05 October 2005

Matched Pair Testing in Antibody Selection

The strength of the micro-array is the ability to test multiple biomarkers within one well. The key to this technology is to have antibodies that are highly sensitive to a specific biomarker. To achieve high sensitivity two antibodies are selected as a matched pair.

Matched pair antibodies have different binding sites for the same selected target. The capture antibody recognizes a specific epitope and the detection recognizes another. If the antibodies recognize the same bind spot or overlap binding locations then the signal will be reduced and the sensitivity lowered. if the antibody recognizes the same overlapping epitope, they are not a matched pair and are not suitable for a sandwich ELISA.

A collection of antibodies is identified as potential capture/detection antibodies. All of the potential capture antibodies are then printed into every well on the plate. After the plate is processed an antigen solution is added to the plate. The antigen is added to the plate in a standard curve. In the standard curve the antigen concentration in each well is half of the concentration of the previous well. The last well is left blank as a negative control. The plate is then incubated and then washed. The antibodies selected as potential detection antibodies undergo the process of biotynilation. After the antigen incubation the detection antibodies are added to the plate. Only one detection antibody is added to each standard curve. The plate is then incubated and washed again. A streptavidin HRP solution is then added to the plate. After another incubation and wash a substrate solution is added to the plate. The plate is imaged with a CCD gel doc system. The response is then measured by analyzing the luminescence intensity using the Quansys Array software.

This experimental set up allows us to determine what is the best matched pair out of our potential antibodies. By having only one detection antibody on each standard curve we can see what detection antibody is the best match for each capture antibody. The antibodies with no overlap of binding sites produce a higher signal than those antibodies with overlapping binding regions. Each standard curve is analyzed and the best matched pair is selected. The selection criteria consists of the largest dynamic range, high sensitivity as determined by the lower limit of detection and a high R2 value, and low well and spot background.

Capture Ab	Detection Ab	R2	Max Pixel Intensity
Cap1A	Det1B	0.99	32044.4
Cap1A	Det1C	0.79	60961.5
Cap1A	Det1D	0.99	33360.2
Cap1B	Det1A	0.99	8338.4
Cap1B	Det1C	0.92	24206.8
Cap1C	Det1A	0.98	12893.9
Cap1C	Det1B	0.95	16194.3
Cap1C	Det1C	0.85	54311.8
Cap1C	Det1D	0.97	18563.2

With the system selected above the best choice for the matched pair is by using the antibody 1A as capture and the antibody 1B as the detection. The curve does flat line at the higher antigen concentrations however this can be solved by diluting the detection antibody to a lower concentration.