Detection of endogenous macrophage colony-stimulating factor (M-CSF) in human blood.

We have detected endogenous human macrophage colony-stimulating factor (M-CSF) in blood of normal individuals, using a novel RIA that accurately measures M-CSF concentrations as low as 60 U/ml (1.2 ng/ml) in the presence of serum proteins. The RIA uses an antibody to highly purified recombinant human M-CSF and is calibrated to a mouse bone marrow colony-forming assay. Ten samples of normal human blood plasma contained an average 118 +/- 9 U/ml of M-CSF, and similar concentrations were detected in serum prepared from the same individuals. RIA-positive samples contained biologically active M-CSF, as determined in a colony assay performed on mouse bone marrow cells. The M-CSF biological activity was removed by specific immune precipitation and inhibited by addition of M-CSF antibody. Physical characterization of plasma M-CSF was done by immunoblotting after partial purification on controlled pore glass and immunoaffinity chromatography. The major reduced protein species of plasma M-CSF had an apparent molecular weight of about 24 kd, and minor species of 30, 45, and 60-70 kd were also present. The RIA results on ten normal individuals suggest that endogenous circulating M-CSF is present at a low but detectable concentration. This RIA can be used to measure M-CSF in clinical samples that contain serum proteins and other growth factors that may interfere with accurate bioassay determinations.

Immunoassay for the detection of E. coli proteins in recombinant DNA derived human growth hormone.

An enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitation of part-per-million levels of the most probable E. coli polypeptide (ECP) contaminants of E. coli produced biosynthetic human growth hormone (hGH). The antibody preparation, used for both coat and conjugate in this ELISA, was demonstrated to be reactive with the reference ECPs (a collection of the most probable protein contaminants) by both affinity chromatography and immunoblot analysis. Affinity purification of this antibody preparation using immobilized reference ECPs resulted in an assay with a higher signal-to-noise ratio and also 'normalized' the antibody population to approach stoichiometric equivalence with the immobilized ECPs. Reference ECPs, size fractionated by gel filtration, were quantitated in agreement with their absorbance at 280 nm. The assay was demonstrated to be specific for ECPs obtained from the hGH purification process. Since the purification of each recombinant DNA derived protein from E. coli requires its own unique process, this means that no generic ECP assay can be developed. It is felt that the criteria established for this assay provide a comprehensive approach to the development of quantitative multiple antigen immunoassays.

A sensitive radioimmunoprecipitation assay for the detection of antibody to recombinant human gamma-interferon: comparison to a bioassay neutralization test.

This report describes a specific radioimmunoprecipitation (RIP) assay for the detection of antibodies to recombinant DNA (rDNA) derived human gamma-interferon (rHuIFN-gamma). The assay was shown not to detect antibodies to rHuIFN-alpha, rHuIFN-beta, human lymphotoxin, or E. coli proteins and was reproducible with intraassay and interassay coefficients of variation of 1.6 and 3%, respectively, for the log titer of a high positive control. Comparison of this assay with a standard bioassay for detection of neutralizing antibody (abrogation of the inhibitory effect of rHuIFN-gamma on EMC virus replication in A549 cells) demonstrated that the RIP assay was more sensitive for detection of HuIFN-gamma neutralizing monoclonal antibody. Nonneutralizing monoclonal antibody was detectable in the RIP assay but not in the bioassay neutralization test. Examination of polyclonal antisera (rabbit and monkey) that contained neutralizing antibodies also demonstrated the RIP system to be a more sensitive indicator of the presence of antibodies than the bioassay neutralization test. In preliminary studies of human samples (86 patients) from clinical trials using an assay precipitation system capable of detecting antibody of the IgG, IgM, IgA, and IgE classes, no antibody to rHuIFN-gamma was observed. These patients were also found negative for neutralizing antibody to rHuIFN-gamma.

The isolation and characterization of the V-H domain from rabbit heavy chains of different a locus allotype.

The Fd fragment of rabbit gamma-chains was split by papain to yield a smaller fragment with a molecular weight of approximately 14,000 and dialyzable small peptides and amino acids. The domain size fragment was identified as intact variable region from its amino acid content, its blocked amino-terminus, and two characteristic cysteine-containing peptides, while the small peptides and amino acids were accounted for by the degradation of the C-H1 region. The variable regions isolated from Aa1 and Aa3 Fd fragments not only reacted quantitatively with immunoadsorbents conjugated with the homologous anti-a allotype antibody, but also completely inhibited the binding of the parent Fd fragment to the homologous antibody as measured by radioimmune assay. These data provide direct evidence that the group a allotypic determinants are contained entirely in the variable portion and are independent of the constant portion of rabbit heavy chains.