254 Modern Food Microbiology
rather than a radioactive isotope. Essentially synonymous with ELISA are the enzyme-multiplied immunoassay technique (EMIT) and the indirect enzyme-linked antibody technique (ELAT). A typical
ELISA is performed with a solid-phase (polystyrene) coated with antigen and incubated with antiserum. Following incubation and washing, an enzyme-labeled preparation of anti-immunoglobulin is
added. After gentle washing, the enzyme remaining in the tube or microtiter well is assayed to determine the amount of specific antibodies in the initial serum. A commonly used enzyme is horseradish
peroxidase and its presence is measured by the addition of peroxidase substrate. The amount of enzyme present is ascertained by the colorimetric determination of enzyme substrate. Variations of this
basic ELISA consist of a “sandwich” ELISA in which the antigen is required to have at least two
binding sites. The antigen reacts first with excess solid-phase antibody, and following incubation and
washing, the bound antigen is treated with excess labeled antibody. The “double sandwich” ELISA is
a variation of the latter method, and it employs a third antibody.
The ELISA technique is used widely to detect and quantitate organisms and/or their products in
foods, and synopses of some of these applications are presented below.
Salmonellae
1. Employing a polyclonal EIA with the immunoglobulin G (IgG) fraction of polyvalent flagellar
antibodies and horseradish peroxidase, 92.2% agreement was found with the classic culture
method on 142 food samples. False positives by the EIA were 6.4%, and a 95.8% agreement
with FA was achieved.
201
2. A polyclonal EIA was used with polystyrene microtiter plates, a capture antibody technique,
and a MUG assay. The sensitivity threshold was 10
7 cells/ml, and results could be obtained in 3
working days.
143
3. The Salmonella-TEK micro-ELISA method with monoclonal antibodies could detect 1–5 cfu/25
g with results in 31 hours. The sensitivity threshold was 10
4 –10
5 cells.
213
4. Monoclonal antibodies were used in a microtiter plate antibody capture method, and 10 cells/25
g could be detected in 19 hours with no cross-reactions with other organisms.
123
S. aureus and its enterotoxins
1. A double-antibody EIA was developed for staphylococcal enterotoxin A (SEA) that detected 0.4
ng in 20 hours in wieners, 3.2 ng/ml in 1–3 hours in milk, and 1.6 ng/ml from mayonnaise.
178
2. With polystyrene balls coated individually with respective antibodies, SEA, SEB, and SEC were
detected at 0.1 ng or less per milliliter.
196
3. A standard ELISA for SEA, SEB, SEC, and SEE was used for ground meat, and the method
detected <0.5 µg/100 g.
149
Molds and mycotoxins
1. Both viable and nonviable molds could be detected with an ELISA method, which produced
comparable or better results than the Howard mold count method.
127
2. For the detection of aflatoxin B 1 (AFB 1 ), an ELISA method employing monoclonal antibodies
could detect 0.1 ng/ml,
166 0.2 ng/ml,
26 and 0.5 ng/ml.
44 A commercially available kit could
detect 5 ppb (Environmental Diagnostics); a tube ELISA could detect <10 pg/ml; a polystyrene
microtiter plate method could detect 25 pg per assay,
161 and a nylon bead or Terasaki plate
method could detect 0.1 ng/ml.
160
rather than a radioactive isotope. Essentially synonymous with ELISA are the enzyme-multiplied immunoassay technique (EMIT) and the indirect enzyme-linked antibody technique (ELAT). A typical
ELISA is performed with a solid-phase (polystyrene) coated with antigen and incubated with antiserum. Following incubation and washing, an enzyme-labeled preparation of anti-immunoglobulin is
added. After gentle washing, the enzyme remaining in the tube or microtiter well is assayed to determine the amount of specific antibodies in the initial serum. A commonly used enzyme is horseradish
peroxidase and its presence is measured by the addition of peroxidase substrate. The amount of enzyme present is ascertained by the colorimetric determination of enzyme substrate. Variations of this
basic ELISA consist of a “sandwich” ELISA in which the antigen is required to have at least two
binding sites. The antigen reacts first with excess solid-phase antibody, and following incubation and
washing, the bound antigen is treated with excess labeled antibody. The “double sandwich” ELISA is
a variation of the latter method, and it employs a third antibody.
The ELISA technique is used widely to detect and quantitate organisms and/or their products in
foods, and synopses of some of these applications are presented below.
Salmonellae
1. Employing a polyclonal EIA with the immunoglobulin G (IgG) fraction of polyvalent flagellar
antibodies and horseradish peroxidase, 92.2% agreement was found with the classic culture
method on 142 food samples. False positives by the EIA were 6.4%, and a 95.8% agreement
with FA was achieved.
201
2. A polyclonal EIA was used with polystyrene microtiter plates, a capture antibody technique,
and a MUG assay. The sensitivity threshold was 10
7 cells/ml, and results could be obtained in 3
working days.
143
3. The Salmonella-TEK micro-ELISA method with monoclonal antibodies could detect 1–5 cfu/25
g with results in 31 hours. The sensitivity threshold was 10
4 –10
5 cells.
213
4. Monoclonal antibodies were used in a microtiter plate antibody capture method, and 10 cells/25
g could be detected in 19 hours with no cross-reactions with other organisms.
123
S. aureus and its enterotoxins
1. A double-antibody EIA was developed for staphylococcal enterotoxin A (SEA) that detected 0.4
ng in 20 hours in wieners, 3.2 ng/ml in 1–3 hours in milk, and 1.6 ng/ml from mayonnaise.
178
2. With polystyrene balls coated individually with respective antibodies, SEA, SEB, and SEC were
detected at 0.1 ng or less per milliliter.
196
3. A standard ELISA for SEA, SEB, SEC, and SEE was used for ground meat, and the method
detected <0.5 µg/100 g.
149
Molds and mycotoxins
1. Both viable and nonviable molds could be detected with an ELISA method, which produced
comparable or better results than the Howard mold count method.
127
2. For the detection of aflatoxin B 1 (AFB 1 ), an ELISA method employing monoclonal antibodies
could detect 0.1 ng/ml,
166 0.2 ng/ml,
26 and 0.5 ng/ml.
44 A commercially available kit could
detect 5 ppb (Environmental Diagnostics); a tube ELISA could detect <10 pg/ml; a polystyrene
microtiter plate method could detect 25 pg per assay,
161 and a nylon bead or Terasaki plate
method could detect 0.1 ng/ml.
160