Colorimetric or Chromogenic Susbstrate

The colorimetric method involves a substrate color change that can be detected by the naked eye or by optical density using a specific wavelength of light detected by a spectrophotometer.

Latex agglutination is a photometric immunoassay that is used more in antigen detection than in antibody detection and thus is not covered in this chapter.

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA is an indirect or colorimetric enzyme immunoassay (EIA). Solid-phase enzyme-coupled reagent assays were developed 30 years ago (Engvall and Perlmann, 1971). In principle of indirect ELISA (Fig. 4.1), antibody, if present in test sample, forms immune complex first with the capture antigen affixed to the solid phase (plastic microwell plate or tube). The primary or target antibodies in serum sample can bind to the target or capture antigens immobilized on plate wells by using enzyme-linked detector (or secondary, conjugate) antibodies, such as goat, mouse, or rabbit anti-human IgG antibodies. Secondary antibody labeled by chemical conjugation of an enzyme binds the immune complex. The enzyme "fixed" on the solid phase through immune complex interacts with the substrate, then catalyzes a chemical reaction, and yields a colored product. The colored product can then be visualized and measured by optical density measured by a spectrophotometer. The intensity of substrate color change is proportional to the amount of enzyme-linked secondary antibodies present in the sample wells, which is proportional to the amount of primary antibodies in the sample that are bound to the immobilized antigen.

Use of indirect ELISA can reduce or eliminate the nonspecific Ab binding and interfering serum factors (e.g., rheumatoid factors), thus providing low background and high sensitivity and specificity. Some indirect ELISA use avidin-biotin complexes between antibodies and antigens to increase assay sensitivities. The most commonly used enzymes for EIA are alkaline phosphatase (AP) and horseradish peroxidase (HRP). These are effective detection enzymes because of their stability, turnover number, and lack of interferences. HRP is a relatively small enzyme with a high turnover and is derived from nonmammalian sources. When used with a

Table 4.1. Types of antibody detection methods.


Lable conjugated on


Capture antigen (Ag)

target antibody

detector (secondary) antibody

Detection method

Colorimetric or chromogenic

Usually goat, mouse, or rabbit anti-human IgG

Latex agglutination

Ag on bead suspension


Visible agglutination


Ag on solid phase or microparticle


Enzyme (HRP or APa)

Visible color, optic density (OD) by spetrophotometer


Ag on nitrocellulose membrane


Enzyme (HRP or APa)

Visible band

Lateral flow diffusion

Ag colloidal gold-labeled on

Serum, blood,

Colloidal gold (chromatographic lateral flow)

Visible line


nitrocellulose or nylon membrane

oral fluid

Radioimmunoassay (RIA)

Ag on bead (or radiolabeled)


radiolabeled (1251,14C, 3H)

Radioactivity by gamma counter

Chemiluminescence (CLIA)

Ag on solid phase or


Luminol (dioxetane through HRP or APa) or

Photon output or light by

Enhanced CLIA


acridinium ester



Ag on magnetic beads


Chelate ruthenium (Ru) as electron carrier

Photon output by flow cell


(TPA as substrate)

with photon detector

Fluorescence Indirect

Ag bound on slide or


Fluorescein isothiocyanate (FITC) conjugated

Fluorescence by microscope

fluorescence (IFA)


under UV light or fluoromter


Ag bound on microparticle


Fluorescein biotinylated with lanthanide

Fluorescence by fluorometer

fluoroescence (TRF)

chelate (europium) in low pH

Flow cytometry (FC)

Ag-coated dyed microsphere


Fluorescein (through biotin-Ab to streptavidin)

Fluorescence cell scanner

Multianalyte profile

(flow cytometer), or with


FlowMetrix (Luminex)

a Horseradish peroxidase (HRP) or alkaline phosphatase (AP).

a Horseradish peroxidase (HRP) or alkaline phosphatase (AP).

Ln 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 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.

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