Control of antigen mass transfer via capture substrate rotation: an absolute method for the determination of viral pathogen concentration and reduction of heterogeneous immunoassay incubation times.
Immunosorbent assays are commonly employed as diagnostic tests in human healthcare, veterinary medicine and bioterrorism prevention. These assays, however, often require long incubation times, limiting sample throughput. As an approach to overcome this weakness, this paper examines the use of rotating capture substrates to increase the flux of antigen to the surface, thereby reducing the incubation time. To assess the capability of this approach, porcine parvovirus (PPV) was selectively extracted from solution by systematically varying the rotation rate of a gold substrate modified with a layer of anti-PPV monoclonal antibodies. The captured PPV were then directly imaged and quantified by atomic force microscopy. The benefits of substrate rotation are demonstrated by comparing an assay performed under stagnant conditions to one carried out with substrate rotation at 800 rpm, both for 10 min incubations at 25 degrees C. The use of rotation lowered the limit of detection to 3.4x10(4)TCID50/mL (approximately 80 fM) from 3.2x10(5)TCID50/mL (approximately 800 fM) under stagnant conditions. Results are also presented that show this strategy can be used: (1) to determine antigen concentrations without standards and (2) to establish the numerical relationship between quantal concentration units (e.g., 50% tissue culture infective dose (TCID50)) and quantitative concentration units (e.g., viruses/mL) The potential to broadly apply this technique to heterogeneous immunoassays is also briefly discussed.
Driskell
Jeremy D
JD
Iowa State University, Institute for Combinatorial Discovery, Department of Chemistry, The Ames Laboratory-USDOE, Ames, IA 50011-3020, United States.
Kwarta
Karen M
KM
Lipert
Robert J
RJ
Vorwald
Ann
A
Neill
John D
JD
Ridpath
Julia F
JF
Porter
Marc D
MD
D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000032:analysis*; D007118:Immunoassay; D018625:Microscopy, Atomic Force; D028323:Parvovirus, Porcine / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000956:Antigens, Viral