Chemical, Biological, and Physical Methods
253
immunoband develops, indicating antigen–antibody reaction. Following nonselective enrichment, test
results can be obtained in 8–14 hours.
37
When compared to a culture method on 196 food and feed samples, the 1–2 test detected 34 positive
samples and the culture method detected 26 positive samples.
145 With the addition of a tetrathionate
brilliant green broth enrichment step for the 1–2 test, 84 of 314 samples were salmonellae positive—3
more than the culture method—and results could be obtained 1 day before the culture method.
145 That
this method produces better results with a pre-enrichment step was shown by others on foods that
contained large numbers of nonsalmonellae.
Radioimmunoassay
This technique consists of adding a radioactive label to an antigen, allowing the labeled antigen to
react with its specific antibody, and measuring the amount of antigen that combined with the antibody by the use of a counter to measure radioactivity. Solid-phase radioimmunoassay (RIA) refers to
methods that employ solid materials or surfaces onto which a monolayer of antibody molecules binds
electrostatically. The solid materials used include polypropylene, polystyrene, and bromacetylcellulose. The ability of antibody-coated polymers to bind specifically with radioactive tracer antigens is
essential to the basic principle of solid-phase RIA. When the free-labeled antigen is washed out, the
radioactivity measurements are quantitative. The label used by many workers is
125 I.
Johnson et al.
100 developed a solid-phase RIA procedure for the determination of S. aureus enterotoxin B and found the procedure to be 5–20 times more sensitive than the immunodiffusion technique.
Employing polystyrene and counting radioactivity with an integral counter, these investigators found
the sensitivity of the test to be in the 1–5-ng range. Collins et al.
34 employed RIA for enterotoxin B
with the concentrated antibody coupled to bromacetylcellulose. Their findings indicated the procedure to be 100-fold more sensitive than immunodiffusion and to be reliable at an enterotoxin level
of 0.01 µg/ml. Staphylococcal enterotoxin A was extracted from a variety of foods, including ham,
milk products, and crabmeat, by Collins et al.
33 and measured by RIA, all within 3–4 hours. They
agreed with earlier workers that the method was highly sensitive and useful up to 0.001 µg/ml and
quantitatively reliable up to 0.01 µg/ml of enterotoxin A.
By iodination of enterotoxins, solid-phase RIA can be used to detect as little as 1 ng of toxin per
gram.
17 When protein A was used as immunoabsorbent to separate the antigen–antibody complex
from unreacted toxin, a sensitivity of <1.0 ng/g for staphylococcal enterotoxin A (SEA), SEB, SEC,
SED, and SEE was achieved within 1 working day.
17,142 In another study, 0.1 ng/ml of SEA and
0.5 ng/ml of SEA and 0.5 ng/ml of SEB could be detected when protein A was used.
3 A sensitivity of
100 pg for SEC 2 was achieved by use of a double-antibody RIA.
171
The RIA technique lends itself to the examination of foods for other biological hazards such as
endotoxins, paralytic shellfish toxins, and the like. The detection and identification of bacterial cells
within 8–10 minutes have been achieved
198 by use of
125 I-labeled homologous antibody filtered and
washed on a Millipore membrane. Because of its requirement for an isotope and its lack of portability,
the RIA method is rarely used now in the context of food microbiology.
ELISA
The enzyme-linked immunosorbent assay (ELISA, enzyme immunoassay, or EIA) is an immunological method similar to RIA but employing an enzyme coupled to either an antigen or an antibody
253
immunoband develops, indicating antigen–antibody reaction. Following nonselective enrichment, test
results can be obtained in 8–14 hours.
37
When compared to a culture method on 196 food and feed samples, the 1–2 test detected 34 positive
samples and the culture method detected 26 positive samples.
145 With the addition of a tetrathionate
brilliant green broth enrichment step for the 1–2 test, 84 of 314 samples were salmonellae positive—3
more than the culture method—and results could be obtained 1 day before the culture method.
145 That
this method produces better results with a pre-enrichment step was shown by others on foods that
contained large numbers of nonsalmonellae.
Radioimmunoassay
This technique consists of adding a radioactive label to an antigen, allowing the labeled antigen to
react with its specific antibody, and measuring the amount of antigen that combined with the antibody by the use of a counter to measure radioactivity. Solid-phase radioimmunoassay (RIA) refers to
methods that employ solid materials or surfaces onto which a monolayer of antibody molecules binds
electrostatically. The solid materials used include polypropylene, polystyrene, and bromacetylcellulose. The ability of antibody-coated polymers to bind specifically with radioactive tracer antigens is
essential to the basic principle of solid-phase RIA. When the free-labeled antigen is washed out, the
radioactivity measurements are quantitative. The label used by many workers is
125 I.
Johnson et al.
100 developed a solid-phase RIA procedure for the determination of S. aureus enterotoxin B and found the procedure to be 5–20 times more sensitive than the immunodiffusion technique.
Employing polystyrene and counting radioactivity with an integral counter, these investigators found
the sensitivity of the test to be in the 1–5-ng range. Collins et al.
34 employed RIA for enterotoxin B
with the concentrated antibody coupled to bromacetylcellulose. Their findings indicated the procedure to be 100-fold more sensitive than immunodiffusion and to be reliable at an enterotoxin level
of 0.01 µg/ml. Staphylococcal enterotoxin A was extracted from a variety of foods, including ham,
milk products, and crabmeat, by Collins et al.
33 and measured by RIA, all within 3–4 hours. They
agreed with earlier workers that the method was highly sensitive and useful up to 0.001 µg/ml and
quantitatively reliable up to 0.01 µg/ml of enterotoxin A.
By iodination of enterotoxins, solid-phase RIA can be used to detect as little as 1 ng of toxin per
gram.
17 When protein A was used as immunoabsorbent to separate the antigen–antibody complex
from unreacted toxin, a sensitivity of <1.0 ng/g for staphylococcal enterotoxin A (SEA), SEB, SEC,
SED, and SEE was achieved within 1 working day.
17,142 In another study, 0.1 ng/ml of SEA and
0.5 ng/ml of SEA and 0.5 ng/ml of SEB could be detected when protein A was used.
3 A sensitivity of
100 pg for SEC 2 was achieved by use of a double-antibody RIA.
171
The RIA technique lends itself to the examination of foods for other biological hazards such as
endotoxins, paralytic shellfish toxins, and the like. The detection and identification of bacterial cells
within 8–10 minutes have been achieved
198 by use of
125 I-labeled homologous antibody filtered and
washed on a Millipore membrane. Because of its requirement for an isotope and its lack of portability,
the RIA method is rarely used now in the context of food microbiology.
ELISA
The enzyme-linked immunosorbent assay (ELISA, enzyme immunoassay, or EIA) is an immunological method similar to RIA but employing an enzyme coupled to either an antigen or an antibody