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We performed inhibition ELISAs to further investigate antibody cross-reactivity between sPLA2s. Sera from two beekeepers and two allergic individuals are pooled and checked for inhibition of binding to solid-phase-coated sPLA2s by soluble sPLA2s (21). Solid-phase sandwich ELISAs for IFN-��, IL-4, IL-10, and IL-13 have been previously described (22). Briefly, 96-well microtiter plates (Maxisorb, Roskilde, Denmark) were coated with mAb 43�C11 to human IFN-��. Sensitivity was 30?pg/ml. To detect IL-13, mAb JES 10-5A2 (Pharmingen) was used for coating. Detection limit was 50?pg/ml. IL-10 was determined using mAb JES3 19F1 for coating. Sensitivity was 50?pg/ml. IL-4 was measured by coating with mAb 8F12 and biotin-conjugated mAb 3H4. Detection limit was of cytokines and stored at ?20��C until the analyses were performed. Specific proliferative responses were determined in triplicate and pulsed with 1?��Ci/well [3H] thymidine on day 5 for 8?h. Incorporated radioactivity was measured in Pharmacia Wallac beta plate reader (Turku, Finland) (20). Three T-cell clones (TCC) generated against BV sPLA2 were tested in response to different sPLA2s from PBMCs of allergic and healthy individuals. TCC specific to Api m1 and T-cell line specific to Api m2 were derived from hyper-immunized healthy individual as described (23). Clone 5.5E7 recognizes an epitope spanning amino acids 94�C111, 5.6B8 recognizes an epitope spanning amino acids 81�C98, and 5.6H2 recognizes an epitope spanning amino acids 113�C124 of Api m1. In addition, Api m1-nonspecific clones 5.1F1 and 7.3B4 were used as controls. Epstein�CBarr virus-transfected autologous B cells were used as APC (23). Experiments were performed in dose and time kinetics. Optimum proliferation and cytokine production at 0.6?��M antigen doses are shown. Data were compared by variance analysis. If homogeneity of variances is not available, then comparison of groups was analyzed by Kruskal�CWallis test and Z-test. To verify homogeneity of variances, Levene��s test was used. Student��s t-test was used for the comparison of two different groups. Multiple alignments for the amino acid sequences of AM, hGIII, DR, NM, and BT sPLA2s are shown in Fig.?1(A�CC). We have found a relatively high amino acid sequence identity between AM, HsGIII, and BT. The identity and similarity of the corresponding amino acid region of GIII PLA2s reach up to 88% and 94% between Homo sapiens (HS) and BT, and 31% and 48% between HS and AM, respectively. Furthermore, the identity and similarity of the corresponding amino acid region of GI/II PLA2s are 41% and 48% between NM and DR, respectively.