ELISA for quantitation of tumor necrosis factor-alpha in serum.

A sensitive and precise ELISA has been developed for the quantitation of recombinant Tumor Necrosis Factor-Alpha (rTNF-alpha) in undiluted sera. Affinity purified rabbit antibody was used as capture antibody and mouse monoclonal antibody labelled with horseradish peroxidase was used as the second antibody in a sandwich ELISA. The assay range was from 50 to 2000 pg/ml and the relative standard deviation was 8% or less for both interassay and intra-assay precision studies. Recovery of rTNF-alpha added to 10 different human and 10 different monkey sera ranged from 81 to 102% and 100 to 120% of the expected value, respectively. This ELISA has been used to measure serum rTNF-alpha levels in over 60 patients in Phase I Clinical Trials treated with rTNF-alpha. The levels in a representative, pharmacokinetic study showed low variability between 8 patients receiving intravenous bolus administration of 100 mu rTNF-alpha/m(2). The ELISA results correlated well with TNF bioassay data with a mean specific activity of 2.5 x 10(7) U/mg.

Mutants of Escherichia coli that are resistant to certain beta-lactam compounds lack the ompF porin.

Carbenicillin-resistant mutants of Escherichia coli K-12 and B/r were found to produce greatly diminished levels of the porin coded by the ompF gene. Physiological and ecological implications of these findings are discussed.

The receptor-binding domain of human apolipoprotein E. Monoclonal antibody inhibition of binding.

To investigate the potential of monoclonal antibodies as probes to determine the receptor-binding domain of apolipoprotein E (apo-E), five apo-E antibodies were tested to see if any of them inhibited 125I-apo-E3 . dimyristoylphosphatidylcholine binding to apo-B,E receptors on cultured fibroblasts. Only one of the five antibodies, referred to as 1D7, was found to inhibit binding, blocking greater than 90% of the receptor-binding activity of apo-E3 dimyristoylphosphatidyl-choline. The 1D7 Fab fragments were also effective inhibitors. The 1D7 bound to a Mr = 22,000 NH2-terminal thrombolytic fragment of apo-E (residues 1-191) and to a 93-residue cyanogen bromide fragment of apo-E (residues 126-218). The four noninhibitory antibodies bound only to the NH2-terminal thrombolytic fragment. These results suggested that the 1D7 epitope is contained between residues 126 and 191, and that the epitopes of the other antibodies are not contained in this region. The use of synthetic apo-E fragments, which cover various lengths of the sequence from residues 129-169, and human apo-E variants with substitutions at residues 145, 146, or 158, narrowed the location of the 1D7 epitope to residues 139-169 and, most likely, to the immediate vicinity of residues 140-150. It is of interest that 1D7 was found to bind to the same region of apo-E that has been implicated as the receptor-binding domain in receptor-binding studies using human apo-E variants and apo-E3 fragments.