Titertek immunoassay plates (ICN Biomedical, Inc

Titertek immunoassay plates (ICN Biomedical, Inc., Aurora, Ohio) had been coated immediately with 0.5 g of recombinant MSP-119 per ml in bicarbonate coating Tfpi buffer (50 l per well) (pH 9.6). sera mainly contained IgG1 and IgG2b antibodies with negligible amounts of IgG3 Aminocaproic acid (Amicar) antibodies. Figure ?Number11 illustrates the parasitemias of the mice in all organizations. All mice within the control Aminocaproic acid (Amicar) organizations (NMS, PBS, and control MAb) developed a rapidly ascending parasitemia, and many succumbed to illness. This is similar to the parasite denseness curves in normal, untreated mice infected with YM. Mice that recovered from infection were able to clear Aminocaproic acid (Amicar) the infection by day time 30. Enzyme-linked immunosorbent assays (ELISAs) of serum from control mice showed no detectable anti-MSP-119 antibody throughout the course of the Aminocaproic acid (Amicar) experiment. Open in a separate windows FIG. 1 Parasitemias (packed circles) and MSP-119-specific-antibody titers (open symbols) in mice that received different antibody preparations as indicated. Mice received the antibodies via the i.p. route at days ?1, 0, and +1 relative to the day of challenge with 104 YM-infected RBCs i.p. IgG-specific reagents were used to determine ELISA titers in recipients of antibodies. ELISAs were performed as previously explained (8). Titertek immunoassay plates (ICN Biomedical, Inc., Aurora, Ohio) were coated immediately with 0.5 g of recombinant MSP-119 per ml in bicarbonate coating buffer (50 l per well) (pH 9.6). Plates were clogged with 3% nonfat skim milkCPBS for 1 h at 37C. Sera and secondary antibodies were diluted in 0.5% skim milkCPBS with incubation times of 1 1 h at 37C each. Serum dilutions began at 1/3,200 and were continued serially 1:2 across the plate. The substrate used was ABTS [2,2-azinobis(3-ethylbenzthiazoline sulfonic acid]. Plates were go through at 405 nm after a half-hour incubation at space temperature. Titers were determined by end point dilution. Sign: ?, death of a mouse. Mice (both +/+ and ?/?) that were given anti-MSP-119 antisera showed a marked delay (6 to 8 8 days) in the onset of parasitemia compared to mice that received NMS or PBS. The delay in patency is the most defining feature of the effectiveness of passively transferred MSP-119-specific antibodies (9). The similarity of efficacies of MSP-119-specific sera in +/+ and ?/? mice was expected since even though these mice have undamaged Fc receptors for the isotypes present in these sera (IgG1 and IgG2b), Fc receptors are not required for the manifestation of immunity mediated by MSP-119-specific antibodies of these isotypes (15). However, these receptors are not practical for binding IgG3 (7). The most potent MSP-119-specific antibody defined to day, MAb 302, is an IgG3 antibody; the receptor for this antibody is definitely Fc-RI. The data relevant to this antibody and receptor will also be demonstrated in Fig. ?Fig.1.1. However, +/+ and ?/? recipients of MAb 302 experienced very similar parasitemia curves, and for mice both having and lacking Fc-RI, there were significant delays in patency compared to recipients of the control IgG3 MAb. The entire experiment was repeated, and the data again showed the course of parasitemia was related in control and Fc-RI KO Aminocaproic acid (Amicar) (?/?) mice. A Mann-Whitney test showed no significant difference between maximum parasitemias for the MAb-treated normal and KO mice in either experiment. In order to determine whether mice which received specific antibodies and resolved their patent parasitemia were able to sterilize (completely get rid of) their illness, we transferred blood (0.2 ml) from each mouse into a naive mouse. In all instances the recipient mice failed to develop illness, indicating that the donor mice experienced cleared their illness. Figure ?Number11 also displays the anti-MSP-119 antibody titers while shown by ELISA over the course of the experiment. IgG-specific reagents were used to determine MSP-119-specific titers in recipients of MAb 302 or MSP-119-specific sera. Antibody levels in these mice were high just after the transfer of the sera or MAb (105 to 106), but as the parasitemia started to increase, the titers decreased. That transfer of immune sera can protect against and even treat malaria infections has been observed repeatedly. The exact mechanism of action of antibodies, however, remains incompletely explained. Quinn.