Expression, immunochemical characterization and localization of the Mycobacterium tuberculosis protein p27.

Rv2108 is a gene of the PPE family of Mycobacterium tuberculosis specific for this bacterial complex and that may encode a putative protein p27. This gene was amplified, inserted into bacterial vectors, sequenced, and expressed as a recombinant protein. Specific antibodies to this protein were generated and used for immunochemical characterization and cellular localization. Mass spectrometric analysis of the expressed protein revealed a molecule that corresponded to the p27 putative protein. The expressed protein was immunologically active, and reacted with antibodies from tuberculosis patient sera. Specific immunoblot analysis confirmed the presence of the p27 antigen in Mycobacterium bovis BCG strain and in human clinical isolates of M. tuberculosis, but not in other mycobacteria tested. Western blot and immunoelectron microscopic analysis of BCG strain indicated that the p27 protein is localized in the membrane of the cell. The specific expression of the p27 protein in the M. tuberculosis complex could provide a novel specific complimentary diagnostic test for the presence of and infection with M. tuberculosis.

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.

Lectin immuno tests: quantitation and titration of antigens and antibodies using lectin-antibody conjugates.

We have investigated the possibility of using lectin-antibody conjugates as general reagents in immunological procedures requiring a labeled antigen or antibody. Using these conjugates, labeling is achieved through saccharide binding sites of lectins which operate as acceptors for glycoconjugate marker substances added secondarily. Marker substances used in this work were enzymes, radioactively labeled glycoconjugates and erythrocytes, but other markers can also be used. Using the first two markers, antigens and antibodies were determined with accuracy and sensitivity equal to those of conventional enzyme or radioimmunoassays. Using erythrocytes as a marker, a simple erythro-adsorption procedure, possibly followed by hemolysis, has been developed which allowed the titration of antigens and antibodies to be carried out with a sensitivity at least equal to enzyme or radioimmunoassays.

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.

An amplification system using BSA-antibody conjugate for sensitive enzyme immunoassay.

A procedure is described for sensitive titration of antibodies, macromolecular antigens and haptens by enzyme immunoassay. It involves using first antigen or antibody labelled with bovine serum albumin (BSA) and then an anti-BSA antibody conjugated with an enzyme. The performance characteristics of this assay are indicated and compared with those for conventional enzyme immunoassay. The present procedure allowed fast sensitive titration of human IgE, rabbit type III anti-streptococcal antibody and cAMP.

Magnetically responsive polyacrylamide agarose beads for the preparation of immunoabsorbents.

Glutaraldehyde-activated magnetically responsive polyacrylamide agarose beads have been employed to bind bovine serum albumin and human, sheep and rabbit IgG. These were tested for their effectiveness as immunoabsorbents and were found to allow isolation of pure antibodies in high yields. The use of magnetically responsive beads as the solid support in immunoabsorption procedures renders isolation of antibodies easy and rapid.

Escherichia coli heat-stable enterotoxin (STa)-biotin conjugates for the titration of STa antisera by an enzyme-linked immunosorbent assay.

The development of a new approach to the diagnosis of infectious diarrhoea, caused by Escherichia coli heat-stable enterotoxin (ST), was preceded by a preliminary study. The purpose of the latter was to establish whether three preparations of ST produced by a human isolate of enterotoxigenic E. coli (STa), obtained at different steps of the purification procedure (involving Amberlite XAD2 resin chromatography (P3), a gel filtration chromatography on a Biogel P4 (P2) or a disc-gel electrophoresis (P1)), could be employed to titrate antisera to STa using an ST-biotin enzyme-linked immunosorbent assay (ELISA). The solid-phase STa was obtained by first coupling the toxin to biotinyl-N-hydroxysuccinimide and then binding this conjugate to avidin adsorbed to flat-bottomed polystyrene microtitre plates. Using these reagents, the assay conditions were examined. Checkerboard tests determined optimal biotin-P3, P2 or P1 toxin conjugate concentrations to be used as the immunosorbent for P3, P2 and P1 antiserum titration. The immunosorbent prepared with STa purified only on Amberlite XAD2 resin was unable to differentiate significantly between P3, P2 or P1 antisera. Immunosorbent prepared with P2 or P1 detected widely differing titres between the three antisera and gave more sensitive results. Only small but questionable differences were observed between P2 and P1 toxin preparations.

Development of a bispecific monoclonal antibody for use in molecular hybridisation.

A mouse hybrid hybridoma (tetradoma) was prepared by fusing hybridomas producing monoclonal antibody to acetyl-aminofluorene with hybridomas producing antibody against calf intestine alkaline phosphatase. The tetradoma line established secreted immunoglobulin manifesting parental and bispecific binding characteristics. Bispecific monoclonal antibody was purified and used for a one-step immunodetection assay of non-radioactive DNA and RNA probes. The immunoassay developed was able to detect 5 pg DNA within 2 h and gave low background noise.

Enzyme immunoassay for the measurement of histamine.

This paper reports a competitive solid-phase enzyme immunoassay for measuring histamine in various biological samples. In this assay, the histamine to be quantified is chemically modified by 1,4-benzoquinone treatment and allowed to compete with a histamine-peroxidase conjugate for binding to a limited amount of an anti-histamine monoclonal antibody which was used to coat the wells of a microtitration plate. After incubation and washing, peroxidase activity associated with the solid phase is measured. With this method the histamine concentration in blood or various tissues may be determined easily, safely and reproducibly. Histamine concentrations from 0.3 to 20 ng/ml may be measured with the procedure reported here.

In situ enzyme immunodetection of surface or intracellular bacterial antigens using nitrocellulose sheets.

We describe an immunological method which allows the in situ colorimetric detection of translated DNA fragments in bacteria. In the absence of lysis only cell surface proteins are detected. For cytoplasmic proteins, lysis is required. The procedure comprises the following steps: bacteria are lysed, the proteins are transferred onto a disc of nitrocellulose sheet, the remaining protein sites are blocked, the disc is successively soaked in a solution of antibodies specific for the protein to be detected and in a solution of peroxidase-labelled anti-IgG antibody solution. Finally, the immune complexes are made visible by enzyme substrate incubation. We describe the application of this method to the detection of the LamB protein, the LacZ protein, and a LamB-polio VP1 chimera translated from cloned DNA fragment in E. coli.

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.

Solid-phase sandwich enzyme immunoassays of human chorionic gonadotropin using monoclonal antibodies.

A two-site sandwich enzyme immunoassay for human chorionic gonadotropin (hCG) employing monoclonal antibodies directed against beta- and alpha-subunits is described. Monoclonal anti-beta-hCG antibody was used for coating microtitration plates and monoclonal anti-alpha-hCG antibody labelled with 1 of the 3 enzymes namely horseradish peroxidase, alkaline phosphatase or beta-galactosidase was used as tracer. The assay is able to detect up to 1 ng hCG/ml. No significant difference was observed with respect to sensitivity and range of assay with the 3 enzymes. The assay can be performed as a 'two-step' assay or reduced to a 'one-step' procedure with a linear relationship between absorbance and hormone concentration up to 31.25 ng hCG/ml. Beyond these concentrations an inflection of the dose curve was observed. This can, however, be avoided by increasing the concentration of antibody-enzyme conjugate. A higher sensitivity enabling detection up to 0.25 ng hCG/ml was attained in the sandwich enzyme immunoassay with the use of biotin-avidin interface. The hCG values obtained on 47 human urine samples either by the 'one-step' or 'two-step' procedure were similar with a correlation coefficient of 0.996. Results obtained by 'two-step' sandwich enzyme immunoassay on 22 human urine samples correlated well (r = 0.968) with the values obtained by radioimmunoassay.

TerELISA: the ELISA test performed in Terasaki plates.

The use of Terasaki (10 microliter samples) and microtitration (100 microliter samples) plates as the solid phase in enzyme immunoassays was compared. Various antigens were used for coating the plates and antibodies present in human sera were evaluated using the same anti-human Ig antibody labelled with either beta -galactosidase, alkaline phosphatase, peroxidase or glucose oxidase. The results obtained, either by scoring with the naked eye or by absorbance reading with appropriate densitometers, showed that both plates were equally suitable and that the 4 enzymes were equally effective in detecting the same lowest quantity of antibody. A comparative evaluation using either Terasaki or microtitration plates for the quantitation of human anti-Echinococcus granulosus antibody ii 50 sera demonstrated that there was a good correlation between the two procedures (r = 0.8097). Finally, the use of glucose oxidase as the enzyme marked allowed a clear-cut distinction to be made between positive and negative samples with the naked eye alone.

Miniaturization of beta-galactosidase immunoassays using chromogenic and fluorogenic substrates.

Enzyme immunoassay techniques are widely use to quantify various antigens and antibodies. The final step of these techniques (i.e. enzyme reaction) may be carried out in several ways (e.g. chromogenic, fluorogenic, or radioactive substrate and thermometric measurement). This paper compares the effectiveness of the chromogenic and the fluorogenic substrates in the beta-galactosidase immunoassay. Using microtitration plates (150 microliter samples) coated with anti-human IgE, and anti-human IgE labeled with E. coli beta-galactosidase, the lowest concentrations of IgE that one could detect employing either the chromogenic (o-nitrophenyl-beta-D-galactopyranoside) or the fluorogenic (4-methyl-umbelliferyl-beta-D-galactopyranoside) substrate were determined. It was found that both substrates were almost equally effective in measuring the lowest concentration of IgE (0.075-0.13 IU/ml) under the optimal conditions. But, using fluorogenic substrate and suitable apparatuses the enzyme immunoassay can be miniaturized. Thus by using decreasing volumes of reagents, progressively smaller amounts of antigen were quantified: as the sample volumes were reduced from 150 to 10 microliter and finally to 0.3 microliter a progressive decrease from 7 x 107 molecules of IgE to 2.9 x 107 molecules and to 1.5 x 106 molecules was observed. The corresponding lowest detection limits were 0.075 IU/ml, 0.46 IU/ml and 0.8 IU/ml.

Antibody chimera technique applied to the detection of Escherichia coli heat-labile enterotoxin.

Covalently prepared chimera antibodies were tested in a ganglioside GM1 erythro-immunoassay (CERIA) for E. coli heat-labile enterotoxin (LT) detection. The antibody specific for LT was conjugated with a polyclonal antibody specific for sheep erythrocytes. The assay is based on the specific binding of LT to polystyrene-adsorbed GM1 and subsequent erythro-adsorption via chimera antibody by which the bound toxin is visualized. Enterotoxin titers determined with this CERIA method were similar to those obtained with the Vero cell assay and with ELISA. 5 ng of cholera toxin/ml may be detected with the assay. The CERIA, as described, may be used either qualitatively or quantitatively and is well suited for routine laboratory diagnosis of LT in a culture supernatant of E. coli.

Monoclonal anti-nucleoside antibodies. Characterization and application in an enzyme immunoassay of single-stranded DNA.

Two mouse monoclonal antibodies were raised against adenosine and guanosine coupled to bovine serum albumin (BSA) by periodate oxidation. They were named A-16 and G-K21 respectively and selected for their ability to recognize single-stranded DNA. Their epitope specificities were assessed and their dissociation constants determined by an indirect ELISA method. The KD values for adenosine and guanosine coupled to BSA were 9.9 X 10(-7) M and 1.1 X 10(-10) M for G-K21 respectively, and 2.5 X 10(-8) M and 1.0 X 10(-6) M for A-16. These monoclonal anti-nucleoside antibodies were used to develop a sandwich enzyme immunoassay for single-stranded DNA. The purified IgG antibodies were coupled to beta-galactosidase and alkaline phosphatase by the one-step glutaraldehyde method and used in a test optimized for pH, saturating proteins, coating antibody, nature of the conjugate and protein concentrations. Less than 100 pg/well of single-stranded DNA could be detected, and the detection was linear over a DNA concentration ranging from 0.34 to 34 ng/ml. The assay could quantitate single-stranded DNAs of differing origin, but not RNAs. The test was compared to another titration method, and used to calibrate target DNA amounts in non-radioactive hybridization experiments.

Solid-phase competitive and sandwich-type erythro-immunoassays for human chorionic gonadotropin.

A simple '1-step' competitive erythro-immunoassay for human chorionic gonadotropin (hCG) employing V-shaped well microtitration plates coated with monoclonal anti-beta-hCG antibody has been described. hCG of the test sample competes with the antigen-coupled sheep erythrocytes for binding to the antibody on the solid surface. The assay is able to detect up to 31.25 ng hCG/ml. A higher sensitivity enabling detection up to 0.25 ng hCG/ml is attained by the sandwich erythro-immunoassay using a chimera antibody prepared by coupling monoclonal anti-alpha-hCG antibody to an affinity-purified polyclonal antibody specific for sheep erythrocytes. This assay is amenable to the qualitative as well as quantitative use as described. The urinary components do not interfere in the assay. Results obtained by this assay on 47 human urine samples correlated well with the values obtained by '2-step' sandwich enzyme immunoassay and radioimmunoassay.

Monoclonal anti-histamine antibody. Preparation, characterization and application to enzyme immunoassay of histamine.

An enzyme immunoassay to measure histamine has been developed. A histamine-bovine serum albumin conjugate was prepared using 1,4-benzoquinone as the coupling agent and was employed to immunize mice for the preparation of monoclonal antibodies against histamine. After an initial screening to identify antigen-binding monoclonal antibodies the clones were isolated by limiting dilution cloning, grown in ascites and antibodies which had been secreted into the ascitic fluid were precipitated by ammonium sulphate at 50% saturation. A systematic approach for the determination of epitope specificities of monoclonal antibodies was performed. It was found that for the most specific antibody the main epitope encompassed the 2-histaminyl-1,4-benzoquinone moiety and that the KD value determined by indirect ELISA was 1.5 X 10(-8) M for the hapten part of the immunogen and 4.6 X 10(-10) M for a histamine-Bq-ovalbumin conjugate. The selected monoclonal antibody could not recognize histidine or methyl-histamine. Using this antibody, we developed an enzyme immunoassay for histamine and pg amounts could be detected. The same assay was used to quantify the allergic release of histamine from guinea pig lung mast cells. Results obtained either by the present enzyme immunoassay or by a fluorometric assay were closely correlated (correlation coefficient r = 0.9702, n = 37).

An antibody chimera technique applied to enzyme immunoassay for human alpha-1-fetoprotein with monoclonal and polyclonal antibodies.

A 2-step enzyme immunoassay (EIA) for human alpha-1-fetoprotein (AFP) is proposed, which uses covalently coupled anti-AFP IgG and anti-horseradish peroxidase (HRP) IgG (antibody chimera) binding HRP as the marker enzyme immunologically. The use of polyclonal and monoclonal anti-AFP linked to anti-HRP antibodies was compared with a conventional 2-site binding EIA with HRP covalently bound to anti-AFP IgG. The sensitivity of the conventional EIA is increased by the use of an antibody chimera comprising a molar ratio of anti-AFP IgG: anti-HRP IgG of 1:8, especially if monoclonal antibodies are employed. This improved sensitivity may be achieved by a very simple coupling procedure without purification of conjugate and with very crude HRP preparations.

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.