- Monkey serum or plasma samples
- ANA ELISA Kit (including pre-coated plates, standards, controls, and reagents)
- Sample diluent or buffer
- Wash buffer
- Enzyme-conjugated secondary antibodies
- Substrate solution
- Stop solution
- Pipettes and pipette tips
- Microplate reader
Here's a step-by-step guide on how to proceed with the ANA ELISA Kit:
- Allow the kit components and samples to come to room temperature (if refrigerated) before starting the assay.
- Prepare the samples: Dilute the monkey serum or plasma samples with the sample diluent or buffer provided in the kit. Follow the kit instructions for the recommended dilution ratio. Mix the samples thoroughly to ensure homogeneity.
- Prepare the standards and controls:
- The kit will provide standards of known ANA concentrations and positive/negative controls. Follow the instructions to reconstitute or dilute them as specified.
- It's important to include both positive and negative controls in the assay for accurate interpretation of the results.
4. Plate set-up:
- Take the pre-coated plates from the kit and place them on a clean surface.
- Determine the number of wells needed for your samples, controls, and standards. Reserve a few wells for blank measurements.
- Label the wells accordingly to keep track of the samples, controls, and standards.
5. Add the standards, controls, and samples to the plate:
- Pipette the standards, controls, and diluted samples into the designated wells of the plate according to the manufacturer's instructions.
- Ensure accurate and consistent pipetting while avoiding cross-contamination.
6. Incubation :
- Cover the plate with a plate sealer or lid to prevent evaporation.
- Incubate the plate at the recommended temperature and duration as instructed by the kit manufacturer.
- This step allows the ANAs in the samples to bind to the pre-coated antigens.
7. Wash the plate :
- After the incubation period, carefully remove the liquid from the wells.
- Wash the plate multiple times with the provided wash buffer to remove any unbound substances.
8. Secondary antibody addition :
- Add the enzyme-conjugated secondary antibodies to each well of the plate, ensuring complete coverage.
- Incubate the plate again at the recommended temperature and duration as instructed by the kit manufacturer.
- The secondary antibodies will bind to the captured ANAs.
9. Wash the plate again :
- Repeat the washing steps described in Step 7 to remove any unbound secondary antibodies.
10. Substrate addition:
- Add the substrate solution to each well of the plate, covering all samples.
- Incubate the plate for the specified time period, allowing the enzymatic reaction to occur.
11. Stop the reaction:
- Add the stop solution to each well to halt the enzymatic reaction.
- he stop solution will typically change the color of the wells.
12. Read the absorbance:
- Using a microplate reader, measure the absorbance at the appropriate wavelength specified in the kit instructions.
- Record the optical density (OD) values for each well.
13. Data analysis:
-Compare the OD values of the samples, controls, and standards to determine the concentration or presence of ANAs.
- Follow the kit instructions for interpreting the results, as.