Rat collagen Ⅳ (Col Ⅳ) enzyme-linked immunosorbent assay kit using method and precautions

This kit is used to quantitatively detect the content of type IV collagen (Col IV) in serum, plasma, tissue, cell supernatant and related liquid samples in vitro

Validity: 6 months

Storage condition: 2-8℃

The kit uses a double-antibody one-step sandwich method enzyme-linked immunosorbent assay (ELISA)

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Note: Hemolysis of the specimen will affect the final test result, so this test is not suitable for hemolyzed specimens

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The kit should be equilibrated at room temperature after being removed from refrigeration before use

Dilution of 20× washing buffer: Dilute with distilled water at 1:20, that is, 1 part of 20× washing buffer plus 19 parts of distilled water

Steps:

1.
Take out the required slats from the aluminum foil bag after equilibrating at room temperature for 20 minutes, and seal the remaining slats in a ziplock bag and return to 4°C
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2.
Set standard wells and sample wells, and add 50 μL of different concentrations of standard to each standard well;

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Add 50 μL of the sample to be tested to the sample well; do not add the blank well
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In addition to blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of standard wells and sample wells, seal the reaction wells with sealing film, and incubate in a water bath or incubator at 37°C 60min
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Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350 μL), let stand for 1 min, shake off the washing solution, pat dry on absorbent paper, and repeat the plate washing 5 times (you can also wash it with a plate washer).
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Add 50 μL each of Substrate A and B to each well, and incubate at 37°C for 15 minutes in the dark
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Add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 minutes
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Precautions:

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Incubate strictly according to the specified time and temperature to ensure accurate results
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All reagents must reach room temperature 20-25°C before use
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Store reagents refrigerated immediately after use
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Improper plate washing can lead to inaccurate results
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Make sure to aspirate as much liquid as possible from the wells before adding the substrate
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Do not allow the wells to dry out during incubation
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Eliminate the remaining liquid and fingerprints on the bottom of the plate, otherwise the OD value will be affected
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The substrate chromogenic solution should be colorless or very light in color.
The substrate solution that has turned blue cannot be used
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Avoid cross-contamination of reagents and specimens to avoid erroneous results
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Avoid direct sunlight during storage and incubation
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After equilibrating to room temperature, open the airtight bag to prevent drips from condensing on the cold slats
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Any reagents should not come into contact with the bleaching solvent or the strong gas emitted by the bleaching solvent
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Any bleaching components will destroy the biological activity of the reagents in the kit
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Expired products cannot be used
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If disease transmission is possible, all samples should be managed, and samples and testing devices should be handled according to prescribed procedures
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Washing method:

Manual plate washing method: suck (do not touch the plate wall) or shake off the liquid in the ELISA plate; lay a few layers of absorbent paper on the laboratory table, and pat the ELISA plate downward several times; put 30 (20 times of 48T) The concentrated washing solution was diluted 30 times with distilled water (20 times of 48T) and used for later use
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Repeat this process as many times as necessary
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Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment after skilled use
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Calculation of experimental results:

Take the OD value of the measured standard as the abscissa and the concentration of the standard as the ordinate, draw the standard curve on the graph paper or with the relevant software, and obtain the linear regression equation, and substitute the OD value of the sample into the equation to calculate the sample.
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