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. Author manuscript; available in PMC: 2010 Nov 5.
Published in final edited form as: J Immunol Methods. 2009 Sep 2;350(1-2):171–182. doi: 10.1016/j.jim.2009.08.013

Figure 2. Protein expression, stability, and specificity of the bead ELISA.

Figure 2

Figure 2

Figure 2

A) Specific detection of 72 captured GST-tagged tumor antigens on beads. DNA was prepared using a high-throughput robotic preparation method, expressed in vitro, bound to anti-GST-coupled beads, and detected using an anti-GST MAb. Background expression (10% above the average of non-antigen containing beads) is shown as a dotted line. B) Anti-GST-coupled beads stored over 11 months showed no change in the stability of bound IgG using donkey anti-goat IgG-PE antibody. C) EBNA-1 antibody-positive serum from a healthy donor was tested for anti-EBNA-1 antibodies using the bead ELISA. EBNA-1-GST and p21-GST fusion proteins were expressed by IVTT and bound to anti-GST-coupled Luminex beads. Serum was added in the titrations shown for either 30 minutes at rt or at 4°C overnight. P21-GST and no DNA controls were tested at 1:20 dilution. Incubation overnight increased the sensitivity of the assay 8-fold to >1:20,000, and optimal serum dilution for maximal detection was at 1:80–1:320.