For Best Results

Copyright © Reddot Biotech Inc. 2018-2019 All rights reserved

We know that your experiments are important, and we want you to succeed when using Reddot Biotech ELISA kits. That’s why we’ve complied a list of important tips and tricks to help you when you perform your ELISA experiment.

     Storage and Expiration

The shelf life for all our products is 12 months from the time of manufacture. To ensure your product stays viable for the entire shelf life, be sure to store it properly as soon as you receive it.

For our traditional kits, the TMB substrate, Wash Buffer, and Stop Solution should be stored at 4oC, while all other components should be stored at -20oC.

For our new Ready-To-Use kits, all components can be stored at 4oC.

    Reagent and Sample Preparation

Some important tips when collecting and preparing your samples:

-Fresh samples can be kept at 4oC for up to 5 days before use with the assay, but samples collected farther in advance should be stored at -20oC or -80oC (long term storage). It is always best to use fresh samples if possible

-Avoid using any samples that have undergone hemolysis

-Be sure to bring samples to room temperature before beginning the assay

-Ensure that the samples used with the kit fall within the detection range, diluting samples if necessary. If you are unsure of the sample concentration, perform a preliminary experiment to determine the best dilution ratio for your samples. Samples should be diluted using 0.01mol/L PBS (pH 7.0-7.2)

-If you are using a chemical lysis buffer to prepare your samples and you are unsure if it will interfere with the kit, please contact us before beginning your experiment

-Cell culture supernatant samples will not always be detected by our kits due to factors such as cell viability and cell volume at the time of collection

Some important tips when preparing the reagents included in the kit:

-Make sure all components are at room temperature before beginning the experiment

-Mix all reagents before beginning the experiment 

-Do not let the prepared standard sit for extended periods of time before use, it is best to prepare the standard within 15 minutes of performing the assay

-We recommend performing the serial dilution in clean microcentrifuge tubes, NOT directly in the wells of the plate

-If you are using a Traditional kit, note that detection reagents A and B are sticky solutions and need to be pipetted slowly

-Be gentle when preparing the standard solutions and detection solutions A and B. Avoid foaming when mixing these solutions for best results

-Ensure any pipettes that are being used have been properly calibrated to prevent volume and concentration errors

-Crystals in the Wash Solution Concentrate are normal after periods of storage. Be sure to bring the solution to room temperate and dissolve all crystals by mixing gently before use

-Be sure to start with clean supplies, including any beakers, tubes, and tips, to ensure there is no contamination

     Assay Procedure

Some important tips to keep in mind when preforming the assay procedure:

-Calculate the amount of wells you will need to complete your experiment, and store any extra strips at -20oC

-Always be careful when adding solutions to the wells and avoid touching the well walls to ensure accurate results

-Ensure the time for each pipetting step is the same for accurate time results (ie, avoid taking breaks in the middle of the plate)

-To avoid contamination, do not let the wells sit uncovered for extended periods of time, and always ensure plate sealers are properly applied during incubation steps

-Once the assay has been started, do not let the wells dry out for the entirety of the experiment

-Washing is one of the most important parts of ELISA assay procedure. For best results, have one experimenter perform the entire assay to ensure the procedure is consistent for all washing steps. Ensure that all liquid in the wells is removed during each wash step

-Monitor the reaction time once the TMB substrate has been added (we recommend once every 10 min). If the colour changes quickly, add the stop solution whenever necessary to ensure accurate results

-Be sure to protect the TMB substrate from light contamination when using it

-Keep the temperature and humidity constant while preforming your assay to ensure environmental changes do not affect your results

    Results Calculations

When calculating your results, construct your standard curve first, then plot your sample OD values to determine their concentration.

Some important tips when calculating the results of your assay:

-We recommend using curve plotting software for the most accurate results. We do not recommend only using Microsoft Excel to calculate your results

-Remember to plot the OD values on the x-axis and the concentration values on the y-axis

-For competitive inhibition kits, note that the results will be reverse proportional to the concentration in your samples

-Make sure to multiply your results by the dilution factor if your samples were diluted

If you have any questions or comments about this article or your ELISA kit, feel free to contact us here or send us an email at support@biohippo.com!