Multicenter evaluation of new enzyme-linked immunoassays of follitropin and lutropin in serum or plasma.

Results from a multicenter evaluation of two new enzyme-linked immunosorbent assays [Enzymun-Test for follitropin (FSH) and lutropin (LH)] are presented and compared with results from 11 other commercial immunoassays, radioactive as well as nonradioactive. Enzymun-Test FSH and LH assays are suitable for automated systems and manual applications. The tests were reproducible (CV less than 5%), highly specific, and sensitive enough (less than 0.5 int. unit/L) to measure the hormones directly in almost all patients' samples, except for LH measurements in prepubertal children. We did not find interference by heterophilic antibodies or other factors. A comparison of assays for FSH found very good agreement among all modern two-site assays; competitive immunoassays almost invariably yielded systematically lower results for FSH, probably because of the heterogeneity of the International Reference Preparation (2nd IRP FSH, 78/549). For LH also we found good agreement, with no systematic differences among the various reagents. Guidelines for reference values with the new reagents are given.

Cross-reactive antigenic determinants present on different Plasmodium falciparum blood-stage antigens.

A gene encoding a previously undescribed antigen of Plasmodium falciparum has been isolated from a genomic expression library by use of a pool of human immune sera. Northern blot analysis indicated that the gene is expressed at the late stages of the intra-erythrocytic cycle. This antigen, 332, contains a series of degenerated amino acid repeats. Human antibodies affinity-purified on the 332 recombinant antigen reacted with a family of parasite proteins that are products of different genes. We identified antigens 11.1 and Pf155-RESA as members of this family and confirmed, using a human monoclonal antibody, the presence of cross-reacting determinants. The sequences of these antigens also share some structural homologies. The significance of this family of blood-stage antigens is discussed.

Immunogenicity of Plasmodium falciparum antigenic determinants produced by Escherichia coli recombinant clones.

The immunogenicity of two parasite antigens produced by Escherichia coli as proteins fused to beta-galactosidase was investigated in three animal species: mice, rabbits and squirrel monkeys. 2L protein carries 71 amino acids of a parasite antigen and 11.1 protein carries 23 repeats of a 9-amino-acid repetitive unit. The humoral response was studied using indirect immunofluorescence and immunoprecipitation. The results indicate that immunization of mice, rabbits and squirrel monkeys using SDS-denatured 2L fusion protein induced antibodies able to bind to parasite antigen 2L in the IFA or in the immunoprecipitation assays. Immunization using the native fusion protein did not induce antibodies able to immunoprecipitate the 2L parasite antigen. The same observation was made for the animals immunized with 11.1 recombinant protein. In this case, the antibody response was also measured by ELISA using synthetic dimers of the repeat as antigen. In mice and rabbits, high titres of anti-11.1 antibodies were found by ELISA. However, when the antigen produced by the parasite itself was used to evaluate the response, low titres were found. This indicates that the animals produced high levels of antibodies to a structure which is not exposed in the parasite. In squirrel monkeys, the same observation was made, but the overall levels of the response to 11.1 antigen were considerably lower than those observed in mice or rabbits.

Characterization of beta-galactosidase--lactose-permease chimaeras of Escherichia coli.

Escherichia coli strains have been isolated in which 3, 39 or 805 5'-end codons of lacZ, the gene for the cytoplasmic enzyme beta-galactosidase are fused to codon 9 of lacY, the gene for lactose permease. Lactose-permease-deficient cells, carrying the lacZ-Y fusions on F' lac pro episomes, are phenotypically positive on eosin/methylene blue/lactose or on melibiose plates, demonstrating that the beta-galactosidase--lactose-permease chimaeras transport lactose and melibiose in vivo. The apparent affinity for beta-D-galactopypanosyl 1-thio-beta-D-galactopyranoside (GalSGal) in cells is similar to that of the wild-type gene product. The maximum velocity of active GalSGal transport is reduced in all three fusion strains. Both lactose and p-nitrophenyl alpha-D-galactopyranoside inhibit GalSGal uptake. As demonstrated by immunoblot experiments the chimaeras cross-react with polyclonal antibodies directed against native lactose permease and they are present in the cell envelope fraction of homogenates. Their apparent molecular weights upon electrophoresis in NaDodSO4/polyacrylamide gels correspond to those expected from their respective primary sequences, taking into account the migration properties of wild-type lactose permease. It is proposed that substitution of eight N-terminal lactose permease residues by N-terminal beta-galactosidase residues neither prevents membrane incorporation of permease nor completely impairs the ability to transport galactosides actively. Alternative interpretations of the experimental results are discussed.

72 residues of gal repressor fused to beta-galactosidase repress the gal operon of E. coli.

An active gene has been constructed which produces a chimera consisting of the N-terminal domain of the gal repressor and all but the first five residues of beta-galactosidase. Seventy two residues of gal repressor fused to beta-galactosidase as tetrameric core are sufficient to repress the gal operon in vivo and to bind to the gal operator in vitro.