Dot-IGSS detection of bovine tuberculosis serum antibodies
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Dot-IGSS detection of bovine tuberculosis serum antibodies
(1) Materials and reagents
1. Antigen bovine tuberculosis PPD.
2. Serum Serum to be tested, standard tuberculosis positive serum and tuberculosis negative serum.
3. The nitrocellulose membrane has a pore size of 0.45µm.
4. Gold chloride (high quality)
5. 0.2Mol / L PB liquid
6. 0.05Mol / L Tris-HCl buffer solution
7. Silver nitrate developer (available now)
Gelatin (20g / L) 6.00ml
Hydroquinone (57.90g / L) 3.00ml
Silver nitrate (25g / L) 0.20ml
pH3.5 citrate buffer 1.00ml
The hydroquinone and silver nitrate solutions need to be protected from light. After the gelatin is heated and dissolved before use, it is mixed in sequence in the above ratio.
Preparation of pH3.5 citrate buffer:
Citric acid (C6H8O7 · H2O) 25.50g
Trisodium citrate (C6H5Na3 · 2H2O) 3.50g
Deionized water to 100.00ml
It can be used after dissolving.
8. Fixer
Sodium thiosulfate (Na2S2O3) 20.0g
Deionized water to 100.00ml
Just dissolve.
9. Rabbit anti-bovine IgG antibody.
(2) Operation method
1. Preparation of colloidal gold The test water requires three distilled waters and passes the ion column. The resistance of the deionized water is greater than 1 million Ω. All containers are finally washed with deionized water.
(1) Prepared by tannic acid-sodium citrate reduction method
Liquid A 1% HAuCl4 2.00ml
Deionized water 158.00ml
Liquid B 1% sodium citrate 8.00ml
0.1% Mol / L K2CO3 0.20ml
1% tannic acid 0.20ml
Deionized water 31.60ml
Put liquid A and liquid B in a 60 ° C water bath for 30 minutes, stirring occasionally. Quickly add liquid B to liquid A, stir, the color is dark blue, continue to heat to 100 ℃, 5 min ~ 10min, the color of the solution becomes dark Red, stored at 4 ℃ after natural cooling.
(2) Identification of colloidal gold: The appearance is deep red, crystal clear, and there is a light band facing the sunlight. Observed by electron microscope, the average size of colloidal gold particles is 10nm, and the particles are uniform in size and evenly distributed.
2. Determination of the ratio of colloidal gold to rabbit anti-bovine IgG The minimum amount of stable protein required per ml of colloidal gold is 30µg, which is increased by 20% according to the amount of reagent, so the minimum amount of stable rabbit anti-bovine IgG required per ml of colloidal gold is 36µg.
3. Preparation of gold-labeled rabbit anti-bovine IgG Take the required amount of rabbit anti-bovine IgG, add a little deionized water, adjust the pH to 9.0 with 0.1 Mol / L K2CO3 solution (measured with precision pH test paper), and the pH of the colloidal gold solution The value was also adjusted to 9.0. Under magnetic stirring, add 20ml of colloidal gold solution to rabbit anti-bovine IgG, continue to stir for 10min, add 3.5ml of 3% polyethylene glycol (molecular weight 20 000) as a stabilizer, so that its final concentration reaches 0.05%, After stirring for another 15 minutes, store in a refrigerator at 4 ° C for future use.
4. Purification of gold-labeled rabbit anti-bovine IgG The gold-labeled rabbit anti-bovine IgG was centrifuged at 2 500 r / min for 15 min to remove the colloidal gold polymer. The supernatant was centrifuged at 65 000 g for 1 h, and the centrifuge was divided into three layers. The upper layer It is light yellow, contains unconjugated rabbit anti-bovine IgG, and the lower layer is nearly black. It is unconjugated colloidal gold particles. The main middle layer is red. The middle layer liquid was collected, mixed with 0.01 Mol / L pH8.2PB containing 0.1% BSA to 1/10 of the original volume, filtered and sterilized, divided into aliquots, and stored at 4 ° C until use.
5. Identification of gold-labeled rabbit anti-bovine IgG
(1) Identification of particle size and uniformity: Take a little gold-labeled rabbit anti-bovine IgG, negatively stain, observe the particle size and uniformity under electron microscope, and measure the particle diameter.
(2) Activity identification: spotting bovine IgG on nitrocellulose membrane, directly adding gold-labeled rabbit anti-bovine IgG antibody, it should show obvious pink spots.
6. Formal test procedure
(1) Spotting The microporous filter membrane is made into a membrane with a diameter of 3 mm using a puncher. Dilute bovine tuberculosis PPD with 0.01Mol / LpH9.0PBS solution to 40μg / ml, add 1μl of antigen to the center of the membrane with a micro sampler, and dry at 37 ℃ for 10min.
(2) Blocking Place the membrane in 0.01Mol / L pH7.4 in 0.2% BSA in PBST solution, and incubate at 37 ° C for 45min with shaking several times. After taking out, it was washed once with distilled water and blotted dry with filter paper.
(3) Add serum to be tested Serum to be tested is diluted 1: 160 with PBST solution. The membrane was immersed in it and acted at 37 ° C for 1 h, during which it was shaken several times. After taking out and washing with PBST solution, the filter paper was blotted dry. In this step, standard positive serum and negative serum controls are set at the same time.
(4) Adding gold-labeled antibody Dilute the gold-labeled solution 1:10 with PBST solution, immerse the membrane in it, and incubate at 37 ° C for 2h, shaking several times in the middle.
(5) Silver stain Remove the membrane from the gold standard solution and wash it in deionized water 3 times for 3 minutes each time. Then the membrane was immersed in the silver staining solution in the dark room for 10 minutes at room temperature, and then moved into the fixing solution for 5 minutes at room temperature. Then rinse with deionized water and observe the results after drying naturally.
(3) Judgment of results:
＋＋＋ ～ ＋＋＋＋＋ ： Brown or black spots, dark coloration, uniformity and contrast with background.
＋＋: The color is darker or darker, but the background color is also darker.
＋: The color is light or dark, but there is a background color.
ï¼: No coloring, or no contrast with the background color.
A positive result appears above "++", otherwise a negative result.