Highly sensitive enzyme immunoassays for antibodies to human tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta).

Two 'inverse sandwich' enzyme immunoassays (ELISAs) were developed for the detection and quantification of antibodies to human tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta), respectively. In these one-step assays, antibodies present in the sample linked antigen which had been covalently coupled to horseradish peroxidase to antigen bound to a solid phase (microtiter plates). The limits of detection of the assays were lower than those of neutralization bioassays; antibodies to TNF-alpha and TNF-beta being detected at concentrations as low as 2 ng/ml and 0.5 ng/ml, respectively, and no cross-reactivity was observed. The advantages of these ELISAs over other assay methods currently in use for the detection of antibodies include: (i) the convenience of the microtiter plate format and its suitability for testing large numbers of samples; (ii) the absence of radioactive tracers and precipitation steps; (iii) the high stability of the reagents; (iv) the avoidance of second antibodies and, thus, the possibility of testing samples from various species without modification of the assay and (v) the ability to detect low-affinity antibodies due to the absence of competitive reactions. The assays may be used without modification for the detection of antibodies in serum samples from both man and laboratory animals as well as in other samples such as hybridoma supernatants.

Highly sensitive enzyme immunoassay for human lymphotoxin (tumor necrosis factor beta) in serum.

We have developed a rapid, simple and highly sensitive 'sandwich' enzyme immunoassay (ELISA) for the detection and quantification of human lymphotoxin (= tumor necrosis factor beta) in serum. The assay, performed in microtiter plates, employs two monoclonal murine antibodies able to neutralize the cytotoxic activity of lymphotoxin. In a one-step procedure, antibody LTX-21 (IgG2b) coated on to the solid phase captures antigen present in the sample; subsequently antibody LTX-22 (IgG1), covalently coupled to horseradish peroxidase, labels the bound antigen. The assay is able to detect lymphotoxin spiked into human serum in concentrations as low as 7 pg/ml, whereas human tumor necrosis factor alpha does not cross-react even at 10(7)-fold higher concentrations. Only biologically active protein is recognized by the antibodies, since inactivation of lymphotoxin measured by bioassay results in a parallel decrease in immunoreactivity. Natural, glycosylated lymphotoxin shows the same reactivity as recombinant, unglycosylated protein. The assay will be useful for the quantification of endogenous human lymphotoxin in serum, other body fluids, and culture supernatants of human cells, and can also be used to monitor levels of recombinant human lymphotoxin in animal studies and clinical trials.

Steroids decrease granulocyte membrane fluidity, while phorbol ester increases membrane fluidity. Studies using electron paramagnetic resonance.

High concentrations of corticosteroids inhibit granulocyte responses and disrupt agonist receptor function. Dose-response and time-course considerations make it unlikely that these effects are mediated via the glucocorticoid receptor, a concept further supported by the ability of sex steroids to work similar effects. We postulated that steroids nonspecifically altered granulocyte membrane fluidity, which we measured directly by electron paramagnetic resonance. As predicted, methylprednisolone caused a dose-dependent increase in order parameter (decrease in fluidity) calculated on the basis of EPR spectra, using 5-doxylstearic acid (5-DS) as a probe of resting PMN membranes. This trend was highly significant (P less than 0.001; P at 0.5 mg/ml less than 0.01). Qualitatively similar results (but with different dose-response features) were obtained with conjugated estrogen. Granulocyte agonists (such as PMA) showed an opposite effect, which was not oxidatively mediated and which was steroid-inhibitable. 16-DS showed less prominent effects, suggesting that the membrane leaflets were more strongly affected than was the deep region of the membrane. Ibuprofen, which has similar effects to those of methylprednisolone on PMN aggregation and receptor function, caused a fluidizing rather than a stiffening of the membrane; this surprising result may indicate that there is a critical range of membrane fluidity for normal function, outside of which--in either direction--agonist receptor dysfunction occurs. We conclude that the immediate effects of very high doses of steroids are probably not mediated by corticoid receptors; instead, they may be due to changes in membrane fluidity.

Inhibition of granulocyte function by steroids is not limited to corticoids. Studies with sex steroids.

Noting that corticosteroid doses required for protection in shock models exceeded those required to saturate glucocorticoid receptors in mammalian cells, we postulated a nonspecific physicochemical effect of steroids upon the cell membrane, and therefore tested three noncorticoid steroids for their effects on granulocyte function. All three (conjugated equine estrogen, a synthetic progestogen, and a synthetic androgen) behaved in manner analogous to corticoids at similar concentrations, inhibiting granulocyte aggregation, chemotaxis, and chemiluminescence, as well as binding to the granulocytes of the synthetic oligopeptide agonist f-Met-Leu-Phe. Estrogen was further shown to reduce granulocyte membrane fluidity, assessed by electron paramagnetic resonance. We propose that the unique effects of extremely high-dose corticosteroids are not mediated via the glucocorticoid receptor, but result rather from physicochemical effects of the drugs upon the membranes of effector cells.

Development of a simple radioimmunoassay for human C3a.

A radioimmunoassay was devised for the human complement cleavage product, C3a, using charcoal separation and selective precipitation of interfering substances. When compared with the commercially available immunoassay now marketed, the assay reported here was somewhat simpler to perform; furthermore, it overcame delivery and availability problems in Europe. The assay showed a mean recovery of 87% of known amounts of C3a or C3adesarginine and had a sensitivity of 32 ng C3a per milliliter of plasma; coefficients of variance were comparable to other radioimmunoassays in common use. Using this assay in a first clinical application, we were able to document a small but statistically significant rise in [C3a] during cardiopulmonary bypass.