Assay procedures1. After taking out from 2‐8℃ refrigerator, prewarm the kit 30 minutes at room temperaturebefore using.2. Make standard and sample dilutions in a clean 96‐well plate. Dilute the 20 X Wash Solution to 1X Wash Solution with distilled water.3. Set standard wells, testing sample and blank wells on the assay plate/strip. Transfer
diluted standard 50 μl to standard wells, diluted sample 50 μl (10 uL sample plus 40 uL Sample Diluent) to sample wells, sample diluent only to blank wells.Note: Sample dilution varies from kit to kit based on your target concentration and the kit’s detection range. Generally, you should start with your sample with a 2-fold serial dilution if you can not estimate the target concentration range.4. Incubate the plate for 30 minutes in at 37℃ incubator.5. Decant as much liquid as possible, fill the wells with washing solution, oscillate the plate on a oscillating shaker if available for 1 min, decant the washing solution and remove residual liquid with absorbent paper. Repeat wash four timesfile:///C:/DOCUME%7E1/tt/LOCALS%7E1/Temp/msoclip1/03/clip_image001.gif6. Add HRP‐conjugated antibody 50μl to each well, except the blank well. Mix by shaking gently, and incubate for 30 minutes at 37℃.
7. Wash the plate or strips as described in Step5.8. Add chromogenic Substrate A and B: Substrate A 50μl and Substrate B 50μl to each well. Mix gently, incubate for 15 min at 37℃.9. Add Stop Solution 50μl immediately to each well to top the reaction ( the blue color change to yellow ).10. Measure the optical density (OD) at 450 nm within 15 min11. Construct standard curve ( plotting the mean OD450 for each standard on the Y‐axis against concentration on the X‐axis, draw a best‐fit log‐log curve through the points ) and calculate linear regression equation, then use sample OD values and regression equation to calculate the corresponding sample concentration. It should be remembered that the sample has been diluted and its actual concentration should be justified by dilution factor(the measurement and calculation can be accomplished by software like SoftMax).Specimen requirements1. Can’t detect the samples containing NaN3, since NaN3 inhibits HRP (horseradish peroxidase) activity.2. If the specimen cannot be tested immediately, it should be kept < ‐200C and repeatedfreeze /thaw should be avoided.3. The samples should be cleared by extensive centrifugation to remove any particulates.4. For serum samples, allow blood to clot for 2 hrs at room temperature before centrifuging for 20 minutes at 1000 X g. Remove serum for assay immediately or aliquot and store serum at < -20 0C.5. For plasma, using EDTA or heparin as anticoagulant, spin for 20 minutes at 1000 X gwithin 30 minutes of collection. Assay immediately or aliquot and store serum at < -20 0C.Important notes1. The operation should be carried out in strict accordance with the provided instructions.2. To preserve unused strip-wells, it should be stored in the sealed bag.3. Always avoid foaming when mixing or reconstituting protein solutions.4. Pipette reagents and samples into the center of each well.5. The samples should be transferred into the assay wells within 15 minutes of dilution.6. We recommended that all standard, testing samples are tested in duplicate to minimize the test errors.
7. Please justify the results with dilution factor. Two dilution is recommended for eachsample to get the best testing result.8. If the blue color too shallow after 15 minutes incubation with the substrates, it may beappropriate to extend the incubation time.
9. Avoid cross-contamination by changing tips, using separate reservoirs for each reagent, avoid using the suction head without extensive wash.10. Do not mix the reagents from different batches11. Stop Solution should be added in the same order of the Substrate solution.12. Chromogenic Substrate B is light-sensitive, please avoid prolonged exposure to light. The kit should be kept at 2 - 8 0C and cannot be used after expiration date. The Standard should be kept at < -20 0C after receiving.Procedures in summaryPrewarm all reagents to room temperature before assay.Prepare reagents, standard dilutions,sample dilutions in clean tubes or 96‐well plate.
Transfer standard and samples to assay plate/strip, incubate 30 minutes at 37 ℃
Plate‐wash four times, add HRP‐conjugated antibody and incubate 30 minutes at 37 ℃ Plate‐wash four times, add Substrate A and B, incubate 15 minutes at 37 ℃
Add stop solutionMeasure within 15 minutes
Get the OD450 mean value of the duplicate readings for each standard, control, and sample and subtract the average zero standard.
Create a standard curve using computer software capable of generating a log ‐log curve‐fit (Excel, for example). As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y‐axis against the concentration on the x‐axis and draw a best fit curve through the points on the graph. Obtain the linear regression equation, and calculate each sample concentration using this formula.
short 37 degree incubations is a sure way to get edge effects unless your lab is also at 37 degrees