Knowledge share

Response to the question meeted with FineTest Elisa kit testing

Top1: High background. The color reaction is too fast after adding TMB and all the wells turn to blue. The blank OD value is more than 0.5, sometimes is higher than 1.0.

Re:

A.Non-exhaustive washing plate

1)Automatic Washing: Make sure the tips are not been blocked by any obstacle, like crystal. Fill each well completely with 350ul wash buffer.

2)Manual Washing: please comply with the washing process strictly which is stated in the manual. The absorbent paper must be oxidizer free and the tips need to be sterilized and clear. Add 350ul wash buffer.

3)After washing: clap the plate on absorbent papers or other absorbent material to absorb up the liquid of wells before the next step.

4)Attention: the washing before adding TMB is very important. The residual HRP enzyme will directly make the TMB color turn to blue.

 

B. Pollution

1)TMB has been polluted. TMB should be transparent, if it looks blue, then turns out to be polluted.

2)Check if the water used for formulating wash buffer is polluted or not. Because common water contain metal ion such as Fe3+, Cu3+, Co3+ and HClO which will cause TMB oxidation. We require using the double distilled water, three steamed water or deionized water to formulate wash buffer.

3)Check if the Elisa plate is damp or not. Check if any component reagents are expired.

4)Check if the experiment environment is polluted or not. There might be dust or substance in the air causing color.

 

Top2: The standard OD values we get are not as good as it stated on the manual.

Re:

1)Check if all the reagents have been stored under the manual requirement or not. Especially for the detection antibodies and SABC, if stored at -20℃, melting during the experiment will affect their activity severely.

2)Make sure the pipette is accurate. You can calibrate the pipette by testing the deionized water. (1000ul=1.0g 100ul=0.1g 10ul=10mg)

3)Equilibrate the TMB for 30 minutes at 37℃ before their incubation.

4)Do all the operations strictly comply with the shipment manual.

5)Make sure the kit is within expiry date.

 

Top3: The duplicate OD value varies a lot. The CV is high.

Re:

1)The deviation will be low if the duplicate OD values are between 0.5 and 2.5. When OD values are less than 0.5 or higher than 2.5, the CV will tend to high because of the operation deviation. In order to get an accurate CV, we suggest the duplicate well quantity is between 5 and 10.

2)Pay attention to your adding sample operation, Eg: Don’t let the tips touch the bottom and internal of the well. It just needs to let the tips touch the liquid and make the residual flow into the sample well.

3)Adding samples as soon and accurate as you can, don’t let any bubbles occur.

4)The pre-experiment is a must before testing sample. Test sample at different concentration to find out the best dilution rate. The inappropriate dilution rate will increase the deviation of duplicate OD value.

 

Top4: The color reaction is too fast and the blank OD value is less than 0.2. The kit works well but the sample OD value is negative.

Re:

1)Make sure the samples are under right storage. Proteins are easy to degrade. You had better store it at -80℃or test the fresh samples directly.

2)The matrix in the sample will affect the specific binding of antibody and antigen, and then cause the negative result. Based on this, you may need to dilute the sample at 1/5, 1/10, 1/100, 1/1000 to find out the best dilution rate. Diluting sample will reduce the matrix effect to help to get a positive result.

3)Some kits demand incubating samples and standard at 4overnight. If you still don’t get any positive OD values after sample dilution, we suggest you to incubate samples and standard at 4for 16h (Execute other operations comply with the shipping manual).

4)If you need to test special sample, please contact us for the tech support.