Manual of Human Anti-sperm Antibody (AsAb) ELISA Kit

Manual of Human Anti-sperm Antibody (AsAb) ELISA Kit

This kit is for research use only.

Drug Name:

Generic name: Human anti-sperm antibody (AsAb) ELISA kit

purpose of usage:

This kit qualitatively measures anti-sperm antibodies (AsAb) in human blood or other related tissues

Experimental principle:

This kit uses double antigen sandwich enzyme-linked immunoassay (ELISA) to determine human anti-sperm antibody (AsAb) in the specimen. Coated microplate with purified sperm antigen to make solid phase antigen, which can be combined with human anti-sperm antibody (AsAb) in the sample, washed to remove unbound antibody and other components, and then combined with HRP labeled sperm antigen To form an antigen-antibody-enzyme-labeled antigen complex. After thorough washing, the substrate TMB is added for color development. 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 human anti-sperm antibody (AsAb) in the specimen.

Kit composition:

1

20 times concentrated washing liquid

50ml × 1 bottle

7

Stop solution

6ml × 1 bottle

2

Enzyme reagent

6ml × 1 / bottle

8

Standard (positive control)

0.5ml × 1 bottle

3

Enzyme coated plate

12 holes × 8

9

Standard dilution (negative control)

0.5ml × 1 bottle

4

Sample diluent

6ml × 1 bottle

10

Instructions

1 serving

5

Developer A liquid

6ml × 1 bottle

11

Sealing film

2 sheets

6

Developer B liquid

6ml × 1 bottle

12

sealed bag

1

Specimen requirements:

1. Specimen processing: serum and plasma specimens can be directly detected

2. Perform the experiment as soon as possible after the specimen is prepared as required. If it can not be detected in time, the specimen can be stored at -20 ℃ for one month, but repeated freezing and thawing should be avoided.

3. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.

Steps:

1. Numbering: Number the samples corresponding to the microwells in sequence. Each plate should be provided with 2 wells for negative control, 2 wells for positive control, and 1 well for blank control (the blank control wells do not add samples and enzyme reagents, the remaining steps are the same)

2. Add sample: Add 50μl of negative control and positive control (standard) 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 20 times concentrated washing liquid to distilled water to 1000ml

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 5 times and 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 at 37 ℃

10. Termination: Add 50μl of stop solution to each well to terminate the reaction (at this time, the blue color turns 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 cut-off value (CUT OFF): cut-off value = average value of negative control well + 0.15

Negative judgment: the sample with OD value <cut-off value (CUT OFF) is negative for human anti-sperm antibody (AsAb)

Positive judgment: the sample whose OD value ≥ critical value (CUT OFF) is positive for human anti-sperm antibody (AsAb)

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.

specification:

96 servings / box

Storage conditions and validity period

1. Kit storage :; 2-8 ℃.

2. Validity: 6 months

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