How to improve the recovery rate of ELISA

[Guest Yucheng Lin]

Hi everyone,

I used the ELISAbasic kit of mabtech, 3460-1H-6 / 3512-1H-6 / 3420-1H-6 / 3416-1H-6 to set up the ELISA experiment.

when I validate the ELISA, the recovery rate was bad in 3420-1H-6 and 3416-1H-6. 

The condition is on attachment.

In our lab, we optimize diluent buffer contain 6% SAS (saturated ammonium sulfate) to improve the recovery rate. But in these two set of ELISA, it did not work.

Is there a better suggestion that can be provided? 

Thanks for understanding the problem.

Best Regards

Yucheng Lin 

20220608 Human IFN-r & IL-1b Recovery rate test (lab condition).xlsx

[Christian@mabtech.com]

The data shown in the excel file states mouse IFN-g and not human IFN-g. Are you analyzing human IFN-g in mouse sera?

Analysis of human serum/plasma samples with the IFN-g and IL-1b kits requires the use of ELISA diluent for dilution of the samples (at least a 2-fold dilution), standard and detection antibody. The diluent prevents false positive read-outs which may be caused by interference of heterophilic antibodies found in serum and plasma. How much was the serum diluted in the assay? It is possible that matrix interference will affect the spike recovery and that the sample needs further dilution. We have not tested to use saturated ammonium sulfate in the ELISA assays.

To achieve correct calculations of spike recovery (recovery rate) we recommend that you include more points in in the standard curve. The OD value obtained at 0 pg/ml is rather high. Which TMB substrate did you use?

[Guest Yucheng Lin]

Sorry, the sample we analyzed were Human sera / plasma. the excel include information was wrong. 

Before assay, we dilute sample 1:1 with diluent buffer (1% BSA + 0.05% tween-20 + 6% SAS in PBS, pH7.3, 0.2um filtered) 

this diluent buffer formulation used to test recovery rate was better than your datasheet recommanded (1% BSA + 0.05% tween-20 in PBS, pH7.3, 0.2um filtered)

And the TMB we used is TMB One Component HRP Microwell Substrate, Surmodics (Cat No. SUMTMBW-1000-01)

  

[Christian@mabtech.com]

Hi again,

For plasma samples we have validated the ELISA after diluting samples in Mabtech ELISA diluent:

https://www.mabtech.com/products/elisa-diluent_3652-d2

 

Furthermore, we do not recommend using 6% SAS as this can cause the antibodies to precipitate. 

Finally, we strongly recommend you use Mabtech TMB ELISA substrate: https://www.mabtech.com/products/elisa-substrate-tmb-hrp_3652-f10