Use of protein microarrays to screen human serum searching for immune responses against P. aeruginosa and V. cholerae
Investigator: Wagner Montor, Ph.D.
Collaborators: Steve Lory (Harvard Medical School), John Mekalanos (Harvard Medical School), Ed Ryan (MGH)
The NAPPA methodology for protein microarrays developed in our lab is also being used to screen human serum in order to find antibodies against specific pathogens. Pseudomonas aeruginosa is responsible for potentially life threatening infections in individuals with compromised defense mechanisms and those with cystic fibrosis. Although a number of immunogenic proteins are known, no effective vaccine has been approved yet. We have used protein microarrays to execute a proteome-wide study of all in silico predicted outer membrane and exported P. aeruginosa proteins identifying 50 that trigger an adaptive immune response in cystic fibrosis and acutely-infected patients, 12 of which were recognized by numerous patients and show the best potential to be used for diagnostics and vaccine development. We are now expanding this approach to the entire proteome of Vibrio cholerae, an organism that continues to be responsible for pandemic infections in many parts of the world. We have access to serum from infected individuals in Bangladesh acquired on day 2, day 7 and day 21 post infection. This will allow us to use the patients’ serum as their own controls.
Representative protein microarray (NAPPA) results of V. cholerae proteins. ORFs (n = 346) were transferred and arrayed as plasmid DNA onto protein microarrays, and expression on the array was tested.
(A and B) The DNA-to-protein relationships. (Upper) Picogreen detection of DNA. (Lower) The corresponding GST protein.
(A) Controls spotted onto the array; on the left and right are 8 feature for plasmids that do not encode protein, and in the center are 12 feature of purified GST protein.
(B) A comparison of 32 V. cholerae ORFs; all ORFs display DNA, but variation in ORF-specific protein expression/capture.
(C) Examples for the entire set of controls and ORFs tested on NAPPA; left array, DNA detection by picogreen staining; right array, protein expression/capture by anti-GST antibody.