Jean-Luc Guesdon

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.

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

Detection of hepatitis B virus sequences in serum by using in vitro enzymatic amplification

In vitro enzymatic amplification was applied to detect hepatitis B virus (HBV) DNA sequences in serum. This technique, known as the polymerase chain reaction (PCR) was used to amplify a 128 bp DNA fragment including a 112 nucleotide long sequence complementary to a region in the S gene of the HBV genome. Amplified samples were subjected to spot-test hybridization and scintillation counting using a 32P-labeled oligonucleotide probe. A kinetic study, performed for 4 to 32 PCR cycles with a viral particle preparation, showed a time-limited exponential accumulation of the specific amplified DNA fragment. Amplification yield after 32 cycles was at least 4 X 10(6) with a detection limit equal to 3 X 10(2) viral particles per ml of serum. As the reliability of the PCR technique was greatest for 24 PCR cycles, these conditions were used to develop a quantitative test with a detection limit of 4 X 10(4) viral particles per ml of serum. Results of this test were perfectly correlated with those obtained from the classical spot test without amplification. Ethidium bromide stained agarose gel and Southern blot analysis confirmed the specific amplification of the 128 bp HBV DNA fragment.

Identification of dengue sequences by genomic amplification: rapid diagnosis of dengue virus serotypes in peripheral blood

Polymerase chain reaction (PCR) was developed for the in vitro amplification of dengue virus RNA via cDNA. A fraction of the N-terminus gene of the envelope protein in the four dengue serotypes was amplified using synthetic oligonucleotide primer pairs. Amplified products were cloned and used as dengue type-specific probes in gel electrophoresis and dot-blot hybridization. We detected and characterized dengue virus serotypes in blood samples by the three-step procedure DNA-PAH consisting in cDNA priming (P), DNA amplification (A) and hybridization (H) using specific non-radiolabelled probes. Our findings showed that DNA-PAH was more rapid and sensitive in the identification of the infecting serotype than the mosquito cell cultures. Moreover, the failure of cultures to detect virus particles in sera containing few copies of viral genome or anti-dengue antibodies justified the approach of DNA-PAH to the dengue identification in clinical specimens.

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.

Polyacrylamide-agarose beads for the preparation of effective immunoabsorbents.

Gluxaraldehyde-activated polyacrylamide-agarose beads (Ultro-gel) have been employed to bind proteins. The derivatives obtained were found to be effective immunoabsorbents allowing the quick isolation of pure antibodies in high yields.

Acetylaminofluorene-labelled ribosomal RNA for use in molecular epidemiology and taxonomy.

The use of acetylaminofluorene-labelled 16 + 23S rRNA (from Escherichia coli) is described for determining rRNA-gene-restriction patterns. The labelled probe allowed molecular fingerprinting of bacteria belonging to diverse phylogenetic branches (Enterobacteriaceae, Haemophilus, Pseudomonas, Acinetobacter, Brucella, Leptospira, Cytophaga, Campylobacter, Methylophaga). The labelled probe can be stored frozen (-20 degrees C) for at least a year and can endure vacuum dessication, ethanol precipitation or lyophilization.

Magnetic enzyme immunoassay for measuring human IgE

This paper reports a solid-phase sandwich technique for the assay of human IgE using anti-IgE antibodies bound to magnetic polyacrylamide-agarose beads. Following this technique, IgE to be determined is allowed to bind to the magnetic beads bearing the anti-IgE antibodies. After washing on a magnetic rack, the beads are incubated with enzyme-labeled antibodies, washed again, and then the enzyme activity associated with the beads is measured. The use of magnetic polyacrylamide-agarose beads and of enzyme-labeled antibodies makes the measurement of IgE in human sera easy to perform, safe, and reproducible. This procedure allows the measurement of IgE concentration in serum containing more than 2 IU/ml. The values obtained from 104 human sera closely corresponded to those obtained by radioimmunoassay (correlation coefficient, r = 0.9751).

Recombinant single-chain Fv antibody fragment–alkaline phosphatase conjugate for one-step immunodetection in molecular hybridization

Using phage-display technology, a recombinant single-chain Fv antibody fragment (scFv) was rapidly generated from the K16-16 hybridoma secreting mouse monoclonal antibody (MAb) that binds to acetylaminofluorene-labeled DNA (AAF-DNA). The selected A4 phage-scFv specifically bound to AAF-DNA. The anti-AAF scFv gene was then recloned into a fusion vector for the production of a hybrid protein comprising the antibody fragment fused to a potent bacterial alkaline phosphatase variant (PhoAv). The anti-AAF scFv-PhoAv hybrid protein was bifunctional and possessed both antigen binding capacity and PhoA activity. The recombinant conjugate was directly used, without further purification, for one-step immunodetection in dot-blot hybridization. The detection limit was identical and the test was quicker than the conventional two-step procedure with the purified anti-AAF MAb revealed with a secondary enzyme-labeled antibody. To assess the value of this new reagent for the immunodetection of genomic nucleic acids, genomic DNAs of Campylobacter jejuni and Campylobacter coli were then one-step immunodetected with non-purified recombinant scFv-PhoAv conjugate in a Southern-blot hybridization experiment. The present study shows that the genetic fusion with PhoAv provides a new tool for immunodetection which presents easier and quicker production and use with the same sensitivity and specificity as classical reagents. The recombinant anti-AAF scFv-PhoAv conjugate is a promising alternative reagent for applications involving the immunodetection of specific DNA or RNA sequences, such as the detection and characterization of microorganisms.

Immunoenzymatic techniques applied to the specific detection of nucleic acids: a review

Numerous enzymatic and chemical methods are now available for the preparation of non-radioactive nucleic acid probes. Labels, such as enzymes, fluorophores, lumiphores can be attached to the nucleic acid probe either by covalent bonds (direct labelling) or by biospecific recognition after hybridization (indirect labelling). The principle of the latter method is based on the use of a hapten-labelled nucleic acid probe which is generally detected by an immunoenzymatic assay. Indirect labelling has several advantages: this procedure uses multienzyme complexes to increase the number of enzyme molecules associated with hybridization and hence provides an increase in detectability; moreover, haptens (biotin, dinitrophenol, acetylaminofluorene analogues, digoxigenin, brominated or sulphonylated pyrimidines) used to label nucleic acid probes are not sensitive to elevated temperatures (42-80 degrees C), extended incubation times (several hours), detergents and organic solvents currently required in hybridization techniques. The application of the immunoenzymatic and related techniques to nucleic acid probing is reviewed, focussing on the strategies of non-radioactive hybridization, hapten-labelling of nucleic acids and methods for the immunodetection of the hybrids.