**Human Anti-Nuclear Antibody (ANA) ELISA Kit Instruction Manual**
**ELISA Kit Specifications:**
This Human Anti-Nuclear Antibody (ANA) ELISA Kit is available in a 48-well or 96-well configuration, designed for high-throughput and accurate detection of ANA in human serum or plasma samples. The kit includes all necessary reagents for performing the immunoassay.
**Standard Dilution:**
- 1.5 mL × 1 vial of enzyme standard reagent (for 48-well)
- 3 mL × 1 vial of enzyme standard reagent (for 96-well)
**Enzyme Standard Package:**
- 1×48 (for 48-well configuration)
- 1×96 (for 96-well configuration)
- Store at 2–8°C
**Sample Diluent:**
- 3 mL × 1 vial (for 48-well)
- 6 mL × 1 vial (for 96-well)
- Store at 2–8°C
**Developer A Solution:**
- 3 mL × 1 vial (for 48-well)
- 6 mL × 1 vial (for 96-well)
- Store at 2–8°C
**Chromogen B Solution:**
- 3 mL × 1 vial (for 48-well)
- 6 mL × 1 vial (for 96-well)
- Store at -20°C
**Stop Solution:**
- 3 mL × 1 vial (for 48-well)
- 6 mL × 1 vial (for 96-well)
- Store at 2–8°C
**Concentrated Washing Solution:**
- 20 mL × 20 times (for 48-well)
- 20 mL × 30 times (for 96-well)
- Store at 2–8°C
**Sealing Film:**
- 2 pieces (for 48-well)
- 2 pieces (for 96-well)
**Instructions:**
The kit contains a standard with a concentration of 2700 ng/L, provided in 0.5 mL × 1 vial. This standard should be used to generate a standard curve for accurate quantification of ANA levels in test samples.
**Procedure Summary:**
1. Prepare the standard solutions according to the instructions.
2. Add the standards, controls, and samples to the appropriate wells.
3. Incubate and wash the plate as described in the manual.
4. Add the enzyme conjugate, incubate, and then add the chromogen.
5. Stop the reaction using the stop solution.
6. Measure the absorbance (OD) at 450 nm using a microplate reader.
**Data Analysis:**
- Plot the standard curve by plotting the concentration of the standard on the x-axis and the corresponding OD values on the y-axis.
- Determine the sample concentration from the standard curve.
- Multiply the calculated concentration by the dilution factor to obtain the actual sample concentration.
- Alternatively, use linear regression analysis to derive an equation from the standard curve and input the sample OD value into the equation for precise calculation.
**Storage Conditions:**
All reagents should be stored at 2–8°C unless otherwise specified. Chromogen B must be stored at -20°C to maintain stability.
**Important Notes:**
This kit is intended for research use only and not for diagnostic purposes. Follow all safety precautions when handling reagents and biological samples. Always use fresh reagents and avoid contamination during the assay process.
**Note:** For optimal performance, ensure that all reagents are brought to room temperature before use and that the plate is properly sealed during incubation steps.
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