Human antinuclear antibody (ANA) ELISA test kit

This kit can only be used for scientific research, not for medical diagnosis

Human antinuclear antibody (ANA) ELISA test kit

user's Guide

Detection principle

The kit uses double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). To the coated microwells pre-coated with human anti-nuclear antigen, add specimens, standards, and HRP-labeled detection antibodies in sequence, incubate and wash thoroughly. The color is developed with the substrate TMB, which is converted into blue under the catalysis of peroxidase and into the final yellow under the action of acid. The color depth is positively correlated with the human antinuclear antibody (ANA) in the sample. Measure the absorbance (OD value) at 450nm with a microplate reader to determine whether the sample contains human anti-nuclear antibody (ANA).

Sample collection, processing and storage methods

1. Serum: Use a test tube that does not contain pyrogens and endotoxins. Avoid any cell stimulation during the operation. After collecting blood, centrifuge at 3000 rpm for 10 minutes to quickly and carefully separate serum and red blood cells.

2. Plasma: EDTA, citrate or heparin anticoagulation. Take the supernatant by centrifugation at 3000 rpm for 30 minutes.

3. Cell supernatant: centrifuge at 3000 rpm for 10 minutes to remove particles and polymers.

4. Tissue homogenate: Add tissue to the right amount of saline and mash. Take the supernatant by centrifugation at 3000 rpm for 10 minutes.

5. Preservation: If the sample is not tested in time after collection, please aliquot it in one dose and freeze it at -20 ℃ to avoid repeated freezing and thawing. Thaw at room temperature and ensure that the sample is thawed evenly and fully.

Bring your own items

1. Microplate reader (450nm)

2. High-precision sampler and tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL

3. 37 ℃ thermostat

Operation notes

1. Store the kit at 2-8 ° C and equilibrate at room temperature for 20 minutes before use. The concentrated washing liquid taken out of the refrigerator will have crystals, which is a normal phenomenon. The water bath is heated to completely dissolve the crystals before use.

2. The slats not used in the experiment should be immediately returned to the ziplock bag, sealed (dry at low temperature) and stored.

3. The sample after pretreatment does not need to be diluted, just take 50μL and add it.

4. Perform the incubation operation strictly in accordance with the time, amount of liquid and sequence indicated in the manual.

5. Shake all liquid components thoroughly before use.

Kit composition


96-well configuration

48 hole configuration



12 holes × 8

12 holes × 4 strips


Negative control




Positive control




Sample diluent




Detection antigen-HRP




20 × washing buffer



Dilute according to the instructions

Substrate A




Substrate B




Stop solution




Sealing film

2 sheets

2 sheets



1 serving

1 serving


Ziplock bag




Reagent preparation

Dilution of 20 × washing buffer: 1:20 dilution of distilled water, that is, 1 part of 20 × washing buffer plus 19 parts of distilled water.

Washing method

1. Manually wash the plate: throw away all the liquid in the hole, fill each hole with the washing liquid, leave the liquid in the hole after standing for 1 min, pat dry on the absorbent paper, and wash the plate 5 times in this way.

2. Automatic plate washing machine: Inject 350μL of washing liquid into each well, soak for 1min, and wash the plate 5 times.


1. Take out the required slats from the aluminum foil bag after equilibrating at room temperature for 20 min. The remaining slats are sealed with a ziplock bag and put back at 4 ° C.

2. Set negative and positive control wells and sample wells, add 50 μL of negative control and positive control to the negative and positive control wells;

3. Add 10μL of the sample to be tested first, and then add 40μL of the sample diluent;

4. Then add 100 μL of horseradish peroxidase (HRP) -labeled detection antigen to each well of the negative and positive control wells and the sample wells, seal the reaction wells with sealing plates, and incubate for 60 minutes in a 37 ° C water bath or incubator.

5. Discard the liquid, pat dry on the absorbent paper, fill each well with the washing solution, let stand for 1min, shake off the washing solution, pat dry on the absorbent paper, and repeat washing the plate 5 times (you can also wash the plate with a washing machine)

6. Add 50 μL of substrate A and B to each well, and incubate at 37 ° C in the dark for 15 minutes.

7. Add 50μL of stop solution to each well, and measure the OD value of each well at 450nm wavelength within 15min.

Result judgment

1. Test effectiveness: the average value of OD value of positive control well ≥1.00;

The average value of OD value of negative control wells was ≤0.15.

2. Cut-off value (Cut off) calculation: cut-off value = average value of negative control well +0.15

3. Negative judgment: the sample OD value <critical value (Cut off), the sample is negative

4. Positive judgment: the OD value of the sample> cut off value, the sample is positive

Kit performance

1. Accuracy: the average OD value of positive control wells is ≥1.00; the average OD value of negative control wells is ≤0.15, indicating that the test results are valid.

2. Specificity: Does not cross-react with other soluble structural analogs.

3. Repeatability: The coefficients of variation within and between plates are less than 15%.

4. Storage: 2-8 ℃, protected from light and moisture.

5. Validity: 6 months

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