![]() Next, the enzyme conjugate is added and the mixture is incubated at room temperature. During incubation, competition for binding sites on the microplate is taking place. The plate is then washed removing all unbound material. The bound enzyme conjugate is detected by the addition of a TMB based substrate.ĮLISA test results may be obtained by measuring and comparing the absorbance reading of the wells of the samples against the standards with a microplate reader at 650 nm or 450 nm if acid stop is used. The extent of color development is inversely proportional to the amount of analyte in the sample or standard. A sample or control is added to each well.Plates are precoated with the antibody.If acid stop is used to halt the assay then the dark blue color will change to a dark yellow color and the light blue color to no color will change to light yellow to no color.įor example, the absence of the analyte in the sample will result in a dark blue color, whereas the presence of the analyte will result in a light blue color or no color as the concentration of the analyte increases. Qualitative results are obtained by measuring the absorbance reading at 650 nm or 450 nm if acid stop is used.Add TMB substrate to each well and allow the color to develop.The bound materials now remain in the microplate.Wash the plate to remove all unbound materials.Next the drug-enzyme conjugate is added and the mixture is incubated at room temperature. The plate was not properly washed (3 x 300 µL) with the diluted wash buffer.Extreme deep blue color development with samples and standards.The following are common examples of unexpected results when running Neogen's ELISA kits with possible explanations. A poor quality of water was used to dilute the wash buffer.The wash buffer was not diluted correctly before use.Extremely low color development with samples and standards.The enzyme conjugate concentrate was incorrectly diluted.If using an automated washer, ensure the machine is working correctly and increase wash cycle to 5 x 300 µL. The kit prematurely deteriorated, possibly from adverse shipping and storage conditions.Deionized water should be used for dilutions. Investigate the condition of the kit when it was received and how it was stored prior to use. Proper storage conditions are listed in the package insert. Check the expiration dates on the test kit and reagents. No component of the kit should be used past the expiration date. Always use aseptic techniques when opening and removing reagents from vials and bottles. Keep the plate covered except when adding reagents, washing or reading. Improper dilution of enzyme conjugate concentrate.No color development with samples and standards.When pipetting, do not allow the pipette tip to touch any of the reagents already in the well.Īlways use different pipette tips for each reagent. ![]() Check the expiration dates on the test kits and reagents. No components of the kit should be used past the expiration date. Little to no displacement with the standard curve.Do not mix any reagents or components of one kit with the reagents or components of another kit. Standard has deteriorated prematurely.Refer to the dilution scheme in the package insert. Contact your Neogen representative and provide them with the kit name, lot number, expiration date and your OD readings for further investigation.
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