A relatively simple method is to perform subgroup analyses, the different subgroups being defined by the different comparisons being made. More appropriate methods for indirect comparisons are available, but the assumptions underlying the methods need to be considered carefully. This comparison ignores the potential benefits of randomization and suffers from the same (usually extreme) biases as a comparison of independent cohort studies. For example, patients receiving advice from a nurse (in the ‘dietician versus nurse’ trials) should not be compared directly with patients receiving advice from a doctor (in the ‘dietician versus doctor’ trials). One approach that should never be used is the direct comparison of the relevant single arms of the trials. In fact, doctors and nurses can be compared indirectly by contrasting trials of ‘dietician versus doctor’ with trials of ‘dietician versus nurse’. We might then wish to learn about the relative effectiveness of ‘doctor versus nurse’ by making indirect comparisons. For example, suppose that some trials have compared the effectiveness of ‘dietician versus doctor’ in providing dietary advice, and others have compared the effectiveness of ‘dietician versus nurse’, but no trials have compared the effectiveness of ‘doctor versus nurse’. Indirect comparisons are made between interventions in the absence of head-to-head randomized studies. For the current version, please go to /handbook/current or search for this chapter here. Learn more about how methods based on the use of biotin-conjugated antibodies work here.This is an archived version of the Handbook. Biotinylated antibodies offer an extra layer for increased signal amplification. They are also relevant to other techniques that rely on the use of fluorophore-conjugated antibodies such as flow cytometry, ELISA, western blot and immunohistochemistry.ĭetection of low abundance proteins can be sometimes challenging even with indirect methods. Samples with endogenous immunoglobulins may exhibit a high background with indirect methods.ĭirect and indirect methods are not limited to immunofluorescence. Non-specific binding is reduced through the use of conjugated primary antibodies. The use of pre-adsorbed secondary antibodies can prevent cross-reactivity. Secondary antibodies may cross-react with species other than the target. Species cross-reactivity is minimized in direct methods as the fluorophore is already conjugated to the primary antibody. Several secondary antibodies will bind to the primary antibody resulting in an amplified signal. The signal obtained in direct methods may seem weak when compared to indirect methods as signal amplification provided by the use of secondary antibodies does not occur. The possibility of using different conjugated secondary antibodies adds greater flexibility. This is particularly relevant in multiplex experiments where several secondary antibodies, each targeting a different species and conjugated to different dyes, are needed.Ĭommercially available pre-conjugated primary antibodies limit your flexibility. Further cost savings may be made by using the same conjugated secondary antibody to detect different primary antibodies.įewer steps in the protocol simplify direct methods.Īdded complexity in indirect methods may result from having to select the appropriate secondary antibody. Secondary antibodies are relatively inexpensive compared to primary antibodies. The fact that you have to use a conjugated secondary antibody to detect the primary antibody results in additional steps.Ĭonjugated primary antibodies are usually more expensive than their unconjugated counterparts. Protocols for direct IF are usually shorter as they only require one labeling step.
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