Frequently asked questions

Possible causes: Please note that Controls available from Fuller Laboratories have been bottled at “screening dilution”. This dilution depends upon the specific assay, but in all cases Positive Controls are intended to be used initially without dilution to demonstrate a “strong” reaction. These Controls give endpoint titers 8-fold beyond the screening dilution (range 1:4-1:16). If IFA Substrate Slides are purchased as components, the Positive and Negative Controls available from Fuller Laboratories may be used to locate the optimal conjugate titer for use. This optimal titer should give a clear 1+ positive reaction at a Positive Control dilution of 1:8 (see #1 above) and a clearly negative reaction using the undiluted Negative Control. Microscope must be equipped with a filter system for AlexaFluor 488 (maximum excitation wavelength 493 nm, mean emission wavelength 518 nm) and 400X magnification.

Possible causes: Substrate slides must be allowed to equilibrate with ambient temperature prior to being opened. Opening cold slide packets will quickly lead to moisture condensation and produce an antigen suspension in the condensate liquid. Such slides are not useable. Allow at least 15-20 minutes for temperature equilibration, with the slides individually laying on the bench surface (not stacked). Substrate slides are routinely checked for vacuum leakage prior to shipping. If you have received slide packets that are not flat beyond the edges of the substrate slide, please notify your supplier.

Possible causes: It is possible to wash a substrate slide too much. We recommend, especially for IgG assays, that the slides be gently washed using a Wash Bottle containing PBS. Aim the stream of wash buffer to gently strike the middle of the slide, not the antigen wells. Do not immerse in wash buffer for long periods or douse with distilled water, simply use the wash bottle 3 times and applythe conjugate. For IgM assays it is recommended that the wash buffer (after the third wash) be allowed to remain on the slide wells for several minutes before removal and addition of conjugate. If only some of the sera tend to detach the substrate, the problem may be bacterial proteases due to bacterial growth in the sera. If the loss of substrate is in patches or streaks, the substrate has possibly been scratched off by the technician in applying sera or conjugate while performing the assay. Application of sera or conjugate should be done from the edge of the substrate wells.

Possible causes: When serum specimens are initially washed from the substrate wells, the gentle stream of PBS should be aimed to strike the middle of the slide (the mask) to remove the sera from top and bottom rows directly into a waste container or sink. Diluted sera washed across a neighboring well can influence the reaction in that well. This is especially true for high titered sera surrounded by wells containing negative sera. Another possible cause is similar in nature, an inadvertantly sharp movement of the slide or incubation chamber mixes samples from well to well. Careful inspection of the slides after incubation may note different well volumes or liquid on the slide mask between wells.

Antigen density in each antigen spot must be compared with the Positive Control reaction. Fluorescent particles may be seen within antigen spots and elsewhere in the open space, yet the true positive reaction much appear in density like the Positive Control, which is the criterion for judging a particular well as positive.