Performing ELISA or enzyme-linked immunosorbent assays needs multitude assay components and steps, and hence, there is often a need for troubleshooting and optimization. In Elisa troubleshooting guide, Booster Antibody and ELISA experts have listed solutions to most common sources of problems occurring while assay development.

Let’s find the solutions for Elisa Standard Curve Troubleshooting:

Problem 1: In case of no or weak signal

• Possible cause: Error in key reagent

Solution: Make sure to analyze that all reagents have been added in the accurate manner.

• Possible cause: Inaccurately prepared, wrong or incomplete substrate

Solution: Make sure that the substrate chosen is as per for the enzyme conjugate.

Make sure that fresh H2O2 is added in a case it is necessary.

• Possible cause: Washes too harsh

Solution: Make sure to use an automated plate wash if needed

Make sure to remove detergent concentration in washing buffer.

• Possible cause: Incubation times insufficient

Solution: It must be noted that incubation times must be as per the system. Typical considers that substrate development times vary from 10 to 40 minutes.

• Possible cause: Substrate or conjugate is no longer active or is weak

Solution: Make sure to test conjugate and substrate for activity.

• Possible cause: Plate reader settings not optimal

Solution: Make sure to verify the wavelength and filter settings during the process of plate reader.

• Possible cause: Enzyme inhibitor present

Solution: Sodium azide will hinder peroxidase reactions.

• Possible cause: Inaccurate assay temperature

Solution: Make sure to use suggested incubation temperature.

Make sure to bring substrates to room temperature before use.

• Possible cause: Blocking protein comprised in the coating solution

Solution: Make sure to exclude blocking protein from coating solution.

• Possible cause: Insufficient volume of substrate

Solution: Make sure that accurate volume is delivered by pipette.

Problem 2: Superior or High background

• Possible cause: Cross-Reactivity

Solution: Make sure to detect antibody cross-reacting with coating antibody and execute appropriate controls.

• Possible cause: Prolonged Incubation time

Solution: Make sure to decline the incubation time

• Possible cause: Non-specific binding of antibodies

Solution: Make sure to use sufficient blocking buffer

• Possible cause: Concentration of conjugated second antibody too high

Solution: Execute dilutions to know to optimal working concentration.

• Possible cause: Buffers contaminated

Solution: Use fresh buffers

• Possible cause: Insufficient washing

Solution: Make sure to check test sample with substrate alone to analyze the contaminating enzyme activity.

• Contaminating enzymes present in sample

Make sure all wells are filled with wash buffer and are being aspirated accurately.

• Possible cause: Inaccurate assay temperature

Solution: Check that the incubation temperature did not exceed 37°C.

Ensure all wells are filling with wash buffer and are being aspirated completely. Use an automated plate washer, if available.

Problem 3: Poor or bad duplicates

1. Possible cause: Uneven or irregular plate coating

Solution: Make sure to use proper ELISA plate. For this, check coating and blocking volumes

2. Possible cause: Variation in incubation temperature

Solution: Make sure to follow complete processes.

Make sure to avoid incubation near any heat source.

3. Possible cause: Insufficient or not uniform washing

Solution: Make sure to follow uniform washing process.

Make sure to check for any obstructions in washing ports.

Problem 4: Inferior standard curve

• Possible cause: Wells not entirely aspirated

Solution: Make sure the aspirate wells between steps. For this, use an automated plate washer, if require.

• Possible cause: Bad or variable adsorption of reagents to plate

Solution: Make sure to check choice of coating buffer. Basically, it will be PBS, pH 7.4 or carbonate-bicarbonate buffer, pH 9.6. Make sure to extend incubating time.

Consider different plates and check homogeneity of samples as well.

• Possible cause: Plates stacked while incubations

Solution: Make sure to keep the plates away in a case of no rotation.

• Possible cause: Reagents badly or poorly mixed

Solution: Be sure that reagents are entirely and accurately mixed.

• Possible cause: Try to capture antibody that didn’t bind to plate

Solution: Make sure to use proper ELISA plate and dilute in PBS without other proteins

Problem 5: Unexpected results

• Possible cause: Neglect of reagents

Solution: Make sure that reagents were entirely accurate and added in the accurate manner.

• Possible cause: Dilution error

Solution: Make sure to pipetting procedures and calculations.

• Possible cause: Technique problem

Solution: Accurate and proper mixing of reagents and wash, all steps are important.

• Possible cause: Unsuitable ELISA plate used

In a case of using fluorescence detection, suitable plates must be used.

Problem 6: Uneven color development

• Possible cause: Imperfect washing of wells

Solution: Make sure to use an automated plate washer, if needs.

Booster Antibody and ELISA experts is a remarkable provider solutions for Elisa Troubleshooting. To get more help, kindly contact by way of the following addresses:

Email ID: [email protected]
Phone: (888) 466-3604
Fax: (925) 215-2184
Website: https://www.antibody-elisa.com