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 α2(I)-CB4, exclusively. These antibodies, as well as the previously described antibodies with specificity for α1(II)-CB8, were used for the development of an inhibition-ELISA. The sensitivity of the assay is 0.3ug/ml for collagen I and 3ug/ml for collagen II. To evaluate the value and practibility 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.Our previous study demonstrated that cataract Shionogi (CTS) mice, an inbred strain related to non-obese diabetic (NOD) mice, are T lymphocytopenic and that their T cell-mediated in vitro reactions, such as proliferative responses of spleen cells to T cell mitogens and alloantigens or production of IL 2 and IL 2 receptors after stimulation of spleen cells with Con A, are greatly reduced. To confirm these in vitro characteristics, in vivo immune responses of CTS mice to T-dependent and T-independent antigens were compared with those of some reference strains including NOD mice. Antibody responses of CTS mice after one injection of a high dose (108) or one or two injections of a low dose (105) of sheep red blood cells (SRBC) were markedly lower than those of the reference strains. The decrease was particularly striking in the IgM antibody production at primary response to both high and low doses, and the IgG antibody production at the secondary response to low dose. Similar lower antibody production was observed in CTS mice against bovine serum albumin (BSA). Little production of IgE antibody was observed from 1 through 3 weeks after an injection of BSA plus Bordetella pertussis. IgG1 response was observed at high incidence but lower in titer than those in the reference strains. Unexpectedly, in spite of the poor antibody production to BSA, potent systemic sensitization for anaphylactic shock was easily established; incidence of lethal shock being comparable with those in the reference strains. This suggests that CTS mice are highly susceptible to the effector phase of active anaphylactic shock. Cell-mediated immunity was also impaired. Delayed type of hypersensitivity to SRBC was low, and the rejection of the skin graft from NOD mouse did not occur. In contrast to the reduced T cell-mediated responses, no difference was found between CTS and reference strains with regard to the antibody production to LPS, a T-independent antigen. These in vivo findings are consistent with the previous in vitro study.