Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics.

Electrochemiluminescence (ECL) has been developed as a highly sensitive process in which reactive species are generated from stable precursors (i.e., the ECL-active label) at the surface of an electrode. This new technology has many distinct advantages over other detection systems: no radioisotopes are used; detection limits for label are extremely low (200 fmol/L); the dynamic range for label quantification extends over six orders of magnitude; the labels are extremely stable compared with those of most other chemiluminescent systems; the labels, small molecules (approximately 1000 Da), can be used to label haptens or large molecules, and multiple labels can be coupled to proteins or oligonucleotides without affecting immunoreactivity, solubility, or ability to hybridize; because the chemiluminescence is initiated electrochemically, selectivity of bound and unbound fractions can be based on the ability of labeled species to access the electrode surface, so that both separation and nonseparation assays can be set up; and measurement is simple and rapid, requiring only a few seconds. We illustrate ECL in nonseparation immunoassays for digoxin and thyrotropin and in separation immunoassays for carcinoembryonic antigen and alpha-fetoprotein. The application of ECL for detection of polymerase chain reaction products is described and exemplified by quantifying the HIV1 gag gene.

Differences in the B cell interferon responses to lipopolysaccharide and poly I:C.

Two polyclonal B cell activators, lipopolysaccharide (LPS) and poly I:C, have been used to induce interferon (IFN) production by murine B cell populations. The results show that splenic B cell-enriched fractions, isolated by wheat germ agglutination followed by C-dependent anti-Thy 1.2 cytolysis, respond to treatment with poly I:C + DEAE-dextran by IFN production at levels comparable on a per cell basis to unfractionated spleen cells. By contrast, the LPS-stimulated IFN response of these same B cell fractions is either undetectable or substantially lower than that of spleen cells; although the B cell fractions appear fully capable of LPS-induced proliferation. Consistent with this pattern of splenic B cell IFN responses, two antibody-secreting hybridoma lines and two myeloma cell lines (including the parental myeloma of the hybrid) can be stimulated by poly I:C + DEAE-dextran to produce IFN; yet these same B cell lines do not synthesize IFN in response to LPS at doses from 1-100 micrograms/ml. The level of poly I:C-induced IFN secreted by the hybridomas are approximately 10-fold greater than that produced by the unfused parental myeloma cells. Not only do these results directly demonstrate that murine lymphocytes of the B cell lineage produce IFN in response to the B cell activator poly I:C, but these observations also strongly suggest that the IFN responses of the B cell tumor lines model the IFN producing capacity of splenic B cells. Moreover, since the hybridoma cell lines and one of the myeloma lines synthesize specific antibody molecules, these observations show that the progeny of a single B cell clone can synthesize and secrete both IFN and immunoglobulin.

T-lymphocyte activation by immunogenic determinants.

Synthetic antigens have been of great value in elucidating the relationships between antigen structure and lymphocyte activation. The compound RAT behaves as a monofunctional antigen in guinea pigs and mice, inducing T-lymphocyte responses without appreciable circulating antibody, although the ABA-specific B cell population is expanded by immunization with the monovalent molecule. On the other hand, bifunctional antigens composed of one RAT moiety serving as a carrier and a second chemical group, either identical to or different from RAT, serving as a hapten, induced antibody responses. In such responses, T cell specificity was always directed against the RAT component. Using symmetrical bifunctional antigens with rigid or flexible spacers between the two determinants, marked differences in structural requirements for cell triggering, assessed by antigen-induced lymphocyte proliferation, and for cell cooperation, determined by antibody formation, were found. Rigidly spaced bifunctional antigens serve admirably for cooperation but poorly for T cell activation, underscoring the advantage of two-point binding for the latter.

Quantitative studies of phytohemagglutinin-induced DNA and RNA synthesis in normal and agammaglobulinemic leukocytes.

Leukocytes from nine patients with acquired agammaglobulinemia were studied in vitro. Synthesis of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) induced by phytohemagglutinin was measured by determination of the degree of incorporation of labeled precursor. Synthesis of both DNA and RNA was decreased in the agammaglobulinemic cells. The presence of an inhibitor in the patients' sera could not be demonstrated. These results suggest that the basic defect in agammaglobulinemia is cellular rather than humoral.