Food Biochemistry and Food Processing (2 edition)

BLBS102-c44 BLBS102-Simpson March 21, 2012 14:34 Trim: 276mm X 219mm Printer Name: Yet to Come

44 Emerging Bacterial Food-Borne Pathogens and Methods of Detection 843

polyclonal antibody. A range of different immunoassays are

available for detection of pathogens these include the enzyme

linked immunosorbent assay (ELISA), immunochromatic as-

say, and immuofluorescent assay. The sandwich ELISA is a

commonly used technique for the detection of pathogens and is

based on the application of an immobilized antibody on a solid

surface (usually, a microtiter plate) followed by exposure of the

antibody to a sample containing the target cells. The antibodies

bind or capture the target cells. Once captured, the sample is

washed to remove unbound cells and then a second antibody

conjugated to an enzyme is added followed by addition of an

enzyme substrate. A positive result is usually observed through

the colorimetric detection of the product of the reaction between

the enzyme and the substrate (see Fig. 44.1).

Immunoprecipitation (DIPSTICK/LATERALflow)

The lateral flow kit is an immunochromatographic method for

the detection of pathogens. The sample is usually applied to a

well and the sample is wicked from the sample port to an area

containing antibodies conjugated to a precipitable material such

as colloidal gold. If the target pathogen is present in the sample

the antigen antibody complex moves to a second region where

binding to a second antibody occurs resulting in an antibody –

antigen – antibody complex which usually results in a colored

immunoprecipitate (see Fig. 44.2).

Immunomagnetic Separation

Another application of immunologic-based detection is the use

of antibody coated beads for the detection of pathogens in enrich-

ment samples using immunomagnetic separation (IMS). Spe-

cially designed beads are coated with antibody specific to the

pathogen of interest. The magnetic beads themselves are param-

agnetic meaning that they are only magnetic in the presence of

a magnetic field. Usually, the beads are mixed with a sample of

the enrichment broth containing the target pathogen. Following a

period of incubation to allow the antibody to react with the anti-

gen of the target organism the beads are exposed to a magnetic

field which captures the beads at the side of the tube. The re-

maining liquid is removed containing unbound organism, food

components and remaining enrichment broth. The beads con-

taining the bound cells is usually washed and then suspended in

a small volume of diluent, which can then be plated directly on

selective media to detect the target organism or further processed

into a PCR reaction to detect the target pathogen or examined

by other means such as immunofluorescence, and so on.

While the use of immunologic-based assays significantly en-

hances detection of pathogens in foods, there are however, some

drawbacks to the use of immunologic-based assays; the first is

the risk of cross-reactivity, due to the ability of the antibodies

used to cross-react with nontarget antigens, thus leading to false

positive results. In addition, most antibody-based assays have a

minimum detection limit of about 10^4 CFU/ml; therefore, lev-

els of the target antigen below this threshold may not be easily

detected leading to false negative results.

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Figure 44.1.Direct enzyme-linked immunosorbent assay for the

detection of pathogens.1.The plate is coated with capture antibody.

2.The test sample is added and the antigen (or target organism)

binds to the capture antibody.3.An enzyme linked capture antibody

is added as a detecting antibody and binds to the antigen.4.A

substrate is added.5.The substrate is converted by enzyme to a

detectable form (usually, a color reaction).