Unintended anti-Ig "cross-reactions"

From Dr. Leonore A. Herzenberg:

CytoGenie guards against the construction of stain sets in which a fluorochrome-coupled second-step reagent that is meant to specifically detect a first step antibody also recognizes other antibodies in the stain set.  This can occur, for example, when a second-step fluorochrome-coupled anti rat IgG reagent is used to reveal the binding of an unlabeled first-step rat IgG antibody.  If another rat IgG antibody is inadvertently added to the stain set, perhaps as a fluorochrome-coupled reagent intended to detect to a marker on a different cell type, it can also react with the second-step antibody.

This unintended reativity will have different consequences, depending on the order of addition of the first and second step reagents and how much washing is done between the steps.   However, because it can seriously compromise data interpretation, CytoGenie by default prevents the construction of stain sets that contain more than one antibody reagent that can react with an anti Ig second step.

This caution stated, there are certain situations in which second step antibodies are commonly used in stain sets containing more than one reagent that can react with a second step antibody.  Therefore, CytoGenie provides methods that allow users to suspend the protection against this type of error.  You can click on the insert to see how to do this.  But before you do, may we suggest that you read the following discussion of the types of errors that can be introduced and what must be done to minimize or avoid them.  

Minimizing errors with second-step staining.

A labeled second step anti Ig reagent can rightly be expected to reveal the unlabeled first step antibody it is intended to reveal.  However, depending on the method used for staining, there are three ways in which second-step antibody interact with other monoclonal or polyclonal first-step reagents to compromise data interpretation.

1) First step and second step antibodies are added together.  Obviously not a good idea since complexes can form that will stick to each other and to cells resulting in major non-specificity.

2) Several antibody reagents detectable by the second-step reagent are used for first-step staining.  When the first step staining is intended to be specific, CytoGenie's single selection mode should be used.  CytoGenie will then protect against inadvertently selecting another reagent detected by the same second step antibody.  "Multi" selection mode (2+ reagents per color button in the Stain set window) can be used to avoid this protection, but should obviously used with extreme caution since the amount of fluorescence due to binding of the second step antibody cannot be reliably assigned to one or the other first step antibodies except under quite special circumstances (e.g., when other reagents identify cell types known not to express one or the other detected determinants).

3)  First and second step staining is completed and the cells are washed well before addition of one or more fluorochrome-coupled reagents that the second step antibody can detect. This method is commonly used to enable use of an unlabelled reagent in combination with one or more fluorochrome labeled reagents that identify particular cell types.  The error creates is more subtle than in the preceding case.  However, it can nonetheless result in significant problems in data interpretation, of which users should be aware. 

That is:   if the cells are washed well after the second-step reagent is applied, there should not be any free second step reagent in solution that can bind the newly added reagents. There are likely, however, to be free combining sites on the second step antibodies that are bound to the first step antibody on the cells. In principle, up to half of the combining sites on an IgG second step antibody could be free, although the number of free sites is probably much smaller.  In any event, those combining sites that are available can bind the newly added fluorochrome labeled antibodies, resulting in binding of these antibodies to cells that do not necessarily display the determinants these antibodies were added to detect.  

Often, this unintended binding to the second-step reagent is interpreted as "stickiness" or "background" binding of reagents to cells.   However, it is identifiable because it will only occur when the second-step antibody is present, and will increase in proportion to the amount of second-step reagent that has specifically bound to the cells.  Thus, it can be recognized and controlled for by staining with fluorochrome-coupled isotype controls, which can be used to some extent to determine the level of "non-specific" staining to use as a threshold above which cells can be considered positive.  This is not a definitive method for setting such thresholds since they will vary according to the amount of the second step reagent bound to the cell, i.e., , it will be higher for cells that have bound the most second-step reagent and will fall at true background levels for cells that don't bind the second step.  In addition, some or all of the isotype control binding can be due to real "stickiness" of the reagent, which often varies even between preparations of the same fluorochrome-coupled antibody. 

Overall, it is probably better to avoid using second-step antibodies in stainsets where additional reagents can react with the second step antibody.  However, when used with caution these methods can be very powerful, and are sometimes the only methods available.  Therefore, the default for CytoGenie is set to protect against this type of stain set construction, but allows users to suspend the protection when they choose to do so.