**Rabbit Lipoprotein Phospholipase A2 (Lp-PLA2) ELISA Kit – User Manual**
**Purpose:**
This kit is intended for research use only and is designed to quantitatively measure the levels of Rabbit Lipoprotein Phospholipase A2 (Lp-PLA2) in rabbit serum, plasma, and other related biological samples. It utilizes a sandwich ELISA format to detect the target protein with high specificity and sensitivity.
**Principle of the Assay:**
The assay is based on an indirect ELISA method. The microtiter plate is pre-coated with a specific antibody against Lp-PLA2. After incubation with standards and samples, a biotinylated secondary antibody is added, followed by streptavidin-HRP conjugate. The enzyme reaction is developed using TMB substrate, which changes color in the presence of HRP. The intensity of the color is directly proportional to the concentration of Lp-PLA2 in the sample. Absorbance is measured at 450 nm, and the results are calculated using a standard curve.
**Kit Components:**
- 20× Washing Solution: 50 mL × 1 bottle
- Standards: S1 (180 μg/L), S2 (120 μg/L), S3 (60 μg/L), S4 (30 μg/L), S5 (15 μg/L) – 0.5 mL × 1 each
- Streptavidin-HRP Conjugate: 6 mL × 1 bottle
- Biotinylated Anti-IgG Antibody: 6 mL × 1 bottle
- TMB Developer A & B: 6 mL × 1 each
- Stop Solution: 6 mL × 1 bottle
- Sample Dilution Buffer: 6 mL × 1 bottle
- Microplate: 96-well strip plate × 1
- Sealing Membrane: 3 sheets × 1
- Instruction Manual: 1 copy
**Sample Requirements:**
- Avoid samples containing NaN3 as it may inhibit HRP activity.
- Samples should be processed immediately after collection. If not tested right away, store at -20°C and avoid repeated freeze-thaw cycles.
**Procedure Summary:**
1. Determine the number of wells needed, including standards, blanks, and samples.
2. Add 50 µL of standard or sample diluent, then 10 µL of sample and 40 µL of diluent to achieve a 5-fold dilution.
3. Incubate at 37°C for 45 minutes.
4. Wash 4 times with 20× diluted washing solution.
5. Add 50 µL of biotinylated anti-IgG antibody and incubate for 30 minutes.
6. Wash again.
7. Add 50 µL of streptavidin-HRP and incubate for 30 minutes.
8. Wash once more.
9. Add 50 µL of TMB A and B, incubate for 15 minutes.
10. Stop the reaction with 50 µL stop solution.
11. Measure OD at 450 nm within 15 minutes.
**Data Analysis:**
Plot the standard curve using OD values vs. concentrations. Calculate sample concentrations from the curve and multiply by the dilution factor if applicable.
**Notes:**
- Allow the kit to reach room temperature before use.
- Store unopened strips in sealed bags.
- Avoid mixing reagents from different batches.
- Use fresh pipette tips and ensure accurate timing.
- For high-concentration samples, perform a preliminary dilution.
- Discard all waste as biohazardous material.
- Follow the manual strictly for accurate results.
**Storage Conditions:**
Store the kit at 2–8°C. Shelf life is 6 months from the date of manufacture.
**Linear Range:** 10–200 µg/L
**Specifications:** 96 wells per box
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