Essay Evaluation Of A Research On Patient Serum

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Initial assays aimed to optimise conditions for each bead separately in order to find conditions that could be utilised for all three antigens. Patient serum was diluted to 1:1000, 1:5000 and 1:10,000 in Assay Diluent and secondary detection reagent was diluted to either 1:500 or 1:100 in Detection Reagent Diluent. Two patients were used for this optimisation, Waikato ARF patient A1, previously shown to have a high titre against SpnA using ELISAs, and lab donor patient U41 previously shown to have a low titre.

Table 3.3: The mean fluorescence values for serum A1 and U41 at different concentrations, with different concentrations of secondary IgG-PE, against SpnA-conjugated beads. A1 U41 Secondary 1:100 Secondary 1:500 Secondary 1:100 Secondary 1:500
Serum 1:1000 730,564 295,227 4,441.96 1,488.47
Serum 1:5000 635,229 379,887 3,045.44 1,485.87
Serum 1:10000 467,243 347,272 1,921.62 1,578.37

The data shown in Table 3.3 indicate that the bead based assay for SpnA was working, as the fluorescence values decreased as the concentration of serum decreased for both patients. This meant that there was decreased serum antibody binding to SpnA on the beads. At the lowest serum concentration tested (1:10,000) the assay was sensitive and could distinguish between high and low titre patients. The lower, unordered fluorescence values for a 1:500 secondary concentration showed that this concentration was not sufficient to saturate the bound serum antibodies for both patients but a…

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