Advanced Techniques in Diagnostic Microbiology

46 Y. F. Wang
variety of substrates such as 2,2 ′ azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)
or ABTS, HRP generates large signals from the production of the colored products
(a deep-green color) in the presence of hydrogen peroxide, which can be seen
without a spectrophotometer. The amount of color generated is then measured
after a fixed incubation time at a specific wavelength such as 405 nm. The optical
density obtained is then related back to the concentration of the antigen in the
sample.
Immunoblotting
Immunoblotting which includes Western blot, is another technique for antibody
detection. Capture antigens such as proteins are electrotransferred to a nitrocellulose
membrane. If target antibodies are present in the specimen, they will bind
to the antigens present on the nitrocellulose strips. Visualization of the antibodies
bound to antigen is accomplished using a series of reactions with goat anti-human
IgG conjugated with biotin, avidin conjugated with HRP, and the HRP substrate.
The bands corresponding to the antigens will be seen on the nitrocellulose strip.
Lateral Flow Diffusion (Handheld, Portable Device) Method
Lateral flow diffusion (handheld, portable device) method has been designed more
for the antigen-specific immunoassay than for antibody detection. It uses colloidal
gold, carbon, paramagnetic, or color latex beads to create a visible line in the
capture zone where there is a nitrocellulose or nylon membrane. Labeled capture
antigen–antibody complex migrates by capillary action.
Immunochromatographic lateral flow assay can be used for antibody detection.
Typical handheld assay devices contain a colloidal gold (or other)-labeled antigen
dried onto a filter pad affixed to a nitrocellulose strip. A capture antibody is applied
in a line.
Lateral flow assays have been available on the commercial market since the
assey was developed for drug and pregnancy testing 20 years ago (Zuk et al.,
1985). Also known as “handheld” assays, they are simple to use, require minimal
training, and require no special storage conditions. In most cases, the manufacturer
provides simple instructions that include pictures of positive and negative results.
The assays are typically designed on nitrocellulose or nylon membranes contained
within a plastic or cardboard housing. In the antibody detection format, a capture
antigen is bound to the membrane, and a second labeled antibody is placed on a
sample application pad. As the sample migrates down the membrane by capillary
action, antibody present in the sample binds to the labeled antigen and is captured as
the complex passes. Colloidal gold, carbon, paramagnetic, or colored latex beads
are commonly used particles that create a visible line in the capture zone of the
assay membrane for a positive result.
Radioimmunoassay (RIA)
RIA uses radiolabels for measurement of antigen-binding antibody in a fluid phase.
Antibody in a test serum binds radiolabeled antigen to form antigen–antibody