Immunohistochemical Micromethods for the Measurement of Specific Collagen Types in Human Liver Biopsies

Abstract

Micromethods are needed for measuring the amount of collagen and other matricial proteins in human liver biopsies. Recently, a colorimetric method for the quantitative estimation of total collagen and protein has been developed. This method is sensitive, can be applied to fresh and fixed tissues and provides the absolute value of total collagen per section. Results are expressed as µg of collagen per mg of proteins or per section. We now report two micromethods for the determination of specific collagen types in fresh frozen liver sections. The first method is a direct method utilizing the tissue section as an ELISA plate. Results are expressed as the amount of color reaction read at 450 nm per slide. After cleaving the antigen antibody complex with dilute acetic acid, the amount of total collagen can be measured by the colorimetric microchemical method and the amount of specific collagen type is expressed as the amount of color reaction per µg of total collagen. The second method is an indirect method: the floating tissue section is incubated with an optimal amount of specific anti-collagen antibody and the residual amount of antibody present in the supernatant is measured in an ELISA plate coated with the specific collagen type. The amount of specific collagen type present in the tissue section is determined by comparing the optical deviation obtained with a standard curve prepared with known amounts of specific collagen, and values are expressed as µg of collagen per tissue sections. As with the direct method, the amount of total collagen per section can be measured by the colorimetric method and then, the amount of specific collagen type can be expressed as µg of specific collagen type per µg of total collagen.