Stratis Avrameas

The use of avidin-biotin interaction in immunoenzymatic techniques.

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.

The Cross-linking of Proteins with Glutaraldehyde and its use for the preparation of immunoadsorbents

Abstract Insoluble antigen and antibody derivatives were obtained or antibody. Evidence has been obtained to show that was added to solutions of antigen or antibody. Evidence has been obtained to show that the protein molecules were cross-linked covalently to one another. The immuno-adsorbent properties of these insolubilized proteins were investigated. They 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.

Coupling of enzymes to proteins with glutaraldehyde: Use of the conjugates for the detection of antigens and antibodies

Abstract 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. The conjugates retained a substantial part of their immunological and enzymatic activity. They were utilized for the intracellular detection of antigens and antibodies and they were employed for the characterization of the antibodies after immunoelectrophoresis.

Coupling of Enzymes to Antibodies and Antigens

The sensitivity of enzyme immunoassays, and of enzyme immunohistochemical techniques in general, depends mainly on the preparation of enzyme-antigen or enzyme-antibody conjugates possessing high enzymatic and immunological activity. The purpose of this paper is to review and to compare the various procedures described to date for the preparation of such conjugates. Coupling of small organic compounds with enzymes is a special procedure, and in this paper only the preparation of enzymelabelled proteins will be considered. The coupling of an enzyme with a protein involves the use of a cross-linking agent. The cross-linking agent reacts via its active groups (at least two) with the functional groups present in enzymes and proteins, and this is how coupling is achieved. Functional groups in proteins include amino, imino, hydroxyl, phenol, and thiol groups; their reactivity depends mainly on their microenvironment. These functional groups may be part of or contribute to the active site of enzymes, antibodies, and antigens. If this is the case, coupling via such a group can abrogate biological activity. These functional groups also contribute to the maintenance of the native conformation of the molecules; a crucial modification by interor intramolecular binding may lead to loss of biological activity. Furthermore, over-substitution can lead either to inaccessibility of the active site or to an increase in the nonspecific adherence of the conjugated macromolecules. Since functional groups present in proteins, including enzymes, are identical, it is evident that selective coupling producing an exclusive homogeneous enzyme-protein conjugate is too difficult to achieve. In fact, all coupling reactions reported so far have always produced a mixture of heterogeneous enzyme-protein conjugates and enzyme-enzyme and or protein-protein conjugates. Even under conditions where the preparation of a homogeneous 1:l molar ratio of peroxidase-antibody conjugate was highly favoured, the simultaneous preparation of minor amounts of other conjugates and of peroxidase dimers could not be avoided. From the above, it is evident that an effective coupling procedure is certainly not easy to devise and that even when a procedure has been shown to produce satisfactory results in any given system, its extrapolation to another system calls for circumspection. Two types of coupling reactions have been described. The first involves one-step reactions and the other two-step reactions. In one-step procedures the enzyme, the cross-linking agent and the protein are all mixed together and allowed to react. In this procedure the reaction is difficult to control, chiefly because the reaction rates of the functional group in enzyme and protein are different. This may lead to selective polymerization of either enzyme or protein. Conjugates prepared with one-step procedures are basically heterogeneous. Theoretically, a wider variety of enzyme-protein conjugates can be prepared with the one-step than with the two-step procedures. In two-step procedures the enzyme (or antigen or antibody) is first treated with the cross-linking agent and then the antigen or the antibody (or enzyme) is added. Alternatively, the enzyme is treated with an excess of cross-linking agent, then the excess reagent is removed, and finally the activated enzyme is allowed to react with the antigen or antibody. Two-step reactions make use of reagents possessing two potentially active groups, the reactivity of which can, however, be

Glutaraldehyde, cyanuric choloride and tetraazotiexe O-dianisidine as coupling reagents in the passive hemagglutination test

Abstract Using tetraazotized o -dianisidine, cyanuric chloride or glutaraldehyde, antigens were coupled to erythrocytes. The sensitized cells could be used in passive hemagglunation tests. With the three named reagents the agglutination reaction was specific and clear cut pictures of positive or negative hemmagglutination were obtained. The maximum hemagglutination titers were at least as high as those obtained with erythrocytes sensitized with bis-diazotized benzidine or tannic acid. The cells coated with the acid of glutaraldehyde could be lyophilized and used again without any appreciable loss of maximum agglutination titer.

Quantitation of 5-bromo-2-deoxyuridine incorporation into DNA: an enzyme immunoassay for the assessment of the lymphoid cell proliferative response☆

As an alternative to the measurement of radiolabeled thymidine incorporated into DNA, a method is presented in which thymidine has been replaced by its analogue, 5-bromo-2-deoxyuridine (BUdR). BUdR incorporated into DNA (BUdR-DNA) is measured by a sandwich-type enzyme immunoassay using a monoclonal anti-BUdR antibody. This method allows the quantitation of 4 ng of BUdR-DNA. Comparative experiments with myeloma cells and LPS stimulated spleen B-cells have shown that this technique is at least as sensitive as the traditional counting of [3H]thymidine.

Polyacrylamide-protein immunoadsorbents prepared with glutaraldehyde

The preparation of biologically active waterinsoluble derivatives of antigens and antibodies using glutaraldehyde as the cross-linking agent has been reported [ 11. The present paper describes a method where antigens and antibodies are coupled to glutaraldehyde activated beads of polyacrylamide gels [2]. The derivatives obtained were examined for their effectiveness in the use as immunoadsorbents. The results obtained proved that these derivatives behave as highly specific immunoadsorbents which allow the isolation of antigens and antibodies in high yields. Polyacrylamide beads Bio-Gel P of different classes and sizes were products of Calbiochem. Luzern, Switzerland. The rabbit or sheep antisera employed in the present work were prepared as already described [l, 31. The concentration of protein was determined by a modified biuret method [4]. The peroxidase content of different preparations was estimated from the extinction coefficient at 403 nm E:Frn = 22. The antibody content of the different preparations was determined by the quantitative precipitation reaction of Heidelberger and Kendall [5]. Immunoelectrophoresis was performed according to the method described by Grabar and Williams [6] using gels of agarose at 0.8% concentration. 2. Experimental 2.2. Polyacrylamide-protein derivatives 2.1. Materials and methods Crystallized human and bovine serum albumin and human and rabbit gamma globulin fraction II were purchased from Pentex (Kankakee, Ill., USA); horse radish peroxidase RZ 3 and cytochrome c were products of Sigma Co. (St. Louis, MO., USA); bovine trypsin and chymotrypsin were purchased from Choay, France. 25% Aqueous solutions of glutaraldehyde was obtained from Schuchardt (Miinchen) and TAAB Laboratories, Reading, Eng land, which were used without further purification.

Natural antibodies against tubulin, actin, myoglobin, thyroglobulin, fetuin, albumin and transferrin are present in normal human sera and monoclonal immunoglobulins from multiple myeloma and Waldenström's macroglobulinemia may express similar antibody specificities

Sera from a pool of 800 healthy donors and from 3 individual healthy donors were passed through tubulin, actin, thyroglobulin, myoglobin, fetuin, transferrin and albumin immunoadsorbents. Proteins were eluted in all the immunosorbents and were found to be essentially composed of the three major Ig classes and albumin. The isolated Ig fractions were shown to react specifically, via their Fab fragment with the antigens and were specifically inhibited by them. These results strongly suggest that natural antibodies against the seven antigens are present in normal human serum, and probably against a high variety of self antigens. These results prompted us to search in the sera of patients with monoclonal gammapathies, paraproteins having natural antibody-like function. Among the 62 sera studied 3 were shown to react with actin and 1 with tubulin. Most important, these 4 monoclonal immunoglobulins exhibited similar specificities to that found with natural antibodies. This seems to indicate, that at least for some patients, the monoclonal immunoglobulins produced may represent the expansion of a clone producing a natural antibody.

Magnetic solid phase enzyme-immunoassay

Abstract A sandwich non-competitive enzyme-immunoassay procedure using antigen or antibody cavalently linked to magnetic polyacrylamide-agarose beads has been developed. A magnetic rack was used to separate the beads from the liquid phase and therefore time-consuming multiple centrifugation was avoided. Comparative studies using various conjugates of peroxidase, glucose oxidase and alkaline phosphate with antibodies were performed. In regard to the accuracy and reproducibility of the enzyme immunoassay, best results were obtained with alkaline phosphatase labelled antibodies. The procedure allowed the measurement of the following lowest quantities of proteins: 8 ng IgG, 40 ng IgA, 70 ng IgM, 1 i.u of IgE and 20 ng of specific antibodies against BSA.

Polymerization and Immobilization of Proteins Using Ethylchloroformate and Glutaraldehyde

By using several cross-linking agents, water insoluble protein polymers have been prepared and employed as immunoadsorbents (14). Of these reagents, ethylchloroformate (1) and glutaraldehyde (2) allowed the preparation of the most effective, stable, and specific immunoadsorbents. Glutaraldehyde is a dialdehyde known to react mainly with &-amino groups of peptides, especially lysine (5). Ethylchloroformate forms covalent bonds between free amino and carboxyl groups of polypeptide chains (1). Addition of one of these cross-linking agents to a solution of low protein concentration produces, in general, protein polymers of low molecular weight which are water-soluble, while its addition to a solutiop of high protein concentration yields protein polymers of high molecular weight which are generally water-insoluble. Our experiments showed that this insolubilization was optimal around the isoelectric point of each protein, while the antigenor antibodybinding capacity was best preserved when insolubilization was carried out at pH 5. Therefore, in order to have the possibility of rendering insoluble all proteins, at optimal conditions, even when their isoelectric point was far from 5 or when their concentration was low, the use of bovine serum albumin (BSA) as an auxiliary protein was introduced. This protein was chosen because its isoelectric point is 4.9, it possesses many lysine residues, and it is relatively cheap and easily available. When glutaraldehyde or ethylchloroformate is added to a solution containing various proteins and BSA, a cross-linking between these various protein molecules and those of BSA occurs. If the reaction is carried out at pH 5, BSA is insolubilized along with the other proteins. In that case BSA acts as the insoluble supporting matrix. Cross-linking agents can also be employed for ,the introduction of active groups into an insoluble matrix which can subsequently be used for the immobilization of proteins. Thus, when there is an excess of glutaraldehyde, one of its two aldehyde groups reacts with free amino or amide residues present in polyacrylamide gels. After washing, the remaining free active aldehyde group is available for combination with amino groups of proteins added subsequently (8).