Biomedical Engineering Reference
In-Depth Information
The recombinant nucleocapsid protein of the Hanta virus envelope is formed from
expression of plasmid constructs with the complete nucleocapsid (
Escherichia coli
) as pre-
viously described in [16]. The product is a fusion protein with phase T7 gene 10 protein
and is purified by passage through a metal chelation column [16]. Woodward's reagent K
(
N
-ethyl-5—phenylisoxazolium-3
-sulfonate), trypsin inhibitor, AP, and HRP-labeled
antiperomyscus IgG (developed in goat), antihuman IgG antibodies, naphthyl phos-
phatase, and Tween-20 were obtained from Sigma Chemical Company (St. Louis, MO).
Hanta virus positive and negative (control) human blood samples were obtained from
clinical diagnostic specimens at the University of New Mexico Health Sciences Center's
Hanta virus Diagnostic Unit. Positive samples were those that exhibited IgM antibodies to
SN virus N antigen and IgG antibodies to SN virus N and G1 antigens, by Western blot or
strip immunoblot tests [16,17]. Microcentrifuge filters were obtained from Sigma Chemical
Company. Ultralow-temperature isotropic (ULTI) carbon microdispersed powder was
provided as a gift from Carbomedics, Inc., (Austin, TX).
22.3.2.1 For Influenza A Virus and Parainfluenza Virus
Graphite powder was obtained from Fisher Scientific Company (Hampton, NH) Ultrafree-
MC Centrifugal filter units pore size 0.22 µm, used as a basis for the measuring cell
(immunocolumn) construction were obtained from Millipore Corp. (Bedford, MA). Carbon
rods used as counter electrode were a courtesy of DFI Pultruded Composites, Inc. (Erlanger,
KY). Woodward's reagent K (
N
-ethyl-5-phenyliso-xazolium-3
-sulfonate), trypsin inhibitor,
sodium iodide, and Tween-20 were obtained from Sigma Chemical Company. 2-Propanol
(US Industrial Chemicals Co., Division of National Distillers and Chemical Corporation,
New York, NY.) was used without further purification. All chemicals used were of analyti-
cal grade. Goat antirabbit IgG, conjugate of antirabbit IgG with HRP and rabbit IgG, was
obtained from Sigma Chemical Company. Inactivated PIV (Strain Sendai) and IAV
(A/Panama/2007/99, H3N2), affinity-purified antibodies to PIV and IAV, and peroxidase-
labeled affinity purified antibodies to viruses (conjugates) were obtained from Biodesign
International (Saco, Maine). Other chemicals of analytical grade were obtained from the
standard sources. For preparation of the aqueous solutions deionized water was used.
A previously described technique for immobilizing analyte related antibody on graphite
powder has been adapted for immunosorbent preparation on staphylococcal protein A.
All previously stages described in Hanta virus detection of prewashing, first and second
stages of incubation, and amperometric measurement stage are similar to flu viruses
detection.
22.4
Results and Discussion for Hanta Virus Test
A flow-through amperometric immunosorbent assay system was developed based on
disposable sensing elements for rapid detection of Hanta virus in mice blood. The system
utilizes flow-through, immunosorbent and enzyme immunoassay techniques in conjunc-
tion with an amperometric sensor. The optimal amount of RNP on the surface of the
immunosorbent was chosen on the basis of calculated kinetic constants [10,14].
The system has four main components: a pump, five valves, amperometric immunosen-
sor assembly, and an electrochemical/data recorder interface. The immunosensor consists
of a disposable centrifugal filter, which acts as the base for the antibody modified carbon
particles. This disposable sensing element rests at the base of a hollow carbon rod that acts
