The influence of naturally occurring heterophilic anti-immunoglobulin antibodies on direct measurement of serum proteins using sandwich ELISAs.

Sandwich ELISAs have become a widely used method for the quantitative detection of serum proteins. However, they can be biased by a variety of interfering substances. As reported recently, we observed false-positive levels of interferon (IFN)-alpha and -beta in up to 27% of sera from healthy blood donors using commercial ELISAs. We now demonstrate that two different groups of naturally occurring heterophilic antibodies (IgG-type) are responsible for these titers. Group I (representing 85% of positive samples) binds to the Fab region of IgG from goat, mouse, rat, horse, and bovidae (but not rabbit). Group II (15%) recognizes an epitope in the Fc region of mouse, horse, bovine, and rabbit (but not goat or rat) immunoglobulins. The antibodies did not crossreact with human IgG subclasses but contributed to false-positive IgG rheumatoid factor levels obtained using a commercially available ELISA. To investigate the susceptibility of assays to these artifacts, various combinations of capture and detection antibodies have been tested. On this basis, we defined the relative risks that standard ELISAs might be influenced by heterophilic anti-immunoglobulin antibodies. In general, assays that use monoclonal antibodies for both capture and detection are less susceptible than others which include at least one polyclonal antiserum. However, only systems utilizing rabbit F(ab')(2) fragments have been found to be immune to this interference.

Exclusion of bioactive contaminations in Streptococcus pyogenes erythrogenic toxin A preparations by recombinant expression in Escherichia coli.

The streptococcal erythrogenic exotoxin A (SPEA) belongs to the family of bacterial superantigens and has been implicated in the pathogenesis of a toxic shock-like syndrome and scarlet fever. Concerning its biological activity, mainly T-cell-stimulatory properties, conflicting data exist. In this study, we show that most of the SPEA preparations used so far contain biologically active contaminations. Natural SPEA from the culture supernatant of Streptococcus pyogenes NY-5 and recombinant SPEA purified from the culture filtrate of S. sanguis are strongly contaminated with DNases. We show that natural SPEA induces more tumor necrosis factor alpha (TNF-alpha) than recombinant SPEA, but we also show that DNases are able to induce TNF-alpha. In commercial SPEA preparations, we identified a highly active protease, which was shown not to be SPEB. To exclude these contaminations, we overexpressed SPEA cloned in the effective high-level expression vector pIN-III-ompA2 in Escherichia coli. The expressed SPEA shows the same amino acid composition as natural SPEA, whereas functional studies reported so far were carried out with toxins containing an incorrect amino terminus. We describe the rapid purification of lipopolysaccharide-, DNase-, and protease-free SPEA in two steps from the host's periplasm and its structural characterization by circular dichroism. Our results represent for the first time the production in E. coli of recombinant SPEA with the authentic N-terminal sequence and a proven superantigenic activity. Collectively, our results indicate that immunological studies of superantigens require highly purified substances free of biologically active contaminations.

Microbial superantigens stimulate T cells by the superantigen bridge and independently by a cytokine pathway.

Superantigens cross-link the MHC II molecule on accessory cells with the Vbeta region of the T cell receptor (TCR). In this study, we compared the capacity of established superantigens for inducing cytokine release. The experimental protocol was generated to answer the question whether all superantigen effects are transmitted by the MHC/TCR cross-linkage and induce mainly a T cell response. We found that TSST-1, ExFTA, and SEC3 differed from all other superantigens tested because they stimulated a stronger monokine release. T cell proliferation after challenge with these superantigens was mainly mediated by a cytokine pathway and not by the cross-linkage of MHC and TCR. For the other superantigens, we were able to demonstrate that major immunomodulatory effect is mediated by the superantigen bridge. With the exception of these three superantigens, the proliferative response of superantigens correlated with their Vbeta specificity. Interleukin-1 (IL-1) and IL-6 were induced in monocytes by all superantigens, whereas tumor necrosis factor-alpha (TNF-alpha) was induced in T cells and by some superantigens, also in monocytes. IL-2 was always induced by the superantigen bridge, whereas interferon-gamma (IFN-gamma) was also induced indirectly by monokines. Collectively, our results indicate that not all superantigens are suitable for investigating superantigen-specific effects, as they show indirect (mitogenic) side effects. Observations for an individual superantigen are, therefore, not transferable to all other superantigens.

Biophysical characterization of the c-Myb DNA-binding domain.

We have examined proteins containing the DNA-binding domain of c-Myb with biophysical methods. This DNA-binding domain consists of three imperfect repeats (R1, R2, and R3) conserved among many species. Our results indicate that the DNA-binding domain forms unspecific and specific complexes with oligodeoxynucleotides. In the presence of R1, DNA sequences related to a canonical c-Myb-binding site are better discriminated. Furthermore, although R2 and R3 are sufficient for sequence-specific DNA binding, a structural change of the DNA-binding domain upon specific complex formation is induced only when R1 is present. Therefore, R1 might serve as an important element required for secondary structure alteration upon binding and its stabilization as well as for better discrimination between specific and related DNA sequences.

Determination of the DNA bend angle induced by the restriction endonuclease EcoRV in the presence of Mg2+.

We have used the method of Zinkel and Crothers (Zinkel, S.S., and Crothers, D.M. (1990) Biopolymers 29, 29-38) to determine the degree of bending induced by the binding of the restriction endonuclease EcoRV to its recognition sequence (-GATATC-). A set of four calibration DNA fragments was constructed that contained zero, two, four, or six phased A-tracts in their centers and an EcoRV site at the 5'-end to account for the electrophoretic influence of the bound protein. The mobilities of these calibration molecules complexed with EcoRV were compared to that of a test DNA containing a central EcoRV site also complexed with EcoRV. The EcoRV-induced bend angle was found to be 44 degrees +/- 4 degrees. These experiments were performed with a catalytically inactive EcoRV mutant that still binds DNA specifically in the presence of Mg2+. In the absence of Mg2+, which is necessary for specific binding, there is no difference in the mobilities of the fragments with a peripheral or a central EcoRV site complexed with EcoRV, indicating that nonspecific binding on average does not lead to measurable DNA bending.