Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies.
TL;DR: Conjugation of peroxidase, glucose oxidase, tyrosinase and alkaline phosphatase to human immunoglobulin-G, human serum albumin, sheep antibody and rabbit antibody was carried out with glutaraldehyde to characterize the antibodies after immunoelectrophoresis.
About: This article is published in Immunochemistry.The article was published on 1969-01-01. It has received 1453 citations till now. The article focuses on the topics: Immunoelectrophoresis & Glutaraldehyde.
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3,724 citations
TL;DR: In this paper, the specificity of the DNP system was assessed by inhibition with hapten, and the reaction of immune serum against DNP with DNP-protein, adsorbed to the tubes, was completely inhibited by haptens in solution.
3,349 citations
TL;DR: Biotin was covalently attached to antibodies, antigens and enzymes, and the effects of this labeling on the antigen and antibody binding capacity and on enzymatic activity were tested.
Abstract: Biotin was covalently attached to antibodies, antigens and enzymes, and the effects of this labeling on the antigen and antibody binding capacity and on enzymatic activity were tested. Based on avidin-biotin interaction, the labeled proteins were used in quantitative enzyme-immunoassay and enzyme-immunohistochemical staining procedures. Two procedures were developed. In the first procedure, named the Bridged Avidin-Biotin (BRAB) technique four steps were used sequentially in order to quantify or detect an immobilized antigen: 1) incubation with biotin-labeled antibody; 2) incubation with avidin; 3) incubation with biotin-labeled enzyme; 4) measurement or histochemical staining of the enzyme. The technique is based on the observation that avidin possesses four active sites. In the second procedure, named the Labeled Avidin-Biotin (LAB) technique, biotin-labeled antibody and enzyme-labeled avidin are used sequentially. Enzyme-associated antigen is then quantified or revealed immunohistochemically. The optimal conditions for enzyme-immunoassay and enzyme-immunohistochemical staining using BRAB and LAB procedures were established.
1,745 citations
Cites methods from "Coupling of enzymes to proteins wit..."
...were obtained by hyperimmunizing animals following procedures already described (1)....
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...Cytocentrifuged mouse lymphoid cells were prepared, fixed, and histochemically stained following procedures described in detail elsewhere (1, 4)....
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...In general, these conjugates were prepared by covalently coupling the enzyme marker to the antibody (1, 5, 20, 22), but the noncovalent immunological reaction has also been used for antibody-enzyme binding (2, 19, 21)....
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TL;DR: An overview of glutaraldehyde as a crosslinking reagent is given by describing its structure and chemical properties in aqueous solution in an attempt to explain its high reactivity toward proteins, particularly as applied to the production of insoluble enzymes.
Abstract: Glutaraldehyde possesses unique characteristics that render it one of the most effective protein crosslinking reagents. It can be present in at least 13 different forms depending on solution conditions such as pH, concentration, temperature, etc. Substantial literature is found concerning the use of glutaraldehyde for protein immobilization, yet there is no agreement about the main reactive species that participates in the crosslinking process because monomeric and polymeric forms are in equilibrium. Glutaraldehyde may react with proteins by several means such as aldol condensation or Michael-type addition, and we show here 8 different reactions for various aqueous forms of this reagent. As a result of these discrepancies and the unique characteristics of each enzyme, crosslinking procedures using glutaraldehyde are largely developed through empirical observation. The choice of the enzyme-glutaraldehyde ratio, as well as their final concentration, is critical because insolubilization of the enzyme must result in minimal distortion of its structure in order to retain catalytic activity. The purpose of this paper is to give an overview of glutaraldehyde as a crosslinking reagent by describing its structure and chemical properties in aqueous solution in an attempt to explain its high reactivity toward proteins, particularly as applied to the production of insoluble enzymes.
1,515 citations
TL;DR: Insoluble antigen and antibody derivatives were obtained and were found to be efficient, specific and stable immunoadsorbents, and were used, either in column procedure or in batchwise operation, for the isolation of antigens or antibodies.
1,396 citations
References
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Book•
01 Jan 1961
TL;DR: The new Pearse bids fair to become the leader, even amongst so notable a collection of books devoted entirely or largely to histochemical techniques.
Abstract: A. G. E. Pearse is a relative newcomer to the field of histochemistry. He holds an M.D. degree from the University of Cambridge and is now Lecturer in Histochemistry at the Postgraduate Medical School, University of London. One of his first contributions to histochemistry consisted of a critical review of its methodology and interpretation, written for pathologists and appearing in the British Journal of Clinical Pathology 4: 1 (1951). He has also introduced a number of new or modified techniques. Perhaps those having the greatest interest for endocrinologists are presented in a series of papers concerning the demonstration of the glycoprotein hormones of the anterior pituitary gland (Nature 162: 651, 1948; J. Path. & Bad. 61: 195, 1949, 64: 791 & 811, 1952; Stain Technol. 25: 95, 1950). The present volume comprises 17 chapters, beginning with a brief but fairly inclusive history of the development of this branch of science and continuing with 2 chapters on methods of fixation and sectioning, 3 on the staining of proteins, 4 on the demonstration of enzymes, 1 on the use of enzymes as histochemical reagents, and 1 each on carbohydrates, lipids, aldehydes and ketones, pigments, inorganic substances, and physical methods. The book concludes with appendices which give the detailed steps of the histochemical techniques which Dr. Pearse has himself found most useful for demonstrating various tissue constituents. Complete author and subject indices follow. A further valuable feature of the book consists of numerous black-and-white and a few colored photomicrographs illustrating the appearance of sections prepared by some of the histochemical methods discussed. A rash of books devoted entirely or largely to histochemical techniques has appeared in the last few years. Outstanding are the treatises by Lillie (1948), Romeis (1948, 15th ed., German), Glick (1949), Gatenby and Beams (1950, 11th ed.), Gomori (1952), and Lison (1953, 2nd ed., French). In this reviewer's opinion, the new Pearse bids fair to become the leader, even amongst so notable a collection. In each chapter, Dr. Pearse reviews the chemistry and biological importance of the substances in question and then goes on to discuss critically the methods used for their demonstration and differentiation. The bibliographic references are numerous and appear to include most of the major contributions in each area. Pearse has omitted some references which appear fundamental, for example, the definitive experimental paper and review by Marcus Singer concerning the nature of acidic and basic staining of proteins (/. Biol. Chem. 75: 133, 1948; Internal. Rev. Cytol. I: 211, 1952). Likewise, the techniques which he has selected to give in detail represent his particular preferences, whereas numerous methods which are possibly equally satisfactory are barely mentioned. While Pearse's omissions in this respect are not serious, the usefulness of some methods which he has overlooked should not be lost sight of. Needless to say, in a field which is growing so rapidly and to which several journals are now entirely devoted, no book can long remain up-to-date.
7,499 citations
TL;DR: The early stages of absorption of intravenously injected horseradish peroxidase in proximal tubules of mouse kidney were studied with a new ultrastructural cytochemical technique, which gives sharp localization and is sensitive to protein transport.
Abstract: The early stages of absorption of intravenously injected horseradish peroxidase in proximal tubules of mouse kidney were studied with a new ultrastructural cytochemical technique. In animals killed as early as 90 sec after injection, reaction product was found on the brushborder membranes and in the apical tubular invaginations. From the latter structures it was transported to the apical vacuoles, in which it was progressively concentrated to form protein absorption droplets. The method, which employs 3,3'-diaminobenzidine as oxidizable substrate, gives sharp localization and is sensitive. This system is advantageous in studying the early stages of renal tubular protein absorption, since small amounts of protein on membranes and in tubules and vesicles can be detected easily. The method also appears promising for studying protein transport in a variety of other cells and tissues.
6,495 citations
TL;DR: A postfixation in osmium tetroxide, even after long periods of storage, developed an image that—notable in the case of glutaraldehyde—was largely indistinguishable from that of tissues fixed under optimal conditions with osmia tetroxides alone.
Abstract: The aldehydes introduced in this paper and the more appropriate concentrations for their general use as fixatives are: 4 to 6.5 per cent glutaraldehyde, 4 per cent glyoxal, 12.5 per cent hydroxyadipaldehyde, 10 per cent crotonaldehyde, 5 per cent pyruvic aldehyde, 10 per cent acetaldehyde, and 5 per cent methacrolein. These were prepared as cacodylate- or phosphate-buffered solutions (0.1 to 0.2 M, pH 6.5 to 7.6) that, with the exception of glutaraldehyde, contained sucrose (0.22 to 0.55 M). After fixation of from 0.5 hour to 24 hours, the blocks were stored in cold (4°C) buffer (0.1 M) plus sucrose (0.22 M). This material was used for enzyme histochemistry, for electron microscopy (both with and without a second fixation with 1 or 2 per cent osmium tetroxide) after Epon embedding, and for the combination of the two techniques. After fixation in aldehyde, membranous differentiations of the cell were not apparent and the nuclear structure differed from that commonly observed with osmium tetroxide. A postfixation in osmium tetroxide, even after long periods of storage, developed an image that—notable in the case of glutaraldehyde—was largely indistinguishable from that of tissues fixed under optimal conditions with osmium tetroxide alone. Aliesterase, acetylcholinesterase, alkaline phosphatase, acid phosphatase, 5-nucleotidase, adenosine triphosphatase, and DPNH and TPNH diaphorase activities were demonstrable histochemically after most of the fixatives. Cytochrome oxidase, succinic dehydrogenase, and glucose-6-phosphatase were retained after hydroxyaldipaldehyde and, to a lesser extent, after glyoxal fixation. The final product of the activity of several of the above-mentioned enzymes was localized in relation to the fine structure. For this purpose the double fixation procedure was used, selecting in each case the appropriate aldehyde.
3,914 citations
3,439 citations
TL;DR: Small amounts of antibody were occasionally visible in cells in the lymphoid follicles of the spleen and lymph nodes, so that a minor contribution by lymphocytes to antibody synthesis cannot be excluded.
Abstract: A method for the specific histochemical demonstration of antibody in cells and parts of cells is described. It consists of carrying out a two stage immunological reaction on frozen sections of tissues: (a) allowing reaction between antibody in the tissue and dilute antigen applied in vitro, and (b) the detection of those areas where this antigen has been specifically absorbed by means of a precipitin reaction carried out with fluorescein-labelled antibody. Examination under the fluorescence microscope reveals the yellow-green fluorescence of fluorescein over those areas where a precipitate has formed. A study of the hyperimmune rabbit on the first few days after the last of a series of intravenous antigen injections reveals that antibody against human gamma-globulin or ovalbumin is present in groups of plasma cells in the red pulp of the spleen, the medullary areas of lymph nodes, the submucosa of the ileum, and the portal connective tissue of the liver. Because of extensive non-specific reactions, the bone marrow could not be examined. Small amounts of antibody were occasionally visible in cells in the lymphoid follicles of the spleen and lymph nodes, so that a minor contribution by lymphocytes to antibody synthesis cannot be excluded.
1,531 citations