AEGiS-13IAC: Improving HIV-1 western blot: modification of a sensitive western blot for detection of HIV-1 antibodies in multiple specimen matrices, e.g. sera/plasma, DBSs, oral fluids, and urines.

13th International AIDS Conference

Durban, South Africa - July 9-July 14, 2000

Improving HIV-1 western blot: modification of a sensitive western blot for detection of HIV-1 antibodies in multiple specimen matrices, e.g. sera/plasma, DBSs, oral fluids, and urines.

Int Conf AIDS 2000 Jul 9-14; 13:(abstract no. MoOrA111)

Kay JW, Tan PL
J.W.D. Kay, Organon Teknika Corporation, 100 Akzo Avenue, Durham,, North Carolina 27613, United States, Tel.: +1 919 620 2430, Fax: +1 919 620 4216, E-mail: jkay@orgtek.com

Background/

OBJECTIVE: The Western blot (Wb) technique for detection of antibodies to HIV-1 has been the most commonly used supplemental test system for visualization of antibody reactivity to specific HIV-1 viral proteins. Each Wb system generally has focused on testing a single type of specimen matrix. The objective of these feasibility studies was to develop procedures with a single sensitive Wb system that were applicable for multiple specimen matrices and that could be run manually or with instrumentation. Experimental: The OraSure Western blot system is effective for detection of HIV-1 antibodies in oral fluids collected with the OraSure device. This system was modified using specimen diluent (spec.dil) and wash solution used in other of OTCs retroviral antibody microelisa systems. The specific procedures were identical after specimen incubation and were applicable to MedTecs Autoblot 2000 and 6000. Panels of specimens representing historical challenges to HIV-1 system specificity and sensitivity were selected in the specimen matrices. Sera/plasma specimens were diluted 1:401 (5uL into 2ml spec.dil) and incubated for 2-3 hours. One 1/8" DBS disk was eluted for 1 hour (with agitation) in 2ml of spec.dil, the spot removed and the Wb strip incubated in the eluate for two hours. OraSure specimens were diluted 1:7.6 (150uL into 1ml spec.dil) and incubated for 3 hours. Urine specimens were diluted 1:2 (1ml into 1ml spec.dil) and incubated for 3 hours. Incubation and wash steps were done with agitation. After specimen incubation, each specimen matrix was washed 2-3 times, incubated with HRPO-anti-human conjugate, washed 2-3 times, incubated with BCIP/NBT substrate, washed 2-3 times, and dried. Results/

CONCLUSIONS: These feasibility studies showed acceptable Wb protocols for sera/plasma, DBS, oral fluid, and urine matrices and comparability between manual and automated procedures.

Keywords: AEGIS, HIV Antibodies, Blotting, Western, HIV-1, Plasma, Mouth, Sensitivity and Specificity, Human, urine, Blood, immunology
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MoOrA111