A step-wedge standard for the quantification of immunoperoxidase techniques

This report describes the preparation and use of an artificial tissue containing differing amounts of an antigen covalently bound to gelatine (step wedge). The use of this step wedge enables the variables in the immunoperoxidase technique to be studied separately. The step wedge may be used as a standard in immunocytochemical quantification. By performing all the steps of the immunoperoxidase procedure on the step-wedge section and the tissue section simultaneously, valid comparisons can be made regarding changes in the amount of antigen in tissues. Absolute measurements of amounts of antigen in tissue cannot be made with this technique at present because of the loss of antigenicity involved in the preparation of the step wedge, differences of penetration by reagents between step wedge and tissue, and non-uniform distribution of antigens in this tissue. Use of the step wedge has shown that, under some circumstances, an increase in amount of antigen may be associated with a reduction in the amount of reaction product, demonstrating the impossibility of making any deductions about variations in the amount of antigens without control of the technique employed by a method such as the one presented here.