林大学 | Mycobacterium tuberculosis (TB) recombinant protein and preparation method thereof |
| CN102707052A (en) * | 2012-05-11 | 2012-10-03 | 中国农业科学院北京畜牧兽医研究所 | Bovine tuberculosis detection reagent containing recombinant protein mixture |
| CN102866254A (en) * | 2012-09-21 | 2013-01-09 | 潍坊市康华生物技术有限公司 | High-sensitivity antenatal screening kit of Down's Syndrome in pregnant women at second trimester as well as preparation and detection methods of kit |
| CN103063841A (en) * | 2012-12-24 | 2013-04-24 | 潍坊市康华生物技术有限公司 | Epidemic hemorrhagic fever (EHF) virus immunoglobulin M (I g M) antibody enzyme-linked immunoassay detection kit and preparation and use method thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114966055A (en) * | 2022-05-26 | 2022-08-30 | 北京美联泰科生物技术有限公司 | Application of buffer solution in detection kit for acidic protein in glial fibers |
| CN115097138A (en) * | 2022-05-26 | 2022-09-23 | 北京美联泰科生物技术有限公司 | Application of buffer solution in PGP9.5 detection kit |
| CN114966055B (en) * | 2022-05-26 | 2023-08-18 | 北京美联泰科生物技术有限公司 | Application of buffer solution in colloidal fiber acidic protein detection kit |
| CN115097138B (en) * | 2022-05-26 | 2023-11-17 | 北京美联泰科生物技术有限公司 | Application of buffer solution in PGP9.5 detection kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103323604B (en) | 2015-12-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Naot et al. | An enzyme-linked immunosorbent assay for detection of IgM antibodies to Toxoplasma gondii: use for diagnosis of acute acquired toxoplasmosis | |
| AU2002248996B2 (en) | Detection of candida | |
| CN207516379U (en) | A kind of colloidal gold immuno-chromatography test paper strip | |
| CN101377504A (en) | Chemiluminescence immune analysis determination reagent kit for detecting Toxoplasma Gondi IgM antibody | |
| CN1877331B (en) | Foot-and-mouth disease virus detecting test paper tape and its preparation method and using method | |
| CN101377493A (en) | Kidney syndrome blooding diagnosis test paper strip, preparing method and detection reagent kit thereof | |
| CN103091500A (en) | Antibody detection kit (OmpC-IgA) | |
| CN101178404B (en) | Human immunodeficiency virus antibody chemiluminescence immune analyzing diagnose reagent box and method of producing the same | |
| Silva et al. | Development of a new lateral flow assay based on IBMP-8.1 and IBMP-8.4 chimeric antigens to diagnose Chagas disease | |
| CN101592660B (en) | Brucellosis indirect enzyme-linked immunosorbent assay milk liquid antibody reagent kit | |
| CN103063841B (en) | Epidemic hemorrhagic fever (EHF) virus immunoglobulin M (I g M) antibody enzyme-linked immunoassay detection kit and preparation and use method thereof | |
| CN103969447A (en) | Vascular endothelial growth factor quantitative determination kit and light-emitting substrate solution thereof | |
| CN103323604B (en) | A kind of tubercle bacillus IgG antibody enzyme-linked immunologic detecting kit and preparation and application thereof | |
| CN103048477A (en) | Nanometer magnetic particle chemiluminescence detection kit for triiodothyronine as well as preparation method and detecting method of same | |
| CN105866409A (en) | Candida mannan antigen immunoassay kit and preparation method and application thereof | |
| Teimouri et al. | Assessment of an in-house enzyme-linked immunosorbent assay and IgG avidity test based on SAG1 and GRA7 proteins for discriminating between acute and chronic toxoplasmosis in humans | |
| CN102368068A (en) | Kit for detecting chlamydia pneumoniae IgM antibody | |
| CN102749446A (en) | Chlamydia pneumoniae IgM (immunoglobulin M) colloidal golden method kit and preparation method thereof | |
| CN102809653A (en) | Preparation and application of ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting novel bunyavirus antigen | |
| CN109633162A (en) | S-100 β protein detection kit | |
| CN102095858A (en) | Western blot kit for cardiolipin and specific IgM antibodies of syphilis and preparation thereof | |
| CN109738642A (en) | The kit and its detection method of dual-antigen sandwich method detection immunoglobulin M | |
| CN201926664U (en) | Kit for immunoblot assay of specific IgG antibody against Treponema pallidum | |
| CN108761091A (en) | Double-antibody method quickly detects the test strips of HPV antibody, test card and preparation method thereof | |
| CN103149356B (en) | A kind of Test paper card utilizing sandwich method to detect Brucella abortus antigen |
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 | ||
| CP03 | Change of name, title or address | ||
| CP03 | Change of name, title or address |
Address after: No.699, Yuehe Road, Weifang Economic Development Zone, Weifang City, Shandong Province Patentee after: Shandong Kanghua Biomedical Technology Co., Ltd Address before: 261023 Shandong city of Weifang Province River Road Economic Development Zone No. 699 Patentee before: WEIFANG KANGHUA BIOTECH Co.,Ltd. |