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Cat.No: EK-DB-0054 Datasheet Instruction for Use
Specification Quantities
Price $952.95–1588.25
| Product Name | Human Lp-PLA2 ELISA Kit |
| Catalog No. | EK-DB-0054 |
| Description | This kit employs a double-antibody sandwich ELISA method to detect the concentration of human Lp-PLA2 in samples. The human Lp-PLA2 capture antibody is pre-coated on the microplate; when samples or standards are added, the human Lp-PLA2 in the sample binds to the capture antibody, while other free components are removed during washing. Next, after adding the biotin-labeled anti-human Lp-PLA2 antibody, the anti-human Lp-PLA2 antibody binds to the human Lp-PLA2, forming a sandwich-type immune complex, while other free components are removed by washing. Next, the enzyme complex is added; biotin specifically binds to the enzyme complex, thereby linking the HRP on the enzyme complex to the sandwich immune complex, while other free components are removed by washing. Finally, a chromogen is added; if human Lp-PLA2 is present in the sample, an immune complex will form, and the HRP attached to it will catalyze the oxidation of the colorless chromogen to produce a blue substance. A stopping solution is then added, and the final product appears yellow. Using a microplate reader, the OD value at 450 nm is measured. The concentration of human Lp-PLA2 is directly proportional to the OD450 value. A standard curve is plotted using standard samples, and by comparing the OD value of the unknown sample against this curve, the concentration of human Lp-PLA2 in the sample can be calculated. |
| Kit Components | Human Lp-PLA2 Pre-coated Plates: 8-well strips × 12 Sample Dilution Buffer: 30 mL Recombinant Human Lp-PLA2 Standards (Lyophilized): 2 vials (100 ng/vial) Biotin-labeled Human Lp-PLA2 Antibody: 130 μL (titer 1:100) Antibody Dilution Buffer: 12 mL Enzyme Complex (HRP-labeled streptavidin): 130 μL (1:100 dilution) Enzyme Complex Dilution Buffer: 12 mL Concentrated Wash Buffer (25×): 30 mL Chromogen TMB: 10 mL Stop Solution: 10 mL Plate Sealing Tape:4 sheets |
| Sample Type | Human serum, plasma, body fluids, tissue homogenates, or cell culture supernatants |
| Detection Limit | 0.39 ng/mL |
| Detection Range | 0.78 ng/mL - 50 ng/mL |
| Assay Method | ELISA |
| Reagent Preparation | After removing the kit from a 4°C refrigerator, allow it to equilibrate at room temperature for 20 minutes. If removed from -20°C, ensure all components are completely thawed before allowing the kit to equilibrate for 20 minutes. After testing is complete, promptly store any remaining reagents at 4°C or -20°C. Dilute the concentrated wash buffer (25×) with double-distilled water or deionized water to prepare a 1× wash buffer; Dilution and Use of Recombinant Human Lp-PLA2 Standards. Prepare a 100 ng/mL standard: Add 1 mL of sample diluent to the standard tube, cap it, and let it stand for at least 15 minutes, then invert and swirl repeatedly to aid dissolution. Prepare a 50 ng/mL standard: Add 500 μL of the 100 ng/mL standard to an EP tube containing 500 μL of sample diluent, mix well, and label it. Perform a two-fold serial dilution of the 50 ng/mL standard solution using the sample diluent according to the table below (with the highest concentration being 50 ng/mL). Calculate the total volume required based on the need to add 100 μL of antibody working solution per well (prepare an additional 100–200 μL to account for losses during handling). Prepare the working solution using a ratio of 1 μL of biotin-labeled human Lp-PLA2 antibody to 99 μL of antibody diluent, and mix gently. Calculate the total volume required based on the need to add 100 μL of enzyme complex working solution per well (prepare an additional 100–200 μL to account for losses during handling). Prepare the working solution by mixing 1 μL of enzyme complex with 99 μL of enzyme complex diluent, and mix gently. |
| Sample Preparation | Select the appropriate processing method based on the sample type: A. Cell supernatant: Centrifuge the cell culture supernatant at 100–500×g for 5 minutes; remove any suspended particles, and the sample is ready for use; B. Serum Samples: Allow whole blood to stand at room temperature for 0.5–2 hours until it naturally clots and the serum separates. Centrifuge to collect the yellow supernatant (4°C, 1,000–2,000 × g, 10 minutes). Be careful not to aspirate the precipitate. Store the prepared serum on ice until use; do not add any preservatives or anticoagulants; C. Plasma samples: Anticoagulate whole blood with EDTA, mix thoroughly, and place on ice. Centrifuge to collect the yellow supernatant (4°C, 1,000–2,000 × g, 10 min). Be careful not to aspirate the precipitate. Store the prepared plasma on ice until use; D. Tissue homogenates/body fluids: Centrifuge to remove the precipitate. Dilute the sample. Different dilution protocols should be followed depending on the concentration of the analyte: If the analyte concentration is between 500 and 5,000 ng/mL, generally dilute at a 1:100 ratio; that is, add 3 μL of sample to 297 μL of sample diluent; For analyte concentrations between 50 and 500 ng/mL, a 1:10 dilution is generally used, i.e., add 25 μL of sample to 225 μL of sample diluent; For analyte concentrations between 0.78 and 50 ng/mL, a 1:2 dilution is generally used, i.e., add 100 μL of sample to 100 μL of sample diluent; If the concentration of the analyte is ≤0.78 ng/mL, the sample generally does not need to be diluted. The above guidelines are for reference only; please record the sample dilution method in detail during the experiment. |
| Test Procedure | Calculate the number of strips required for a single experiment, remove the required strips, and place them in the frame. Return any excess strips to the aluminum foil pouch, seal it, and store at 4°C or -20°C. Add samples diluted with the sample diluent and standards of different concentrations (100 μL/well) to the corresponding wells, seal the wells with sealing tape, and incubate at 37°C for 90 minutes. Tap the microplate to remove excess liquid; no washing is required. Invert the plate onto absorbent paper and tap gently to dry; Add the diluted working solution of the biotin-labeled human Lp-PLA2 antibody (100 μL per well), seal the wells with adhesive tape, and incubate at 37°C for 60 minutes; Wash the plate 5 times, using 300 μL of 1× wash buffer per well. Allow 15–30 seconds between adding and removing the wash buffer. After washing, invert the plate onto absorbent paper and tap to dry. The washing process is critical; inadequate washing may lead to significant errors in the results. Add the diluted enzyme complex (100 μL/well), seal the reaction wells with adhesive tape, and incubate at 37°C in the dark for 30 minutes, wash the plate 5 times. Add TMB chromogen (100 μL/well), seal the reaction wells with adhesive tape, and incubate at 37°C in the dark for 10–25 minutes. During storage and use, do not allow TMB to come into contact with oxidizing agents or metals. Due to variations in laboratory conditions, the optimal development time may vary. When the reaction is complete, a distinct blue gradient will be visible to the naked eye in the first 3–4 wells of the standard curve. Add stopping solution (100 μL/well), mix well, and immediately measure OD450 using a microplate reader. Simultaneously set 540 nm or 570 nm as the correction wavelength to calculate the corrected absorbance value. |
| Results | Plot a standard curve. Plot the standard concentration on the x-axis and the OD value on the y-axis. Use computer software to perform a four-parameter logistic (4-PL) curve fit to create the standard curve. The corresponding concentration can then be calculated from the sample’s OD value on the standard curve. Results are valid only if the OD values of duplicate wells differ by no more than 20%; the average of the duplicate OD values can be used as the measured value; If a sample’s OD value exceeds the upper limit of the standard curve, it should be appropriately diluted and retested; when calculating the concentration, multiply the result by the dilution factor. |
| Storage and Transportation | Storage: 2-8°C. |
| Precautions | The concentrated wash solution may crystallize at low temperatures. Please heat it in a water bath until the crystals are completely dissolved before preparing the working solution; Never mix components from kits with different batch numbers; Avoid creating bubbles during sample addition. Ensure that reagents are thoroughly mixed throughout the experiment; otherwise, significant errors in the results may occur; The room temperature specified in the instructions must be strictly maintained between 25°C and 28°C; Please wear a lab coat and disposable gloves during the procedure; This product is for research use only. |
For research use only, not for clinical use.
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