A colorimetric-enzymatic microassay for the quantitation of antibody-dependent complement activation.

In this report we describe a reliable, sensitive, safe, and easy way to assess antibody-dependent complement-mediated hemolysis. The assay is based on the quantitation of hemoglobin (Hb) released from lysed erythrocytes indirectly, through the generation of fluorene blue, a compound formed from 2-7 diaminofluorene in an enzymatic reaction catalyzed by the Hb molecule. The fact that Hb is the most abundant protein within a mature RBC (approximately 10(11) molecules per cell) and possesses pseudoperoxidase activity, makes the fluorene blue-coupled assay more sensitive than the simple estimation of Hb adsorption at 410 nm (Soret adsorption maxima of Hb), and as sensitive and reliable as its radioactive equivalent based on the release of 51Cr from previously loaded RBCs. Using this assay chimeric mouse-human anti-dansyl antibodies, comprising all the human IgG isotypes, were tested for their ability to mediate complement activation. The results obtained agreed with previously reported data, confirming that the fluorene blue-coupled assay is reliable. This assay also has significant advantages over the radioactive-based assay in that the reagents used are inexpensive, and the concerns of using radioactivity and the associated hazards are obviated. Because there is no need for loading the target cells with the analyte to be detected since RBCs are already loaded with Hb, the fluorene blue-coupled assay is simpler and eliminates many steps in comparison to the 51Cr release based assay.

Effects of Ajoene on lymphocyte and macrophage membrane-dependent functions.

Ajoene, (E, Z) -4, 5, 9-trithiadeca-1, 6, 11-triene 9 oxide, is a compound originally isolated from ethanolic extracts of garlic that impairs platelet aggregation by inhibiting the functional exposure of platelet integrins GPIIb/IIIa. In vitro, Ajoene is toxic for several tumoral cell lines, and exert an antiproliferative effect on T. cruzi and murine malaria parasites. Here we show that Ajoene strongly inhibited the proliferation induced in human lymphocytes by the mitogens phytohemagglutinin (PHA), phorbol myristate acetate (PMA) and anti-CD3, and the capping formation induced in B lymphocytes by anti-IgM antibodies. On macrophages, Ajoene was also found to partially inhibit the lypopolysaccharide-induced production of Tumor Necrosis Factor (TNF), and to decrease the phagocytic activity of thioglycolate-elicited mouse peritoneal macrophages for IgG-opsonized, human erythrocytes. Ajoene also partially prevented the lytic effect of human and rabbit TNF on Actinomycin D-treated WEHI 164 cells. These results strongly suggest that Ajoene is a potent modulator of membrane-dependent functions of immune cells.

Iron overload augments the development of atherosclerotic lesions in rabbits.

Iron, a major oxidant in vivo, could be involved in atherosclerosis through the induction of the formation of oxidized LDL, a major atherogenic factor. This study was designed to test this hypothesis experimentally. Four groups of New Zealand White rabbits were included: iron-overloaded/hypercholesterolemic (group A, n = 8), iron-overloaded (group B, n = 6), hypercholesterolemic (group C, n = 6), and untreated (group D, n = 6). Iron overload was achieved by the intramuscular administration of 1.5 g of iron dextran divided in 30 doses. Hypercholesterolemia was produced by feeding rabbit chow enriched with 0.5% (wt/wt) cholesterol. Serum iron, ferritin, cholesterol, triglycerides, and lipoperoxides in serum were measured throughout the study. Lipoperoxides were measured at the end of the study in liver, aorta, and spleen homogenates. Aortas of groups A and C had multiple lesions; however, group A had greater lesional involvement than group C (P < .05). Lesions were not observed in rabbits fed normal chow (group D). As expected, serum iron and ferritin were above normal levels in groups A and B. Serum cholesterol increased in groups A and C. Lipoperoxides in liver and spleen homogenates of iron-overloaded rabbits were increased. Interestingly, iron deposits were seen by ultrastructural studies in the arterial walls of rabbits in groups A and B. Our study suggests that iron overload augments the formation of atherosclerotic lesions in hypercholesterolemic rabbits.

Treatment of ABO hemolytic disease with synthetic blood group trisaccharides.

Thirteen infants, 10 with A-O and 3 with B-O hemolytic disease of the newborn (ABO-HDN), were treated with synthetic A or B blood group trisaccharides (ATS, BTS) which cause dissociation of maternal antibody bound to infant red cells. The clinical outcome was compared with that of a control group of 21 infants treated with phototherapy during the preceding year. Exchange transfusion was required in 2 out of 13 infants in the experimental group and in 7 in the control group. A randomized prospective controlled study is necessary to confirm these results.

Human monoclonal anti-Rh antibodies produced by human-mouse heterohybridomas express the Gal alpha 1-3 Gal epitope.

The presence of the Gal alpha 1-3 Gal structure (Gal epitope) in the carbohydrate component of fifteen human monoclonal antibodies with specificity for the Rh blood group factor and produced by human-mouse heterohybridomas was evaluated. To do that, an antiglobulin-like agglutination test and an enzyme linked immunosorbent assay were performed using an affinity-purified anti-Gal antibody obtained from the serum of an AB blood group donor. Using the antiglobulin reaction, results were obtained showing that only five of the fifteen human monoclonal antibodies tested contained the structure at levels sufficient to allow agglutination. However, all fifteen monoclonals were positive using the more sensitive enzyme linked immunosorbent assay. By means of an indirect immunofluorescence assay the same anti-Gal antibody was used to test the presence of Gal epitopes on the surface of the producer heterohybridomas. Results were obtained indicating that twelve out of the fifteen hybridomas studied do express the Gal structure on its surface. The relevance of these findings is discussed in the context of therapeutic applications of human monoclonal antibodies.

Enzyme-linked immunosorbent assay for the detection of substances that carry blood group A specificity.

An enzyme-linked immunosorbent assay (ELISA) was developed to estimate the amount of material carrying blood group A activity in biologic samples. A soluble synthetic form of the A antigenic determinant (A trisaccharide, ATS) conjugated to peroxidase competes with the blood group A substance present in a biologic sample for anti-A attached to a solid phase by a second antibody coating the plastic micro-wells. A reference curve is constructed by using known quantities of ATS to compete with a fixed amount of ATS-peroxidase conjugate. The A substance activity in a sample is obtained by extrapolating the degree of inhibition of the binding of the ATS-peroxidase conjugate to an equivalent amount of ATS in the reference curve. The assay is reproducible, specific, and sensitive. It has been used in pharmacologic studies to estimate the concentration of ATS in the blood and urine of rats, rabbits, and baboons and in a study with human samples, testing the potential clinical use of ATS to neutralize anti-A when therapeutically indicated. It is also useful for the detection of ABO natural products in secretions, thus allowing the accurate classification of secretor and nonsecretor individuals.