Human Anti-Endothelial Cell Antibody (AECA) ELISA Kit Procedure

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This reagent is for research use only: This kit is used to determine the content of human anti-endothelial cell antibody (AECA) in human serum, plasma and related liquid samples.

Experimental principle :

The kit uses a double antigen sandwich assay to determine the level of human anti-endothelial cell antibody (AECA) in the specimen. The microporous plate is coated with the purified antigen to prepare a solid phase antigen, and the anti-endothelial cell antibody (AECA) is sequentially added to the coated micropore, and then combined with the HRP-labeled antigen to form an antigen-antibody-enzyme-labeled antigen complex. After thoroughly washing, add the substrate TMB to develop color. TMB is converted to blue under the catalysis of HRP enzyme and converted to the final yellow color by the action of an acid. The color depth is positively correlated with the anti-endothelial cell antibody (AECA) in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader, and the human anti-endothelial cell antibody (AECA) content in the sample was calculated from a standard curve.

Kit composition :

Kit composition

48 hole configuration

96-well configuration

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Instruction manual

1 copy

1 copy

Sealing film

2 pieces (48)

2 pieces (96)

sealed bag

1

1

Enzyme label coated plate

1×48

1×96

Store at 2-8 ° C

Standard: 90ng/L

0.5ml × 1 bottle

0.5ml × 1 bottle

Store at 2-8 ° C

Standard dilution

1.5ml × 1 bottle

1.5ml × 1 bottle

Store at 2-8 ° C

Enzyme standard reagent

3 ml × 1 bottle

6 ml × 1 bottle

Store at 2-8 ° C

Sample diluent

3 ml × 1 bottle

6 ml × 1 bottle

Store at 2-8 ° C

Developer A solution

3 ml × 1 bottle

6 ml × 1 bottle

Store at 2-8 ° C

Developer B solution

3 ml × 1 bottle

6 ml × 1 bottle

Store at 2-8 ° C

Stop solution

3ml × 1 bottle

6ml × 1 bottle

Store at 2-8 ° C

Concentrated washing solution

(20ml × 20 times) × 1 bottle

(20ml × 30 times) × 1 bottle

Store at 2-8 ° C

Sample processing and requirements :

1. Serum: The blood is naturally coagulated for 10-20 minutes at room temperature and centrifuged for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if precipitation occurs during storage, it should be centrifuged again.

2. Plasma: EDTA or sodium citrate should be selected as an anticoagulant according to the requirements of the specimen. After mixing for 10-20 minutes, centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again.

3. Urine: Collect with a sterile tube and centrifuge for about 20 minutes (2000-3000 rpm). The supernatant is carefully collected, and if a precipitate forms during storage, it should be centrifuged again. The chest and ascites and cerebrospinal fluid are referred to.

4. Cell culture supernatant: When detecting secreted components, collect them in a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When the components in the cells were detected, the cell suspension was diluted with PBS (pH 7.2-7.4), and the cell concentration reached about 1 million/ml. By 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. Tissue specimen: After cutting the specimen, weigh the weight. Add a certain amount of PBS, pH 7.4. It was quickly frozen and stored in liquid nitrogen for use. The specimen still maintains a temperature of 2-8 ° C after melting. A certain amount of PBS (pH 7.4) was added, and the specimen was homogenized by hand or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. One part of the package is to be tested, and the rest is frozen for use.

6. The specimens should be extracted as soon as possible after collection, and the extraction should be carried out according to the 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 °C, but repeated freezing and thawing should be avoided.

7. Samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.

Steps

  • Dilution and loading of standard products: 10 wells of standard wells are placed on the enzyme-labeled plate, 100 μl of standard is added to the first and second wells, and standard dilutions are added to the first and second wells. 50μl, mix; then add 100μl from the first hole and the second hole to the third hole and the fourth hole respectively, and then add 50μl of the standard dilution solution in the third and fourth holes, and mix; Take 50 μl of each of the third and fourth wells, discard them, add 50 μl to each of the fifth and sixth wells, and add 50 μl of the standard dilution solution to the fifth and sixth wells, mix and mix; Then, 50 μl of each of the fifth and sixth holes are respectively added to the seventh and eighth holes, and then 50 μl of the standard dilution solution is added to the seventh and eighth holes, respectively, and the seventh and eighth holes are mixed after mixing. 50 μl of each was added to the ninth and tenth holes, and 50 μl of the standard dilution was added to the ninth and tenth holes, respectively, and 50 μl of each of the ninth and tenth holes was discarded. (The amount of each well was 50 μl after dilution, and the concentrations were 60 ng/L, 40 ng/L, 20 ng/L, 10 ng/L, and 5 ng/L, respectively.
  • Adding samples: Set blank holes separately (the blank control holes are not added with the sample and the enzyme standard reagent, the other steps are the same), and the sample holes to be tested. Add 40 μl of the sample dilution to the sample well to be tested on the enzyme-labeled plate, and then add 10 μl of the sample to be tested (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, and shake gently to mix.
  • Incubation: The plate was sealed with a sealing film and incubated at 37 ° C for 30 minutes.
  • Solution: 30 (48 times of 20T) concentrated washing solution was diluted with distilled water 30 (20 times of 48T) and used.
  • Washing: Carefully remove the sealing film, discard the liquid, dry it, fill each well with the washing solution, let stand for 30 seconds, then discard it, repeat 5 times, and pat dry.
  • Add enzyme: 50 μl of enzyme labeling reagent was added to each well, except for blank wells.
  • Incubation: The operation is the same as 3.
  • Washing: The operation is the same as 5.
  • Color development: Add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake and mix, and develop color at 37 ° C for 15 minutes.
  • Termination: 50 μl of stop solution was added to each well to terminate the reaction (in this case, the blue color turned yellow).
  • Measurement: The absorbance (OD value) of each well was measured sequentially with a blank air conditioner of zero and a wavelength of 450 nm. The measurement should be carried out within 15 minutes after the addition of the stop solution.

Precautions:

  • The kit should be taken out from the refrigerated environment and should be used at room temperature for 15-30 minutes before use. If the enzyme label is unsealed after opening, the slats should be stored in a sealed bag.
  • Concentrated washing liquid may crystallize out. When diluted, it can be heated and dissolved in a water bath. The washing will not affect the result.
  • The sampler should be used for each step, and the accuracy should be corrected frequently to avoid test errors. It is best to control the loading time within 5 minutes. If the number of specimens is large, it is recommended to use a gun.
  • Please make a standard curve at the same time of each measurement, it is best to make a double hole. If the content of the substance to be tested in the specimen is too high (the OD value of the sample is larger than the OD value of the first hole of the standard well), please first dilute the sample dilution with a certain multiple (n times) and then measure it. When calculating, multiply the total dilution by the total dilution. Multiple (×n×5).
  • The sealing film is intended for single use only to avoid cross-contamination.
  • Please keep the substrate away from light.
  • Strictly follow the instructions of the manual, the test results must be based on the microplate reader reading.
  • All samples, washings and various wastes should be treated as infectious materials.
  • The different batch components of this reagent must not be mixed.

10. In the case of an English manual, the English manual shall prevail.

Calculation :

Taking the concentration of the standard as the abscissa and the OD as the ordinate, draw a standard curve on the coordinate paper, and find the corresponding concentration from the standard curve according to the OD value of the sample; multiply by the dilution factor; or use the concentration of the standard Calculate the linear regression equation of the standard curve with the OD value, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor, which is the actual concentration of the sample.

Kit performance:

1. The linear regression coefficient of the sample and the expected concentration correlation coefficient R value is 0.990 or more.

2. Within and within the batch should be less than 9% and 11% respectively

examination range:

2ng/L -80ng/L

Storage conditions and expiration date:

1. Kit storage: 2-8 ° C.

2. Validity: 6 months

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