Paul Ordronneau

Improved immunoglobulin-enzyme bridge method for light microscopic demonstration of hormone-containing cells of the rat adenohypophysis

SummaryAn improved procedure of high efficiency is described for light microscopic localization of tissue antigens by the unlabeled antibody-enzyme bridge technique with unpurified antiserum to horseradish peroxidase followed by horseradish peroxidase. The method incorporates blocking of nonspecific tissue binding sites with normal serum derived from the species in which the second component of the bridge was made, incubation for extended periods of time (42–65 hrs) with high dilutions (up to 1∶1,000,000) of the primary antibody, and optimal conditions for the histochemical reaction with diaminobenzidine and H2O2. Hormone-containing cells of the rat adenohypophysis can be stained with this technique using dilutions of the primary antisera that may approach, or exceed, those used in radio-immunoassay or in electron microscopic immunocytochemistry with preformed peroxidase-antiperoxidase complexes. Based partly on analysis of experimental data, it is suggested that the term “efficiency” should be used to describe the ability of immunohistochemical methods to work at high dilutions of the primary antisera, while the term “sensitivity” should be reserved to describe the ability of such methods to detect a specified amount or concentration of tissue antigen.

Criteria of reliability for light microscopic immunocytochemical staining

SummaryThe following criteria of reliability are defined and discussed for immunocytochemical staining at the light microscopical level: efficiency, accuracy, precision, sensitivity, and specificity. Whenever practical, tests are suggested for obtaining information on the extent to which these criteria are fulfilled in a given system, and procedures are outlined for improving immunocytochemical staining in terms of these criteria. It is suggested that consideration of reliability criteria will help investigators in their choice of methodology, design of experimental strategy, and valid interpretation of the results.

Specificity in immunocytochemical staining.

Immunocytochemicai (ICC) localization of tissue antigens is being used with increasing frequency in many areas of modern biologic research. As with other techniques, it is important to define the limitations of the method, and the criteria whereby the validity of ICC results can be evaluated. Among these criteria, specificity is perhaps the most significant but also the most difficult to test and define. All of us who routinely use ICC techniques have been questioning ourselves and each other innumerable times: What is specificity in ICC staining? How can it be tested? What should be accepted as proof of specificity? The views that follow have helped us to formulate reasonably clear answers to these questions, and they are described here in the hope that they may be helpful for others as well. These views have been arrived at through a slow and tortuous path. They represent our own synthesis of information gathered from practical experience, discussions in the laboratory, published literature including textbooks and monographs, and from personal contact with other investigators. References to all these sources can not be included here, but they are collectively acknowledged with gratitude. Finally, these views are presented with due reservations and without claims of general and final validity; indeed, we hope that our concepts and understanding will improve as science

An efficient enzyme immunoassay for glutamate using glutaraldehyde coupling of the hapten to microtiter plates

In order to coat microtiter plates for enzyme immunoassays (EIAs), amino acids and other haptens are usually coupled to larger protein molecules. The formation of such conjugates is not always reproducible. This may lead to inconsistent hapten-protein stoichiometries, unfavorable orientation of the hapten on the protein and/or well-to-well variation in the concentration of the available hapten. In the assay described here the excitatory amino acid (EAA) Glu is coupled directly to polystyrene microtiter wells with GA. Each step of the assay was tested for maximum efficiency. The resulting EIA with Glu as a competitor gave excellent reproducibility (coefficient of variation = 5.87%), an EC50 of 2.02 X 10(-5) M and a detection limit of 1.26 X 10(-6) M. This EIA method is generally useful for a variety of antisera to amino acids and small peptides and a wide range of competing substances. It can be used to characterize the conformational requirements for antigen binding, to assay for glutamate or to identify compounds with glutamate-like structure in unknown solutions.

Distribution of immunoreactive androgen-binding protein/sex hormone-binding globulin in tissues of the fetal rat

Androgen-binding protein/sex hormone-binding globulin (ABP/SHBG) is an extracellular carrier protein that binds androgens and estrogens with high affinity. In the adult, ABP/SHBG is thought to function in the male reproductive system and the general circulation in both sexes to modulate the actions of sex steroids. The ABP/SHBG gene is also expressed in the embryonic rat liver, where SHBG is secreted into the fetal blood of male and female rats. The embryo also expresses an alternative SHBG with a unique N-terminal sequence. In this study, the distribution of immunoreactive SHBG in the 17-day-old male fetal rat was determined with six antisera. In general, all of the antisera reacted with the same structures. Specific tissue immunoreactivity was mostly cytoplasmic and/or extracellular. By far the most prominent immunoreactive structures were the mesoderm-derived tissues: connective tissue, striated and cardiac muscle, cartilage, and the liver hematopoietic system. In addition, all regions of the fetal brain contained immunoreactive neurons. In the developing male reproductive system, there was minor reactivity in the testicular cords, whereas the connective tissue in the differentiating Wolffian duct stained with all of the antisera. The Wolffian duct epithelium and epithelia in other developing organs contained small amounts of immunoreactive SHBG, except for the lung, which stained in the epithelial extracellular matrix. An antibody raised against a unique N-terminal peptide specific for the alternative SHBG protein revealed that it was also present in many tissues. These data suggest that SHBG is important for the differentiation of mesodermal tissues. SHBG may modulate the action of androgens in embryonic stroma, thereby regulating development of the epithelium in hormone-dependent tissues.

Molecular requirements for haften binding to antibodies against glutamate and aspartate

Molecular requirements for hapten recognition by antibodies raised in rabbits against glutaraldehyde conjugates of L-glutamate and L-aspartate were determined in enzyme immunoassays by measuring the displacement of binding of glutamate and aspartate, respectively, by a large number of selected haptens to two anti-glutamate and two anti-aspartate sera. The results indicate that N-terminal modifications of the amino acids, such as the presence of an N-acetyl or N-carbamyl group or the addition of a second amino acid to form dipeptides with C-terminal glutamate or aspartate, are tolerated to variable degrees, more so by the aspartate than the glutamate antisera. The antibodies possess point-to-point recognition sites for the two carboxyl groups present in both amino acids. Strong shape complementarity between the amino acids and their respective binding sites is suggested by the lack of recognition of the appropriate D stereoisomers by any of the antibodies. Changes in the distance between the two carboxyl groups, or modification, replacement or loss of either or both carboxyl groups, strongly reduce or eliminate binding. Based on these results, we suggest that other antibodies raised to similar conjugates of these amino acids are likely to share similar recognition characteristics. In addition, the results provide a rational background for the evaluation of antibody specificity and the interpretation of results in immunocytochemical studies using antisera to glutamate and aspartate.

Characterization of an antiserum to glycyl-d-aspartate (GDA) and its use as a probe for endogenous N-methyl-d-aspartate (NMDA)-like compounds☆

The long-term goal of this study is to identify endogenous N-methyl-d-aspartate (NMDA)-like compounds with the help of antibodies. Since NMDA contains a blocked amino group, it cannot be conjugated with glutaraldehyde to a carrier protein for immunization. Thus, we used the synthetic dipeptide GDA as a model hapten. The resulting antisera were characterized in both immunocytochemistry and enzyme immunoassay. A limited survey of immuno-cytochemically stained paraffin and vibratome sections revealed positively stained neurons in several areas of the central nervous system, e.g., the cerebral cortex, the hippocampus, and the trigeminal and spinal ganglia. The specificity of the staining was determined on paraffin sections of trigeminal ganglion. GDA, NMDA, and d-aspartate blocked staining, whereas l-aspartate, l-glutamate, kainic acid, and quisqualic acid had little or no effect. An enzyme immunoassay was developed in which polystyrene plates were coated with 10 muM GDA and A-GDA 641 used at a 1:150,000 dilution. In competition studies GDA had an EC(50) = 8.8 x 10(-7)M and a detection limit of 2.3 x 10(-7)M. Of the 25 other compounds tested only succinic acid, d-aspartate, quinolinic acid, NMDA, and fumaric acid demonstrated significant cross-reactivity (>0.01%) with the antiserum. In addition extracts of both brain and liver displaced the binding of the antiserum to the GDA-coated plate, although there was no immunocytochemical labeling in liver tissue. The results show that the GDA serum has strict and specific binding requirements for a d-aspartate-like chemical configuration and recognizes endogenous brain substance(s). The identity and function of that substance (or substances) remain to be determined.

Gonadotropin-like Immunoreactivity in the Rat Ovary

To devise efficient and safe methods of contraception, it is necessary to gain a firm grasp of the biological processes of reproduction. In the female, the gonadotropic hormones have a central role in regulating the reproductive cycles. Thus, it is of prime importance to understand the effect that these hormones have on their target cells. Labeled gonadotropins have been used in autoradiographic studies of the ovary. The majority of these reports have focused on the cells of the corpus luteum (Espeland et al., 1968; Midgley and Beals, 1971; Ryan and Lee, 1976; Abel et al., 1976; Chen et al., 1978; Conn et al., 1978). Fewer electron-microscopic autoradiographic studies have been reported on the cells of the stratum granulosum (Abel et al., 1976; Han, 1979), and none on the theca interna. Since immunocytochemistry (ICC) has been successfully used to demonstrate gonadotropin immunoreactivity in the male reproductive tract (Hutson et al., 1977; Childs et al., 1978), it was decided to employ this technique at the ultrastructural level to localize immunoreactive gonadotropins in the rat ovary. Three predominant cell types of the ovary, namely, the cells of the corpus luteum, the stratum granulosum, and the theca interna, were studied.