An inhibition ELISA for the quantification of collagens type I and type II in cyanogen bromide-digested tissues using fragment-directed antibodies.
The quantification of connective tissue components in small tissue samples is of great importance for the examination of drug-induced changes in the development of the mammalian embryo. An inhibition-ELISA for the quantification of collagens type I and type II in CNBr-digested tissue samples was developed. Fragments of type I collagen were produced by CNBr-cleavage of the pure collagen and partially purified by gel filtration chromatography. A mixture of fragments was used to immunize rabbits. Antisera with highest titres were absorbed with immobilized fibronectin and collagens type II, III, V and I. The eluted antibodies exhibited specificity for alpha 2(I)-CB4, exclusively. These antibodies, as well as the previously described antibodies with specificity for alpha 1(II)-CB8, were used for the development of an inhibition-ELISA. The sensitivity of the assay is 0.3 micrograms/ml for collagen I and 3 micrograms/ml for collagen II. To evaluate the value and practicability of the ELISA we have estimated the amounts of both collagens in biochemically well characterized tissues (skin, aorta, chondrosarcoma) and have performed an initial determination in mouse embryos.
Schröter-Kermani
C
C
Institut fur Toxikologie und Embryonalpharmakologie, Freie Universität Berlin, FRG.
Ochsner-Welpelo
I
I
Kittelberger
R
R
D000818:Animals; D000831:Animals, Newborn; D000917:Antibody Formation / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D002813:Chondrosarcoma / Q000737:chemistry; D003094:Collagen / Q000032:analysis*; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D010446:Peptide Fragments / Q000276:immunology* / Q000302:isolation & purification; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D010446:Peptide Fragments; D003094:Collagen; D003488:Cyanogen Bromide