Insulin ELISA 试剂盒

• C-Peptide Calibrator A /Sample Diluent
• C-Peptide Calibrators B thru F (Lyophilized)
• C-Peptide Controls I & II (Lyophilized)
• C-Peptide Antibody Coated Microtitration Strips
• C-Peptide Antibody Enzyme Conjugate-Ready-to-Use (RTU)
• TMB Chromogen Solution
• Stopping Solution
• Wash Concentrate A

1. Microplate reader capable of absorbance measurement at 450 nm, 405 nm and 630 nm.
2. Microplate orbital shaker.
3. Microplate washer.
4. Semi-automated/manual precision pipette to deliver 10-250 μL.
5. Vortex mixer.
6. Deionized water.

1. C-Peptide Calibrators B-F: Tap and reconstitute C-Peptide Calibrators B-F with 1.0 mL deionized water. Solubilize for 10 minutes, mix well and use after reconstitution.
2. Wash Solution: Dilute wash concentrate 25-fold with deionized water. The wash solution is stable for one month at room temperature when stored in a tightly sealed bottle.
3. Microtitration Wells: Select the number of coated wells required for the assay. The remaining unused wells should be placed in the resealable pouch with a desiccant. The pouch must be resealed to protect from moisture.

• Serum is the recommended sample type.
• Fasting serum should be used and the usual precautions for venipuncture should be observed. Store serum at -70 °C. Samples should be stable for at least 30 days when stored frozen at -70 °C [15]. Do not use hemolyzed or lipemic specimens.
• For shipping, place specimens in leak proof containers in biohazard specimen bags with appropriate specimen identification and test requisition information in the outside pocket of the biohazard specimen bag. Follow DOT and IATA requirements when shipping specimens.17

Allow all specimens and reagents to reach room temperature and mix thoroughly by gentle inversion before use. Calibrators, controls, and unknowns should be assayed in duplicate. NOTE: All serum samples reading higher than the highest calibrator should be mixed and diluted in the 0 ng/mL Calibrator A/Sample Diluent prior to assay.

1. Reconstitute C-Peptide Calibrators B-F and C-Peptide Controls I & II each with 1.0 mL deionized water. Solubilize for 10 minutes, Mix well.
2. Label the microtitration strips to be used.
3. Pipette 20 μL of the Calibrators, Controls and Unknowns to the appropriate wells.
4. Add 25 μL calibrator A/sample diluent to each well using a repeater pipette.
5. Add 100 μL C-Peptide Antibody Enzyme Conjugate solution to each well using a repeater pipette.
6. Incubate the plate, shaking at a fast speed (600-800 rpm) on an orbital microplate shaker, for 1 hour at room temperature.
7. Aspirate and wash each strip 5 times with Washing Solution (350 μL/per well) using an automatic microplate washer.
8. Add 100 μL of the TMB chromogen solution to each well using a precision pipette. Avoid exposure to direct sunlight.
9. Incubate the wells, shaking at 600-800 rpm on an orbital microplate shaker, for 10-12 min at room temperature. NOTE: Visually monitor the color development to optimize the incubation time.
10. Add 100 μL of the stopping solution to each well using a precision pipette. Read the absorbance of the solution in the wells within 20 minutes, using a microplate reader set to 450 nm. NOTE: While reading the absorbance of the microtitration well, it is necessary to program the zero calibrator as a ""Blank"".

NOTE: The results in this package insert were calculated by plotting the data on a log vs. log scale using a cubic regression curve-fit. Other data reduction methods may give slightly different results.

1. Calculate the mean OD for each calibrator, Control, or Unknown.
2. Plot the log of the mean OD readings for each of the Calibrators along the y-axis versus log of the C-Peptide of Insulin concentrations in ng/mL along the x-axis, using a cubic regression curve-fit.
3. Determine the C-Peptide of Insulin concentrations of the Controls and unknowns from the calibration curve by matching their mean OD readings with the corresponding C-Peptide of Insulin concentrations.
4. Any sample reading higher than the highest Calibrator should be appropriately diluted with the 0 ng/mL (CAL A) and re-assayed.
5. Any sample reading lower than the analytical sensitivity should be reported as such.
6. Multiply the value by a dilution factor, if required.