MHC III/HLA - III imported testing kit brand

Purpose of testing: Used to determine samples such as serum, plasma, and related fluids. For example, suitable for detecting specimens including serum, plasma, urine, pleural and peritoneal fluid, lavage fluid, cerebrospinal fluid, cell culture supernatant, tissue homogenate, etc . Required but not provided

Product full name:MHC III/HLA - III imported detection kit

English name: MHC III/HLA III ELISA Kit

Product Code: A096798

Specification: 48T/96T

Service: We can provide free testing services, as well as product manuals and technical guidance

Storage environment: 2-8 ℃ low temperature, away from light, moisture-proof

Main ingredients: enzyme-linked immunosorbent assay (ELISA) plates, reagents, standards, etc

Kit composition and reagent preparation:

Detection program:

1. Sample addition: Add 100ul of standard or test sample to each well, mix the reaction plate thoroughly, and place it at 37 ℃ for 120 minutes.

2. Plate washing: Wash the reaction plate thoroughly 4-6 times with washing solution and print it dry on filter paper.

3. Add 100ul of antibody working solution to each well. Mix the reaction plate thoroughly and place it at 37 ℃ for 60 minutes.

4. Board washing: Same as before.

5. Add 100ul of enzyme labeled antibody working solution to each well. Place the reaction plate at 37 ℃ for 30 minutes.

6. Board washing: Same as before.

7. Add 100ul of substrate working solution to each well and let it react in the dark at 37 ℃ for 15 minutes.

8. Add 100ul of termination solution to each well and mix well.

Measure the absorbance value at 450 nm using an enzyme-linked immunosorbent assay reader within 30 minutes.

Preparation before sample experiment:

ELISA kit liquid samples: including serum, plasma, urine, pleural and peritoneal fluid, cerebrospinal fluid, cell culture supernatant, etc.

(1) Serum

After natural coagulation of blood at room temperature for 10-20 minutes, centrifuge for about 20 minutes (2000-3000 revolutions per minute). Carefully collect the supernatant. If precipitation forms during storage, it should be centrifuged again.

(2) Plasma:

EDTA, sodium citrate, or heparin should be selected as anticoagulants according to the requirements of the specimen. After mixing for 10-20 minutes, centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant. If precipitation forms during storage, it should be centrifuged again.

(3) Urine:

Collect with sterile tubes. Centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant. If precipitation forms during storage, it should be centrifuged again. Follow this procedure for pleural effusion, ascites, and cerebrospinal fluid.

(4) Cell culture supernatant:

When detecting secretory components, collect them using sterile tubes. Centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant.

(5) Cultivate cells

When detecting intracellular components, dilute the cell suspension with PBS (pH 7.2-7.4) to a cell concentration of around 1 million/ml. By repeatedly freezing and thawing or adding tissue protein extraction reagents, cells are destroyed and intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant. If precipitation forms during storage, it should be centrifuged again.

(6) Organizational specimen

After cutting the specimen, weigh it. Add a certain amount of PBS, pH 7.4. Quickly freeze and store in liquid nitrogen for future use. The specimen remains at a temperature of 2-8 ℃ even after melting. Add a certain amount of PBS (pH 7.4) or tissue protein extraction reagent, and homogenize the specimen manually or with a homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant. After packaging, one portion is to be tested, and the rest is to be frozen for future use.

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Operation process:

(1) Add 100 μ l of the test sample to each well of the test sample, and set up 3 parallel wells for each type of sample; Set up two negative control wells and add 100 μ l of untreated cell lysate to each well; Set up another blank control well and add 100 μ l of pure cell lysate.

(2) Place the enzyme-linked immunosorbent assay (ELISA) plate at 4 ℃ and coat overnight.

(3) Plate washing: Absorb the reaction solution in the well, rinse it once with washing solution (after filling the plate well with washing solution, shake it off), then fill the plate well with washing solution, soak for 1-2 minutes, and shake intermittently. Shake off the liquid inside the hole and pat dry on absorbent paper. Repeat washing 3-4 times.

(4) Add 50 μ l of PBS to each negative control well, and add 50 μ l of 1:500 diluted rabbit anti human AIF antibody working solution to each sample well and blank well.

(5) Place the enzyme-linked immunosorbent assay (ELISA) plate in a wet box at 37 ℃ and incubate for 60 minutes.

(6) Wash the board, same as (4).

(7) Add 100 μ l of HRP labeled goat anti rabbit antibody working solution diluted 1:5000 to each well.

(8) Place the enzyme-linked immunosorbent assay (ELISA) plate in a wet box at 37 ℃ and incubate for 60 minutes.

(9) Wash the board, same as (4).

(10) Add 100 μ l of TMB chromogenic solution to each well, gently mix for 10 seconds, and let it react in the dark at 37 ℃ for 15-20 minutes.

(11) Add 100 μ l of 2mol/L H2SO4 to each well to terminate the reaction.

(12) Measure the absorbance values W1 and W2 at 450nm and 630nm respectively, and the final measured OD value is the difference between the two (W1-W2) to reduce light interference caused by scratches or fingerprints on the container.

(13) Data processing: After obtaining the OD values of the specimen (S) and negative control (N), calculate the S/N value. S/N ≥ 2.1 is the positive judgment criterion.