Thérèse Ternynck

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 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

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.

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).

Natural autoantibodies constitute a substantial part of normal circulating immunoglobulins.

According to Burnet’s clonal selection theory,’ in the early 1960s, explanations for tolerance and autoimmunity were attractively simple. Autoreactive lymphocytes were deleted during embryonic life to provide self-tolerance, and autoantibodies, always noxious, were the products of mutant forbidden clones that were able to circumvent this process of immunological homeostasis. However, during the last two decades, several groups succeeded in challenging clonal deletion as a general explanation for tolerance to self’ by: (a) Inducing autoimmune diseases by injecting organ extracts; (b) Demonstrating the presence of numerous autoantibodies in normal serum coming from a normal population;’ (c) Demonstrating the presence of normal autoreactive B cells? (d) Inducing in an animal numerous autoantibodies after challenging with mitogens. The existence of natural antibodies in normal human serum was first reported by Landsteiner in 1900, when he discovered the presence of natural hemagglutinins in serum directed against blood group determinants of the A-B-0 system. Accordingly, Boyden’ defined natural antibodies as a family of molecules present in the body fluids of normal animals that is able to specifically combine with antigens, but not with the immunologically acceptable molecules normally present in the body fluids. Nevertheless, natural antibodies directed against various antigens have been reported in different animal species and are often directed against various autoantigens. Obviously, a clear distinction between autoantibodies and natural antibodies is difficult to establish. During preparation of specific antisera against cytoskeletal proteins, we also detected the presence of natural antitubulin antibodies in the sera of normal humans and several species of normal nonimmunized animals: We subsequently isolated and characterized these antibodies, which were able to interact with autologous tubulin. For several years, we have been working on natural autoantibodies a t the Unit6 d’lmmunocytochimie of Pasteur Institute. The results of these studies are reported in the present work.

Conjugation of p-Benzoquinone treated enzymes with antibodies and Fab fragments☆

Abstract The introduction of active groups into peroxidase, alkaline phosphatase, glucose oxidase and β-galactosidase was achieved by treating these enzymes with p-benzoquinone. These ‘activated’ enzymes, after elimination of excess benzoquinone, were able to be coupled with antibodies or their Fab fragments. The conjugates obtained were tested for their capacity to stain by immunocytochemical procedures intracellular immunoglobulins. The most suitable conjugates were obtained when (a) the enzyme was activated at pH 6 for 1 hr at room temperature, (b) the activated was coupled with Fab for 48 hr at 4°C in 0.1 M sodium bicarbonate, and (c) the molar ratio of enzyme to Fab was 4. With these optimally prepared conjugates, good immunocytochemical staining was obtained at a concentration of 10 μg Fab per ml. Under the above conditions, about 60% of the added Fab was coupled with 15% of the peroxidase. Ninety per cent of the conjugate consisted of one mole of peroxidase coupled to one mole of Fab. Benzoquinone treatment did not substantially modify the enzymes since 85–100% of their initial enzymatic activity was preserved; however after their conjugation to Fab a decrease of up to 40% of their initial activity was observed, It is concluded that p-benzoquinone is a useful two-step reagent to prepare enzyme-protein conjugates. The mechanism of conjugation is discussed.

Une technique immunoenzymatique pour la mise en évidence de l'hybridation moléculaire entre acides nucléiques

Summary An immunoenzymatic procedure has been developed based on the use of monoclonal antibodies specific for 5-bromodeoxyuridine (BdUr). It allows the detection of BdUr-labelled DNA immobilized on a nitrocellulose filter. Using this procedure, it was possible to detect up to 0.5 pg of mammalian DNA labelled in vivo with BdUr, 5 pg of nick-translated BdUr-labelled PBR-322 and, using this latter probe and dot-blot hybridization, 50 pg of native unlabelled PBR-322.

Comparison of natural antibodies to autoantibodies arising during lupus in (NZB × NZW)F1 mice

Autoantibodies arising in (NZB x NZW)F1 (B/W) mice during the lupus-like syndrome were studied and compared to natural antibodies present in normal mice. The antibody activities were tested in sera, circulating immune complexes (CIC) and kidney eluates, using an enzyme immunoassay against a panel of self and non-self antigens: actin, myosin, tubulin, DNA, myoglobin, spectrin and trinitrophenylated bovine serum albumin (TNP/BSA). In the B/M mouse sera, IgM antibodies reacting with all the panel of antigens (PAg) and comparable to those of normal mice, increased moderately from 5 to 9 months and markedly during the last stage preceding death (10 months), when particularly high levels of anti-DNA, anti-tubulin and anti-myoglobin antibodies were noted. Polyreactive IgM antibodies present in CIC were moderately increased while those present in complexes deposited in kidneys were strongly enhanced after the 8th month. IgG antibodies showed an early increase (2 months) in B/W sera for anti-TNP activity, which remained more or less constant until death, while a later (5-6 months) and greater increase of activity, mainly directed against DNA but also against the other antigens of the panel, was observed. In CIC, IgG, mainly anti-DNA but also anti-TNP, were enhanced at the end of the disease while at the same time IgG reacting with all the PAg were found in kidney deposits. Isolation of antibodies from sera on a DNA-immunoadsorbent demonstrated that eluted IgM reacted with all the PAg but mainly with DNA, while IgG reactivity was more restricted to DNA and to a lesser degree to TNP. The D23 idiotype, characteristics of natural polyspecific antibodies, was expressed on IgM and IgG autoantibodies from B/W mice and was enhanced, particularly in kidneys, at the end of the disease. These results demonstrate that natural antibodies are a part of the population of increased autoantibodies in this disease and could participate with IgG anti-DNA antibodies in lupus.