Antibody Controls

The IHC stain procedure is a multistep staining protocol, the various steps intended to provide amplification of stain results. Therefore, a control system must include elements to control each step of the stain process. Such a control should also include a range of reactivities, and that range ideally would encompass the total expression range expected for the measured component. The control should also monitor each step of the multistep protocol. This author has devoted a number of years to this concept, resulting in a patented control for multistep staining processes.14 Such a control provides sufficient information to monitor every IHC stain run, and when the control is evaluated quantitatively, normalization of data from one stain run to another within the same laboratory, and even between laboratories. A process control is a measure of the stain protocol and does not take the place of a control for the primary antibody. While the primary antibody control should include range of expression level detection, a different primary control must be present for every primary antibody used in a stain run (Fig. 10.4). 

MAb to PCNA and Ki-67 with the appropriate isotypic antibody controls—secondary antibodies if a two step staining is required... 

Ten milligrams of 2,2 -azino-di-(3-ethylbenzthiazoline sulfonic acid) diammonium salt is dissolved in 50 ml of substrate buffer containing 10 pi of 30% hydrogen peroxide. Only freshly prepared substrate should be used. Incubation is carried out for 30 min at 37°C, followed by visual plate reading on a 1+ to 4+ basis or at 540 pm if a microtiter plate reader is available. The optical density of the instrument is set to zero with the antigen control (well 12 in the first row). Any optical density in the antibody controls (bottom row) is subtracted from the corresponding test well above. 

Keywords immunohistochemistry, variables, antibodies, controls, quantitation, pitfalls... 

Cons Require species-specific reagents, which can present problems in terms of suitable assay controls, particularly when using an animal semm as a positive antibody control for the assay. May suffer from nonspecific binding. 

The 1° antibody control is a specificity control that confirms that the ° antibody did bind to the correct epitope on the expected antigen. Antibodies bind to epitopes on antigens, and each antigen can have multiple epitopes (Chapter 4, Antibodies). In addition, single epitope can be found on different antigens. This distinction between epitope and antigen is important in understanding the specificity of the ° antibody. 

The 2° antibody control, also known as the negative control or the technique control in the experimental context, shows specific binding of the 2° antibody to a 1° antibody (Fig. 8.4a, b). 

Fig. 8.4 2° antibody controls. Controls will show that the 2° antibody binds only to the 1° antibody, (a) With several antigens present, the expected binding of the 1 ° antibody is to its antigen (No. 3) and then the fluorescent 2° antibody binds to the 1° antibody, (b) The elimination of the 1 ° antibody does not allow binding of the 2° antibody under correct conditions... 

In experiments with multiple 1° antibody and multiple 2° antibodies, the control must show that (1) each 2° binds with only the correct 1° antibody and (2) each 2° does not bind to other 2° antibodies. Detailed protocols for multiple 1° antibody experiments will vary depending on the number of 1° antibodies, the species used to make the 1° antibodies, and the labeling protocol used. Descriptions of multiple antibody methods in Chapters 11 and 12 will include a discussion of the specific 2° antibody controls needed. 

Table 10.3 Controls for indirect single antibody Controls for indirect single antibody...

The experimental design presented in this chapter will combine the two single 1° antibody procedures into one procedure. Begin with determining the procedure for each 1° antibody separately (Chapter 10 Single 1° Antibodies). Then decide how to combine two P antibody incubations, design 2° antibody controls, and complete the final procedure. 

In previous experiments with single 1° antibodies (Chapter 10), the 2° antibody control showed that 2° antibody bound correctly and that its concentration was appropriate. With multiple P antibody experiments, the possibility exists that the 2° antibodies will bind unexpectedly in unforeseen ways. In the controls with multiple antibodies, an additional set of control conditions must be done each time the experiment is performed to insure that only the correct pair of 1° and 2° antibodies will bind. 

Experimental Design Chart for Block-Between Method. Design the 2° Antibody Control for the Same Species... 

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