Usage of ischemia modified albumin assay kit

The usage method of the Ischemic Modified Albumin (IMA) assay kit is as follows:

Principle and composition of the kit

The IMA kit uses a double antibody sandwich enzyme-linked immunosorbent assay (ELISA). The sample, standard, and HRP labeled detection antibody are added sequentially through a microplate coated with IMA capture antibody. After incubation and washing, the substrate TMB is added for color development. The color reaction changes from blue to yellow under the catalysis of peroxidase, and the color intensity is proportional to the concentration of IMA in the sample. Finally, the absorbance (OD value) was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450nm, and the sample concentration was calculated using a standard curve. A reagent kit typically includes components such as an enzyme-linked immunosorbent assay (ELISA) plate, standard, sample diluent, HRP labeled antibody, substrate solution, stop buffer, and wash buffer.

Sample collection and preservation

Serum: Whole blood samples are left to stand at room temperature for 2 hours or overnight at 4 ℃, then centrifuged at 1000g for 20 minutes to obtain the supernatant;

; Or store at -20 ℃/-80 ℃ to avoid repeated freezing and thawing.

Plasma: Anticoagulate with EDTA or heparin, centrifuge at 2-8 ℃ and 1000g for 20 minutes within 30 minutes after collection, and collect the supernatant;

; Or store at low temperature to avoid repeated freezing and thawing. Other specimens: Cell culture supernatant, tissue homogenate, etc. need to be centrifuged at 1000g for 20 minutes to obtain the supernatant, stored at low temperature, and avoid repeated freezing and thawing. Hemolytic specimens are not suitable for testing.

Reagent Preparation

Reagent Balance: All reagents should be restored to room temperature (20-25 ℃) before use to avoid direct heating and dissolution.

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Standard dilution: Dilute the standard with a gradient of standard dilution solution according to the instructions (such as 120, 60, 30, 15, 7.5, 0 U/mL), and use fresh dilution solution for each experiment.

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Wash buffer dilution: Dilute the concentrated wash solution with distilled water at a ratio of 1:20 (e.g. 1 part 20 x wash buffer plus 19 parts distilled water).

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Operation steps

Sample addition: Add 50 μ L of standard or test sample to each well (if the sample concentration is too high, it needs to be diluted in advance), and do not add blank wells.

. Incubation: After sealing the plate, incubate at 37 ℃ for 60 minutes to fully bind the antigen and antibody. Washing: Discard the liquid, fill each well with washing solution and let it stand for 1 minute before shaking dry. Repeat washing 5 times and pat dry.

Enzyme labeled antibody: Add 100 μ L of HRP labeled detection antibody to each well (excluding blank wells), and incubate at 37 ℃ for 30 minutes after plate sealing.

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Wash again: Repeat the same steps 5 times.

. Color development: Add 50 μ L of substrate A and 50 μ L of substrate B to each well, and incubate at 37 ℃ in the dark for 15 minutes. Termination reaction: Add 50 μ L of termination solution to each well, and the blue color immediately turns yellow.

Determination of OD value: Within 15 minutes, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each well at a wavelength of 450nm.

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Result Calculation

Draw a standard curve: Using standard concentration as the x-axis and OD value as the y-axis, use coordinate paper or software to draw the curve and fit a linear regression equation.

. Calculate sample concentration: Substitute the sample OD value into the equation to obtain the concentration. If the sample is diluted, it needs to be multiplied by the dilution factor.

Precautions

Avoid contamination: Clean tips should be used when adding reagents and samples to prevent cross contamination;

; The sealing film is only for one-time use.

Operating instructions: During incubation, ensure that the micropores are not dry and wash thoroughly to reduce background interference;

; The substrate solution should be stored away from light and cannot be used after turning blue.

Safety protection: Avoid direct contact with the termination solution and substrate solution during the experiment. If in contact, rinse immediately with water.

; Do not suck reagents with your mouth.

Storage conditions: Unopened reagent kits should be stored at 2-8 ℃. After opening, the remaining enzyme-linked immunosorbent assay plates should be sealed and dried, and stored at -20 ℃.

; The expiration date of the reagent kit is usually 6 months.