Hi Tony: Back many, many moons ago (1991?) I developed a similar assay for looking at human antibody responses to Borrelia burgdorferi (Lyme disease) and Treponema pallidum (syphilis) by coating latex beads (from Bangs labs) with the appropriate antigens. I differentiated the two antigens by coating different size beads (5 and 7 um) and detecting IgG antibodies with an anti-human IgG Fitc and IgM antibodies with an anti-human IgM PE. It actually worked well after all the bugs (no pun intended) were worked out. Jolting my memory a bit, here is what I remember: 1) it is critically important to avoid uncoated bead space as antibodies will non-specifically bind to the plastic of the bead. Bangs Labs has some nice technical tips in calculating the amount of "parking space" on the surface of the bead so you know how much antigen to coat with. After coating the bead with antigen it is important to block any unbound surface with a protein such as BSA. Caveat, we found a few patients that had anti-BSA antibodies! 2) you should be able to titer the antibodies in your serum out to endpoint to confirm specificity 3) it is best to coat the beads fresh, however, with the addition of protease inhibitors we were able to increase the storage time. 4) using clinically defined sera, with supportive laboratory testing (traditional ELISAs and Western Blots) we were able to achieve 92% sensitivity and 98% specificity, which at the time was better than any commercial kits available on the market. 5) we were able to determine cross reactive antibodies (between the two spirochetal organisms which have many common proteins) by the mean channel of fluorescence of the response between the two beads sizes (the cross reactive antibodies had a lower mean channel fluorescence). I think this type of assay is very feasible. Due to the increased surface area of the beads you get much better sensitivity compared to a flat well in a plate. I can't remember the specifics of the incubation times but I do remember I was able to get the whole assay done, including acquisition and analysis, in less than four hours. I hope this is helpful, Good Luck! Joanne Thomas ----- Original Message ----- From: "Tony Schountz" <tschount@mesastate.edu> To: cyto-inbox Sent: Friday, April 12, 2002 6:26 PM Subject: Bead assay for detection of viral infection > > I'd like opinions on developing a bead assay for detecting antibody > responses by flow cytometry. This assay would be used for assessing > immune status of mice immunized with hantaviral nucleocapsid antigen, > and eventually we'd like to get it going for humans as well. Right now, > the turnaround time for the current test (ELISA) in humans is 2 days, > and by that time the patient is either dead or on the road to recovery. > > I'm planning to coat polystyrene beads from Bangs Labs with recombinant > nucleocapsid antigen, then testing serum samples for antibody responses > by detecting with anti-IgG-FITC and anti-IgM-PE. My concern lies > primarily with coating the beads with the antigens. I was hoping that if > anyone on the group has experience with this approach that they might > share some of their insights. > > Thanks, > > Tony > -- > Tony Schountz, Ph.D. > Dept. of Biology > Mesa State College >
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