However, inappropriate glycosylation of the recombinant protein can occur in the Pichia expression system [57]. cleared by day 7 and the peak of malaria parasitemia is usually controlled by day 10, the antibody data could be collected after only 20 days of co-infection, a more practical time frame than the 80 days required for the Pcc-Ls experiments. This also allowed us to address whether our observations of antibody cross-reactivity were a more general feature of Pcc-nematode contamination. Given the apparent cross-reactivity observed at a fixed dilution of sera in the Phenoxodiol Pcc-Ls ELISA we used endpoint titres derived from a serial dilution (1:50 – 1:819200) in the Pcc-Nb assays to address whether this readout would overcome cross-reactivity problems. To determine if the specificity of the assay could be improved with the use of recombinant antigens we also included the malaria protein, MSP-119 [39] not available to us for the Ls studies. The antibody responses we observed on Day 20 of Pcc-Nb co-infection (Fig ?(Fig2)2) paralleled those we had seen in the Pcc-Ls experiments at Day 80. For example, as seen in Fig 2Aiii, Nb mice made IgG1 biased responses against NbA, and responses in Pcc-Nb mice were intermediate between Nb and Pcc mice. In addition, Pcc mice mounted a strong MSP-119-specific IgG2a response that was reduced in Pcc-Nb mice (Fig 2Bi). As before, Phenoxodiol levels of polyclonal IgE in Pcc-Nb mice were intermediate (data not shown). We again observed cross-reactivity, whereby Nb mice mounted detectable IgG1 and IgG2a responses to both recombinant and crude malaria antigens (indicated by X1&2 in Fig ?Fig2A2A and X4&5 in Fig ?Fig2B,2B, respectively). The magnitude of the Nb-induced IgG2a cross-reactive response is particularly striking with titres against crude and recombinant malaria antigens reaching 2500 and 200 respectively. Similarly, Pcc mice mounted responses to NbA (X3 in Fig ?Fig2A2A and X6 in Fig ?Fig2B).2B). It is important to note that these titres although low are markedly greater than background responses (mean plus 3 standard deviations of serum responses from control mice), which are represented as zero around the y-axis. The immune bias that is apparent in serum antibody isotype responses is fully supported by cytokine responses in the lymph nodes of Pcc-Nb infected mice as we have recently described [53]. Of interest, no cross-reactivity was observed at the T-cell level. Open in a separate window Physique 2 Antibody isotype responses in contamination and co-infection with Nippostrongylus brasiliensis and malaria. Mice were infected with 200 Nb L3 larvae and/or 105 Pcc-infected RBCs on day 0. Serum antibody titres (A) IgG1 (B) IgG2a to recombinant Pcc antigen MSP-119 (i), crude Pcc antigen (pRBC) (ii) and crude Nb antigen (NbA) (iii) were measured at day 20 post-infection for 8 mice per contamination group. Black bars represent the Pcc mice, white bars the Nb mice and the chequered bars the Rabbit polyclonal to Nucleostemin co-infected mice (Pcc-Nb). Antibody titres are shown around the y-axis and represent the reciprocal of the greatest dilution at which O.D was greater than the mean plus 3 standard deviations of the O.D values observed for control mouse sera at a 1/200 dilution. The letter X highlights those responses that are cross-reactive. Groups not connected by the same letter denote pairs that are significantly different according to Tukey’s Pairwise analysis. Cross-reactive IgG1 responses of malaria-infected mice Phenoxodiol to NbA are lost at higher dilutions but IgG2a responses remain The analysis of both Pcc-Ls and Pcc-Nb co-infection indicates that the issue of cross-reactivity is usually a factor investigators are likely to routinely encounter. Determining the qualitative and quantitative aspects of the cross-reacting antibody responses are not only Phenoxodiol important for the practical analysis of immune deviation but could be of real biological relevance during co-infection. As expected, antibody responses were biased, in terms of isotype, by contamination status. The bias in isotype due to a particular contamination (Th2 associated IgG1 induced during Nb contamination, for example) was extended to non-specific antigens, as seen in the IgG1 response of Nb mice to both MSP-119 and pRBC (X1 and X2 in Fig 2Ai + 2Aii). However, Pcc-specific IgG2a titres in Pcc mice were significantly higher than the cross-reactive response induced in Nb mice (Fig 2Bi). Thus, although Nb mice made cross-reactive IgG2a responses, these were no longer detectable with increasing.