The effect of heterologous antisera on embryonic development. XIII. Lack of effect of antisera to alpha fetoprotein.

This investigation was carried out to determine whether heterologous antisera to alpha fetoprotein (AFP) are embryotoxic to developing rat embryos. Homogeneous rat AFP was isolated and antisera directed against this glycoprotein were produced in rabbits, horse and goat. The effect of the antisera on embryonic development was examined by injecting the antisera intraperitoneally into pregnant rats on the ninth, eleventh and thirteenth days of gestation. The results demonstrated that there was no evidence of increased incidence of fetal abnormalities in 472 surviving fetuses of 42 injected rats. There was no evidence of increase embryonic death or retardation of intrauterine growth following administration of the antisera on the ninth, eleventh and thirteenth days of gestation. The localization of the injected antisera was examined by the indirect immunofluorescent method. The results showed that the heterologous AFP antibodies localized specifically in the visceral yolk sac placenta. No antibody localization was observed in the embryo proper or the chorioallantoic placenta. It is speculated that the localization of AFP antibodies in the visceral yolk sac does not interfere with the embryotrophic function of the visceral yolk sac placenta.     

Factors affecting production of contrasensitizing antisera in mice.

Using four different protein antigens, two different strains of mice, and various immunization protocols, we have studied production in mice of immunological enhancement antibodies that specifically suppress induction of delayed hypersensitivity. Primary assay of these antibodies was in vivo, because no in vitro test used detected them dependably. Any antigen priming that favored initiation of humoral antibody responses prepared mice to make these contrasensitizing antibodies vigorously following appropriate boosting. The method of boosting usually was more important than that of priming, high titers regularly developing only when primed mice were boosted with much antigen in a short time and were bled a few days later. The presence or absence of delayed hypersensitivity was immaterial. CAF₁ mice made these antibodies better than CF-1 mice, and antigen effectiveness correlated with propensity to induce humoral antibody formation in mice, decreasing from ovalbumin through human serum albumin and bovine serum albumin to methylated human serum albumin. In certain antigenmouse combinations (e.g., ovalbumin in CAF₁ mice) immunosuppressive antibody production was vigorous and prolonged; in others (e.g., bovine serum albumin in CF-1 mice) it was moderate and brief. From our results one can predict what conditions should induce formation of strongly enhancing/contrasensitizing antisera, and speculate that these conditions also should elicit strong, active immunologic tolerance for averting induction of delayed hypersensitivity.     

DRw antisera react with activated T cells.

Nylon wool-purified T cells appear to be nonreactive in a lymphocytotoxicity assay with HLA-DRw antisera and complement before cell activation. However, after activation in mixed lymphocyte culture, responder cells express determinants that are strongly reactive with DRw alloantisera after 6 days and gradually disappear by 16 to 18 days. Restimulation of the primed cells resulted in re-expression of the blast determinants. Mitogenic stimulation with Con A or purified PHA (HA-17) also resulted in temporary expression of these determinants; reactivity usually conformed to DRw genetic restriction; however, occasional extra reactions occurred that were variable depending on the method of activation (i.e., MLC, Con A, or HA-17). The results suggest the presence of additional allospecificities within some of the DRw antisera that react with "Ia-like" antigens on activated cells from unique subsets of T cells. Whether these DRw antisera contain antibodies against T cells or agains activation or differentiation T cell antigens is not as yet clear.     

Preparation of specific antisera to 15alpha-hydroxyestrogens.

The synthesis of haptens of 15alpha-hydroxyestrone, 15alpha-hydroxyestradiol, and 15alpha-hydroxyestriol (estetrol) was undertaken, to obtain specific antisera required for enzyme immunoassay. 3-(1-Carboxypropyl) ethers of these 15alpha-hydroxyestrogens were prepared and conjugated with bovine serum albumin and horseradish peroxidase. The specificity of antisera elicited against bovine serum albumin conjugates was checked by the enzyme immunoassay by using horseradish peroxidase-labeled antigen, and proved to be satisfactory in terms of cross-reactivities to related compounds.     

Antilipose antisera activity against negatively charged phosphate amphiphils.

Complement-immune lysis of liposomes loaded with water-soluble spin label 2,2,6,6-tetramethyl-piperidinoxy-choline chloride (TEMPO-choline) was used to measure specificity of rabbit antisera generated by complete adjuvant with liposomes consisting of sphingomyelin; cholesterol; dicetylphosphate; 5-N-thyroxine-2,4-dinitrophenyl -phosphatidylethanolamine (T₄-Dnp-PE) in the ratio 2.0:1.5:0.2:15. Antisera so generated induced complement lysis of the liposomes containing negatively charged amphiphils. No immune lysis of positively charged liposome was observed. This antiliposome specificity is retained in partially purified antibodies.     

Characterization of molecular heterogeneity and multispecificity in homologous idiotypic antisera.

The molecular heterogeneity of homologous anti-idiotypic reagents was characterized by a novel isoelectric focusing procedure. Idiotypic antisera directed against the PC-binding plasmacytoma protein T15 were raised in CE and and A/J mice. These antisera were shown to be highly specific by hemagglutination with myeloma protein-derivatized sheep erythrocytes and by radioimmunoassay. Competition experiments performed with affinity-labeled T15 revealed that about 40% of the pooled CE antibody activity was directed against binding site-associated determinants. Further analysis of anti-idiotypic sera from individual animals with the use of isoelectric focusing disclosed heterogeneous populations of antibody molecules distinguishable by isoelectric point and by subspecificity. Each animal expressed a unique spectrotypic profile. In addition, clones reactive with binding site and non-binding-site determinants as well as some clones with specificity for other PC-binding mouse myeloma proteins were detected. These results emphasize the importance of careful selection and thorough absorption of idiotypic antisera.     

Embryotoxic effects of heterologous antisera against rat Reichert's membrane.

Parietal yolk-sacs of rat embryos at the fifteenth day of gestation were obtained by microdissection. A Reichert's membrane (RM) preparation was isolated by treating the parietal yolk-sacs with the chelating agent tetrasodium salt of ethylenediaminetetraacetic acid (EDTA) combined with mechanical shaking. Less than 1% of the membrane preparation was DNA and phosphorus contaminants. The membrane purity was also evaluated by electrom microscopic examination. Rabbit Ig G directed against the RM preparation when injected ip into ninth day pregnant rats produced malformations, fetal growth retardation and resorption. Fluorescent-labeled antibody localization studies demonstrated that the teratogenic antibodies localized in RM. It is postulated that RM antibodies induce teratogenesis by interfering with the function of RM.     

Absorption of antisera for studies on specific enzyme turnover.

Antisera were raised to acetyl-CoA carboxylase and 6-phosphogluconate dehydrogenase from mammary glands of lactating rabbits, and cytochrome oxidase from rat liver. The enzymes were all highly purified but gave rise to multispecific antisera when tested against tissue extracts. Absorption procedures were devised to free the antisera of contaminating antibodies. Antisera to acetyl-CoA carboxylase and cytochrome oxidase were absorbed with fractions discarded during enzyme purification. The antiserum to 6-phospho-gluconate dehydrogenase was absorbed with a tissue extract from an early stage in mammary-gland differentiation. Monospecific antisera are essential for enzyme turnover studies and therefore antisera should be extensively tested and absorbed before use. A general procedure for the absorption of antisera to purified enzymes has been devised on the basis of accepted principles of antisera absorption.