No abstract

o-Phenylenediamine, 2,2'-azino-di(3-ethylbenzthiazoline sulphonic acid-6) (ABTS), odianisidine and 4-aminoantipyrine were compared as chromogens for the determination of horseradish peroxidase. Highest sensitivity in the determination of horseradish peroxidase-IgG conjugates in dissolved form was obtained with o-phenylenediamine. When these conjugates were used in a two-site binding enzyme immunoassay for hepatitis B surface antigen (HBsAg), the steepest calibration curve and the lowest detection limit were obtained when ABTS was used to determine the immune complexes bound to the solid phase. Non-ionic detergents, such as polyoxyethylene^sorbitol ester, retarded horseradish peroxidase inactivation, resulting in a chromogen-dependent activity rise of horseradish peroxidase. An optimised determination of horseradish peroxidase is reported, in which the sensitivity of the solid phase enzyme immunoassay is doubled by the use of 0-dianisidine. Vergleich von zur Aktivitätsbestimmung von Meerrettich-Peroxidase als Marker im Enzymimmunoassay verwendeter ChromogeneZusammenfassung: Die Empfindlichkeit der Aktivitätsbestimmung von Meerrettich-Peroxidase wurde mit den Chromogenen 0-Phenylendiamin, 2,2'-Azino,di(3-ethylbenzthiazolin-sulfonat-6) (ABTS), o-Dianisidin und 4-Aminoantipyrin verglichen. An IgG gekoppelte Meerrettich-Peroxidase wird in gelöster Form am empfindlichsten mit ö-Phenylendiarnin bestimmt. Nach Einsatz der Konjugate in einem zwei-Seiten Enzymimmunoassay zur Bestimmung des Hepatitis B-Oberflächenantigens (HBsAg) und quantitativer Bestimmung der an die feste Phase gebundenen Immunkomplexe werden die steilste Ständardkurve und geringste untere Nächweisgrenze mit ABTS erhalten. Nichtionische Detergenzien wie Polyoxyethylen*orbitester verzögern die Inaktivierung von Meerrettich-Peroxidase während der Reaktion und fuhren zu einer chromogenabhängigen Aktivitätssteigerung der Meerettich-Peroxidase. Es wird eine .optimierte Bestimmung von Meerrettich-Peroxidase mit <7,Dianisidin vorgestellt, wodurch die Empfindlichkeit des Festphase-OEnzymimmunoassays verdoppelt werden kann.

Core particles of the hepatitis B virus (HBV) potentiate the immune response against foreign epitopes presented on their surface. Potential insertion sites in the monomeric subunit of the HBV core protein were previously identified at the N- and C-terminus and in the immunodominant c/e1 region. In a C-terminally truncated core protein these sites were used to introduce the entire 120 amino acid (aa)-long potentially immunoprotective region of the hantavirus (serotype Puumala) nucleocapsid protein. The N- and C-terminal fusion products were unable to form core-like particles in detectable amounts. However, a suppressable stop codon located between the HBV core and the C-terminally fused hantavirus sequence restored the ability to form particles ('mosaic particles'); in contrast to the C-terminal fusion product the mosaic construct allowed the formation of particles built up by the core protein itself and the HBV core-Puumala nucleocapsid-readthrough protein. The mosaic particles exposed the 120 aa region of the PUU nucleocapsid protein on their surface as demonstrated by ELISA and immuno electron microscopy applying different monoclonal antibodies. Insertion of the hantaviral sequence into the c/e1 region not only allowed the formation of chimeric particles, but again the surface accessibility of the sequence. HBV core antigenicity itself was, however, reduced in the particles carrying insertions in the c/e1 region, probably due to a masking effect of the 120 aa long insert.

The molecular interaction of the Fab fragment of the human monoclonal antibody 3D6, directed against the transmembrane protein gp41 of human immunodeficiency virus (HIV) 1, with its peptide epitope is characterized by a panel of overlapping peptides, a peptide epitope library and molecular modeling techniques. The sequence CSGKLICTTAVPW, corresponding to amino acids 605-617 of gp41, was identified as the best binding peptide (KD = 1 x 10(-8) mol/l). This peptide served as a starting point to prepare a cellulose-bound peptide epitope library in which each residue of the epitope is substituted by all L- and D-amino acids, resulting in 494 epitope peptide variants which were subsequently analyzed for binding 3D6. The library was synthesized to identify residues critical for binding and to obtain information about the molecular environment of the epitope peptide bound to 3D6. Both cysteine residues, as well as isoleucine 6, threonine 8 and proline 12, of the epitope were highly sensitive to substitution. Using the data obtained from the epitope characterization, as well as a low-resolution electron density map of a 3D6 Fab-peptide complex, a 3-D model of the Fab-peptide complex was generated by molecular modeling. The modeling experiments predict binding of the peptide, which is cyclized via the two cysteine residues, to a pocket formed dominantly by the hypervariable loops complementarity determining regions CDR3L, CDR2H and CDR3H.

Determination of percentages of CD4+ and CD8+ T cells from patients with human immunodeficiency virus infection is usually done by flow cytometric analysis. We compared a cell marker ELISA with flow cytometry for quantitation of CD4 and CD8 molecules on T lymphocytes, and correlated the values both with the number of CD4+ and CD8+ T lymphocytes and with clinical data. Results by cell marker ELISA (y) correlated well with those by flow cytometric analysis (x); r = 0.69, P &lt; 0.001 (y = 0.01x + 3.9) for CD4; r = 0.81, P &lt; 0.001 (y = 0.03x + 5.4) for CD8; n = 343. The ELISA detected changes in numbers of CD8 molecules on the cells earlier than flow cytometry recognized changes in CD8+ T-cell counts. The advantages of the ELISA are the small sample volume required (0.5 mL of blood), its internal standardization by a CD4+/CD8+ cell line, and its simple and fast performance. The cell marker ELISA appears to be an efficient alternative to flow cytometry.