**Rabbit Lipoprotein Phospholipase A2 (Lp-PLA2) Enzyme-Linked Immunosorbent Assay (ELISA) Kit – User Manual**
This kit is intended for research purposes only and is not approved for use in diagnostic or clinical settings. It is designed to quantify Rabbit Lipoprotein Phospholipase A2 (Lp-PLA2) in serum, plasma, and other biological fluid samples.
**Purpose**
The Rabbit Lp-PLA2 ELISA Kit provides a reliable method for the quantitative determination of Lp-PLA2 levels in rabbit samples. This assay is based on an indirect immunoassay format using specific antibodies and enzyme detection technology.
**Principle of Operation**
The microtiter plate is pre-coated with a monoclonal antibody specific to Lp-PLA2. Standards and test samples are added to the wells, allowing Lp-PLA2 to bind to the immobilized antibody. After incubation, a biotinylated secondary antibody is introduced, followed by streptavidin-conjugated horseradish peroxidase (HRP). The enzyme catalyzes a color reaction using TMB substrate, resulting in a blue color that turns yellow when the reaction is stopped. 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 concentration is determined by comparing the OD values to a standard curve.
**Kit Components**
- 20× Washing Solution: 50 mL × 1 bottle
- Standard Solutions (S1–S5): 0.5 mL × 1 bottle each (ranging from 15 μg/L to 180 μg/L)
- Streptavidin-HRP Conjugate: 6 mL × 1 bottle
- Biotinylated Anti-IgG Antibody: 6 mL × 1 bottle
- TMB Developer A: 6 mL × 1 bottle
- TMB Developer B: 6 mL × 1 bottle
- Stop Solution: 6 mL × 1 bottle
- Sample Dilution Buffer: 6 mL × 1 bottle
- Microplate: 96-well strip plate × 8
- Sealing Membrane: 3 sheets
- Instruction Manual: 1 copy
**Sample Requirements**
- Avoid samples containing sodium azide (NaN3), as it may inhibit HRP activity.
- Process samples immediately after collection. If storage is necessary, keep at -20°C and avoid repeated freeze-thaw cycles.
**Procedure Overview**
1. Determine the number of wells needed, including standards, blanks, and samples. Use duplicate wells for better accuracy.
2. Add 50 μL of standard solutions and 10 μL of sample to the respective wells, then add 40 μL of diluent to achieve a 5-fold dilution. Cover and incubate at 37°C for 45 minutes.
3. Wash the plate 4 times using 20× diluted washing solution.
4. Add 50 μL of biotinylated anti-IgG antibody and incubate for 30 minutes at 37°C.
5. Repeat washing steps.
6. Add 50 μL of streptavidin-HRP and incubate for 30 minutes.
7. Wash again.
8. Add 50 μL of TMB Developer A and B, incubate for 15 minutes.
9. Stop the reaction by adding 50 μL of stop solution.
10. Measure absorbance at 450 nm within 15 minutes.
**Data Analysis**
Plot the OD values of the standards against their concentrations to create a standard curve. Calculate the sample concentration using linear regression or by reading off the curve. Multiply by the dilution factor if applicable.
**Notes**
- Allow the kit to reach room temperature before use. Store unused strips in sealed bags.
- The washing solution may crystallize; heat gently if needed.
- Use a pipette for accurate measurements and avoid cross-contamination.
- If the sample OD exceeds the highest standard, dilute the sample and retest.
- Do not mix components from different batches. Store reagent B away from light.
- Always follow the manual instructions and rely on the microplate reader results.
- Treat all waste materials as biohazardous.
- In case of conflict, the English version of the manual takes precedence.
**Storage Conditions**
Store the kit at 2–8°C. The shelf life is 6 months from the date of receipt.
**Linear Range**
10 μg/L – 200 μg/L
**Specifications**
96 tests per box
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