Dear experts and colleagues,
I have an lateral flow assay that previously worked but this time the test line did not appear.
1) I have tried different vials of Antibody for test line and different vials of Antigen as the deterioration of protein may happen but nothing changed.
2) I check the reaction between Antigen and antibody in conjugation by dotting antigen on membrane and use conjugation as sample. It worked well.
3) I made another strip and dotted the Antibody on the membrane as test line (no control line). Then, I mixed the Antigen with conjugation and put the strip into this mixture. The dot appeared.
From above experiments, I assumed that the test line did not appear in full strip (having both test line and control line) experiment is due to the the Antigen did not react with the conjugation on the conjugation pad. So, after going conjugation pad, the sample contained Antigen and conjugation instead of Antigen+conjugation.
Have you experience the same situation? Could you please give me some advices and suggestion. Thank you for your help.
For your information: The conjugation pad was treated with sucrose, trehalose, tween 20%, BSA. The membrane was not treated.
Difficult to say. Maybe your strip has gone bad, there was something in the sample buffer that denatured the antibodies, the sample or you had too much antigen (-> Hook Effect).
Can you elaborate on the exact principle of you assay (same antibody or a matched pair, nature of the antigen etc.)?
Wolfgang Schechinger Thank you for your kind suggestion.
We used the same monoclonal antibody for conjugation and test line. The antigen is a recombinant protein paired with the antibody. It worked well previously.
Since we made new conjugation with the same procedure and reagents. It has gone bad.
1) We have tried polyclonal antibody, matched pair antibody at the test line, and changed the concentration of the antigen in the sample but it has still not worked. only a control line appeared.
2) As you suggested, we may change the sample buffer. We currently use water to dilute the antigen. Although it worked we may change it to PBS. Do you have any suggestions for the running buffer?
3) We pretreated the conjugation pad, it helped stabilize and release the conjugation well. However, this time I prolonged the dry time from 2 hours to 6 hours. Do you think it can change the quality of the conjugation pad, leading the failure of the assay?
Thank you so much!
I'd suggest PBS with a little Tween 20 as sample buffer. Running buffer would be the same, possibly include some PBS.
What were your drying conditions? Temperature?
Have you checked if the conjugation was successful?
Wolfgang Schechinger Thank you for your suggestion. I will try it.
1) I dried the conjugation pad following pretreatment in the incubator at 37oC with desiccant.
2) I have checked the conjugation by hand-made half-strip: Antigen was dropped at testline and used conjugation as the sample. This showed that conjugation worked well. Also, the control line appeared in all of my experiments showing that the conjugation was successful.
37°C are fine, time usually is not critical, as long as it gets dry, the desiccant actually is not necessary. It's sufficient (and highly advised) to include a little in the container where you will store the strips, unless you're vacuum packing them, e.g. in assembled cassettes.
You said you are using the same monoclonal antibody for both capture and detection. Is your antigen divalent/multivalent? It is difficult to use the same monoclonal antibody for both capture and detection if your antigen is monovalent as both the detector and the capture antibody will be competing for the same epitope on the antigen molecule. Have you checked for non-specific signal when you did the spot test using the antibody spot by adding just the conjugate without the antigen and showed no signal?
In addition, as Ahmed Jehanli has mentioned: The same monoclonal won't work, unless your antigen is multimeric and in excess. And then it won't work well.
@Ahmed Jehanli: It was my mistake to use the same antibody. But it did work well, luckily. However, this time it did not work whether I tried polyclonal antibody or match paired Antibody at the test line. I did check the non specific binding. It did not occur. One experiment that I check whether the assay works is mix the conjugation with Antigen and used it as sample. Then I dot the same Antibody on test line. It worked well. But when I repeat the experiment but immobilized the conjugation on the pad, it did not work.
what is the buffer that you use for dispensing the gold-antibody conjugate into the glass fibre pad or polyester pad?
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