Elabscience Human CPP(Copeptin) Elisa Kit

Test principle 
This ELISA kit uses the Competitive-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with Human CPP. During the reaction, Human CPP in samples or Standard competes with a fixed amount of Human CPP on the solid phase supporter for sites on the Biotinylated Detection Ab specific to Human CPP. Excess conjugate and unbound sample or standard are washed from the plate, and Avidin conjugated to Horseradish Peroxidase (HRP) are added to each microplate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of stop solution and the color change is measured spectrophotometrically at a wavelength of 450 ± 2 nm. The concentration of Human CPP in the samples is then determined by comparing the OD of the samples to the standard curve.

Sample collection 
Serum: Allow samples to clot for 1 hour at room temperature or overnight at 2-8℃before centrifugation for 20 min at 1000×g at 2-8℃. Collect the supernatant to carry out the assay. 
Plasma: Collect plasma using EDTA-Na2 as an anticoagulant. Centrifuge samples for 15 min at 1000×g at 2-8℃ within 30 min of collection. Collect the supernatant to carry out the assay.

Assay procedure 
1. Determine wells for diluted standard, blank and sample. Add 50 μL each dilution of standard, blank and sample into the appropriate wells (It is recommended that all samples and standards be assayed in duplicate. It is recommended to determine the dilution ratio of samples through preliminary experiments or technical support recommendations). Immediately add 50 μL of Biotinylated Detection Ab working solution to each well. Cover the plate with the sealer provided in the kit. Incubate for 45 min at 37℃. Note: solutions should be added to the bottom of the micro ELISA plate well, avoid touching the inside wall and causing foaming as much as possible. 
2. Decant the solution from each well，add 350 μL of wash buffer to each well. Soak for 1 min and aspirate or decant the solution from each well and pat it dry against clean absorbent paper. Repeat this wash step 3 times. Note: a microplate washer can be used in this step and other wash steps. Make the tested strips in use immediately after the wash step. Do not allow wells to be dry.

Calculation of results 
Average the duplicate readings for each standard and samples. Plot a four parameter logistic curve on log-log graph paper, with standard concentration on the x-axis and OD values on the y-axis. 
If the OD of the sample under the lowest limit of the standard curve, you should re-test it with an appropriate dilution. The actual concentration is the calculated concentration multiplied by the dilution factor.