Here are some common culprits for why your ELISA was unsuccessful
General ELISA Troubleshooting Guide
Q1: My standard curve does not look nice and has a low R2 value.
A1: Make sure you have stored the standard according to the manufacturer’s specifications. Spin down the tube that contains the standard before opening. Ensure your pipettes are calibrated and the standard solution is mixed thoroughly and diluted correctly. If the curve fitting model does not work with the data, a different curve fitting may be needed.
Q2: When I split the ELISA kit for several uses, can the same standard curve be used every time?
A2. No, the standard curve should be made at the same time as the samples for each experiment. The ELISA result of each experiment is affected by multiple factors, such as operator, environment, time, etc. Therefore, it is recommended to make a new standard curve every time you use it to obtain more accurate and reliable test results.
Q3. My CV is higher than 20%.
A3: Make sure you have stored the standard according to the manufacturer’s specifications. Remove any bubbles in the wells before reading the plate. Ensure all the wells are washed thoroughly after each washing step. Handle plates properly to avoid edge effects.
Q4.I don’t detect any signal.
A4. Always add a positive control to make sure the protein of interest is present. Make sure the secondary antibody is compatible with the primary antibody. Also make sure the sample type (for example, use supernatant of cell culture for AAV ELISA kit) is appropriate. Option to add higher concentration of primary antibodies and incubate samples longer. Try to use high binding ELISA plates. Biotinylation may be used to enhance the signal. Ensure the instrument is set to read the correct wavelength or absorbance. Perform washing steps thoroughly but not aggressively, being excessively aggressive could remove detection reagents.
Q5. I got a high background.
A5. Make sure the secondary antibody was raised in a different species to your sample. There is a possibility that the primary antibody concentration or presence of the substrate is too high, dilute the antibody or the substrate if that’s the case. Increasing blocking incubation time may help. Ensure the washing is thorough. Make sure the wells are free of precipitation, if that’s the case, decrease the substrate. Be sure to read the plate within 5 min of adding the stop solution.
PRODUCT SPECIFIC QUESTIONS
Q1. Can the specific serotypes AAV Titration ELISA Kit cross detect with other AAV serotypes?A1. No. Each serotype AAV ELISA kit is specific to that particular serotype. Kactus screened and tested antibodies for ELISA kits to select highly specific monoclonal antibodies. In internal testing, antibodies to specific serotypes are not bound to or cross-detected with other serotypes of AAV.
Q2. Can AAV titration ELISA Kit detect a modified AAV serotype?
A2. Not conclusive. The ELISA kit is developed based on natural AAV virus particles. The customer can determine whether it is suitable for modified AAV particles.
Q3. Will AAV Titration ELISA Kit detect denatured AAV capsid?
A3. No. In Kactus internal testing, the AAV antibodies chosen for ELISA kit specifically recognize native and intact AAV particles without binding to denatured AAV capsids.
Q1. Is the MaxNuclease ELISA kit universal for other endonucleases?
A1. Not recommended. We recommended using ELISA kits developed by the same endonuclease supplier. However, most of endonuclease products share the same key sequence (wild type), and individual testing may be required by the customers.
Q2. Are coated- and detected- antibodies monoclonal?
A2. Yes, the pair of antibodies used in MaxNuclease ELISA kit is recombinant monoclonal antibodies, screened and expressed by Kactus.
For CAS9 ELISA kit
Q1. Can Kactus Cas9 ELISA kit be used to detect Cas9 expressed by myself?A1. Not conclusive. The Cas9 ELISA kit is based on the wild-type Cas9 protein. The coated- and detected- antibodies are developed by Kactus. Customers are encouraged to try.
Q2. Are coated- and detected- antibodies monoclonal?
A2. Yes, the pair of antibodies used in Cas9 ELISA kit is recombinant monoclonal antibodies, screened and expressed by Kactus.