50 OD405, but were higher for strain UCT40a than the other three test strains. Figure 2 Cross-reaction tests of this website indirect ELISAs involving primary antibodies assayed against 4 test antigens, PND-1186 with plant tissue and PBS as controls.

Nine antigens prepared for each test strain were assayed in duplicates. Error bars representing standard errors ranged from 0.001 – 0.006 OD405. Cross-reaction tests using random antigens extracted from three field soils produced less defined readings with a number of distinct cross-reactions (Table 5). The primary antibodies raised against strains UCT40a and UCT61a gave absorbance readings that were unambiguously negative (≤ 0.30 OD405). Optical density readings were higher (≤ 0.50 OD405) for the antibody raised against strain UCT44b, but all readings were distinguishable as negative. The readings for the primary antibody raised

against strain PPRICI3, on the other hand, were ambiguous (≥ 0.50 OD405) as the antibody produced many false positive readings (≥ 1.0 A405). The cross-reactions were more than 50% for each of the three field soils with the primary antibody of strain PPRICI3. Antigens isolated from the soil of Rein’s Farms notably produced 90% false positive readings with the primary antibody raised against strain PPRICI3 in the indirect ELISA test (Table 5). Table 5 Cross-reaction KPT-8602 solubility dmso tests of indirect ELISAs involving primary antibodies assayed against random antigens extracted from 3 different field soils. Antigen (field soil site) 1° antibody   PPRICI3 UCT40a UCT44b UCT61a Waboomskraal 60 0 0 0 Rein’s Farms 90 0 0 0 Kanetberg 55 0 3 0 Data are % antigens tested positive (≥ 1.0 OD405), n = 30, assayed in duplicates. Discussion Suitability of intrinsic antibiotic resistance for identification of Cyclopia rhizobia The four Cyclopia strains fell into two distinct pairs with regard to their

intrinsic natural resistance to the antibiotics streptomycin and spectinomycin. In the 0.0 – 0.1 μg ml-1 range, all four strains were resistant to streptomycin and could therefore not be distinguished Calpain by this technique. Over 0.2 μg ml-1, UCT40a and PPRICI3 were sensitive and did not grow, while UCT44b and UCT61a were resistant and could therefore be distinguished from the other two but not between themselves. However, from 1.2 – 1.8 μg ml-1 streptomycin, only strain UCT44b could grow and this strain could therefore be detected in a mixture with the other three strains. Test strain resistance to spectinomycin was similar in pattern to streptomycin, in that, all strains were resistant to the 0.0 – 0.6 μg ml-1 range, and were therefore not identifiable among them. However, between 1.0 and 10.0 μg ml-1 spectinomycin, only strains UCT44b and UCT61a could grow in the medium and could therefore be distinguished from any one of the other two in a mixture, but again not between themselves.