Bovine Mucosal Diarrhea Antibody (BD-Ab) Enzyme Linked Immunoassay (ELISA)

Kit instruction manual

This reagent is for research purposes only: this kit is used to detect the level of mucosal diarrhea antibody (BD-Ab) in bovine serum, plasma and related liquid samples, and hematological diagnosis of infected people.

Experimental principle:

This kit uses double antigen sandwich enzyme-linked immunoassay (ELISA) to determine the bovine mucosal diarrhea antibody (BD-Ab) in the specimen. Coated microtiter plates with purified bovine mucosal diarrhea disease (BD) to make solid phase antigens, which can be combined with mucosal diarrhea antibody (BD-Ab) in the sample, and then washed to remove unbound antigen and other components Combined with HRP-labeled mucosal diarrhea (BD) to form an antigen-antibody-enzyme-labeled antigen complex. After thorough washing, add substrate TMB to develop color. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm and compared with the CUTOFF value to determine the presence or absence of bovine mucosal diarrhea antibody (BD-Ab) in the specimen.

Kit composition:

Kit composition

48 hole configuration

96-well configuration

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Instructions

1 serving

1 serving

Sealing film

2 pieces (48)

2 pieces (96)

sealed bag

1

1

Enzyme coated plate

1 × 48

1 × 96

Store at 2-8 ℃

Negative control

0.5ml × 1 bottle

0.5ml × 1 bottle

Store at 2-8 ℃

Positive control

0.5ml × 1 bottle

0.5ml × 1 bottle

Store at 2-8 ℃

Enzyme reagent

3 ml × 1 bottle

6 ml × 1 bottle

Store at 2-8 ℃

Sample diluent

3 ml × 1 bottle

6 ml × 1 bottle

Store at 2-8 ℃

Developer A liquid

3 ml × 1 bottle

6 ml × 1 bottle

Store at 2-8 ℃

Developer B liquid

3 ml × 1 bottle

6 ml × 1 bottle

Store at 2-8 ℃

Stop solution

3ml × 1 bottle

6ml × 1 bottle

Store at 2-8 ℃

Concentrated washing liquid

(20ml × 20 times) × 1 bottle

(20ml × 30 times) × 1 bottle

Store at 2-8 ℃

Sample processing and requirements:

1. Serum: room temperature blood coagulates naturally for 10-20 minutes, centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully and centrifuge again if a precipitate appears during storage.

2. Plasma: EDTA or sodium citrate should be selected as the anticoagulant according to the requirements of the specimen, mixed for 10-20 minutes, and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.

3. Urine: collected in a sterile tube and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, centrifuge again. Pleural and ascites, cerebrospinal fluid reference implementation.

4. Cell culture supernatant: When detecting secreted components, collect with a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When detecting the components inside the cells, dilute the cell suspension with PBS (PH7.2-7.4), and the cell concentration will reach about 1 million / ml. Through repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again.

5. Organize the specimen: after cutting the specimen, weigh it. Add a certain amount of PBS, PH7.4. Quickly freeze and save with liquid nitrogen for later use. After the specimen melts, it still maintains a temperature of 2-8 ° C. Add a certain amount of PBS (PH7.4) and homogenize the specimen with a manual or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. After aliquoting, a portion is to be tested, and the rest is frozen for future use.

6. The specimen should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided.

7. The sample containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP).

Steps:

1. Numbering: serially number the corresponding microwells of the sample, each plate should be set with 2 wells for negative control, 2 wells for positive control, and 1 well for blank control (the blank control well does not add sample and enzyme reagent, the rest of the steps are the same)

2. Add sample: add negative control and positive control 50μl to the negative and positive control wells respectively. Then add 40μl of sample diluent to the sample well, and then add 10μl of the sample to be tested. Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, gently shake to mix,

3. Incubation: Seal the plate with a sealing plate and incubate at 37 ° C for 30 minutes.

4. Mixing solution: Add 30 times (20 times of 48T) concentrated washing liquid to 600ml with distilled water and set aside

5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing liquid, let it stand for 30 seconds and then discard, repeat this 5 times, pat dry.

6. Add enzyme: add 50μl of enzyme label reagent to each well, except blank well.

7. Incubation: The operation is the same as 3.

8. Washing: The operation is the same as 5.

9. Color development: add 50μl of developer A to each well, and then add 50μl of developer B, mix gently, and develop for 15 minutes in the dark at 37 ℃

10. Termination: Add 50μl of stop solution to each well to stop the reaction (at this time the blue will turn to yellow).

11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.

Result judgment:

Test effectiveness: the average of positive control wells ≥1.00; the average of negative control wells ≤0.10

Calculation of critical value (CUT OFF): critical value = average value of negative control wells + 0.15

Negative judgment: the sample with OD value <cut-off value (CUT OFF) is negative for mucosal diarrhea antibody (BD-Ab)

Positive judgment: the sample with OD value ≥ critical value (CUT OFF) is positive for mucosal diarrhea antibody (BD-Ab)

Precautions

1. The operation is strictly in accordance with the instructions. The components of different batches of this reagent must not be mixed.

2. The kit should be equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment. If the enzyme label coated plate is unopened, the strip should be stored in a sealed bag.

3. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing.

4. The sealing film is limited to one-time use to avoid cross-contamination.

5. Please keep the substrate away from light.

6. The test results must be determined based on the reading of the microplate reader. When using dual wavelength detection, the reference wavelength is 630nm

7. All samples, washing liquids and various wastes should be treated as infectious agents. The stop solution is 2M sulfuric acid, and you must pay attention to safety when using it.

Storage conditions and validity period

1. Kit storage :; 2-8 ℃.

2. Validity: 6 months

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