Quantitative Evaluation of a Standard for Immunogold Labeling of Collagen Type IV

Quantitative gold labeling studies allow the comparison of tissue antigens only if labeling conditions are the same. When labeling conditions deviate slightly, the labeling efficiency can also shift. Labeling efficiency can be determined on a standard containing known concentrations of the target antigen. When labeling this standard, the labeling density (LD) divided by the antigen concentration (C) gives the labeling efficiency (LE).(l) If the assumption is accepted that labeling efficiency is the same over the standard and tissue, then the antigen concentration in the tissue can be calculated from the labeling density. In this study, a labeling standard was constructed that contained collagen type IV in five concentrations and a blank agarose gel. Our design criteria specified that these six gels measure less than 2 mm across and the zones between protein concentrations be clearly delineated.The labeling standard was constructed by diluting lyophilyzed collagen type IV in water to final concentrations of 1,2,3,4, and 5 mg/ml.