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Niklas

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  1. ELISPOT Membrane coating

    Hello Luc, We have done tests to see how the incubation time affects the coating efficiency. The tests were made at room temp and the amount of mAb bound to the membrane was determined by measuring the free mAb in the PBS (the buffer used for coating) at different times. In the graph shown we added 1.5 ug of mAb in 100 ul and after 8 h close to 100% of the added mAb was bound. Shortening the coating time to less than 8 h yielded less efficient coating and a 4 h incubation resulted in approximately 85% bound mAb. Please note that this was done with EtOH treated PVDF plates; the use of non-treated PVDF will result in much less efficient coating even at longer coating times. To compensate for a shorter incubation time one can add more mAb to each well. Decreasing the volume to e.g. 50 ul/well (without decreasing the amount of mAb) is likely to also improve the coating at shorter incubation times. The data in the graph were obtained using mAb 1-D1K against human IFN-g but I think one can assume that most mAbs will behave in a similar manner. But as Christian said, any deviation from the recommended protocol can impact the assay and should be properly validated. Another way of getting around the issue of shortening the coating time is of course the use of pre-coated plates. Best regards, Niklas Ahlborg Head of R&D at Mabtech
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