Chemical, Biological, and Physical Methods
255
3. A commercially available field kit could detect 5 ppb of AFB 2 and AFG 1 ; T-2 toxin has been
detected at a level as low as 0.05 ng/ml; and ochratoxin A at a level of 25 pg per assay (162).
Botulinal toxins
1. For type A toxin a “double-sandwich” ELISA detected 50–100 mouse LD 50 of type A and
<100 mouse i.p. LD 50 of type E; and a “double-sandwich” ELISA with alkaline phosphatase
and polystyrene plates was shown capable of detecting 1 mouse i.p. median lethal dose of type
G toxin.
125
E. coli enterotoxins
1. A monoclonal antibody specific for enterohemorrhagic strains of E. coli (EHEC) was shown to
be highly specific when used in an ELISA to detect EHEC strains.
154
2. Two “sandwich” ELISAs were developed based on toxin-specific murine monoclonal capture
antibodies and rabbit polyclonal second antibodies specific for the Stx1 and Stx2 genes of E. coli.
The Stx1 ELISA could detect 200 pg of purified Stx1 toxin, whereas the Stx2 could detect 75
pg of Stx2 toxin.
47
Gel Diffusion
Gel diffusion methods have been widely used for the detection and quantitation of bacterial toxins
and enterotoxins. The four most widely used methods are the single-diffusion tube (Oudin), microslide
double diffusion, micro-Ouchterlony slide, and electroimmunodiffusion. They have been employed to
measure enterotoxins of staphylococci and C. perfringens; and the toxins of C. botulinum. The relative
sensitivity of the various methods is presented in Table 11–1. Although they should be usable for any
soluble protein of which an antibody can be made, they require that the antigen be in precipitable form.
Perhaps the most widely used is the Crowle modification of the Ouchterlony slide test as modified
by Casman and Bennett
28 and Bennett and McClure.
15 The micro-Ouchterlony method can detect
0.1–0.01 µg of staphylococcal enterotoxin, which is the same limit for the Oudin test. The doublediffusion tube test can detect levels as low as 0.1 µg/ml, but the incubation period required for such
low levels is 3–6 days. This immunodiffusion method requires that extracts from a 100-g sample be
concentrated to 0.2 ml. Although other methods such as RIA and RPH (reverse passive hemagglutination) are more sensitive and rapid than the gel diffusion methods, the latter has been more widely
used. Their reliability within their range of sensitivity is unquestioned. Recent studies suggest that
results can be obtained in <8 hours when slides are incubated at 45
◦ C.
Immunomagnetic Separation
This method employs paramagnetic beads (about 2–3 µm in size, about 10
6 –10
8 /ml) that are surface
activated and can be coated with antibody by incubating in the refrigerator for varying periods of time
up to 24 hours. The unabsorbed antibody is removed by washing. When properly treated, the coated
beads are added to a food slurry that contains the homologous antigen (toxin or whole cells in the case
of Gram-negative bacteria), thoroughly mixed, and allowed to incubate from a few minutes to several
hours to allow for reaction of antigen with antibody-coated beads. The latter complex is collected by a
magnet followed by elution of antigen or measurement on beads. The concentrated antigen is assayed
255
3. A commercially available field kit could detect 5 ppb of AFB 2 and AFG 1 ; T-2 toxin has been
detected at a level as low as 0.05 ng/ml; and ochratoxin A at a level of 25 pg per assay (162).
Botulinal toxins
1. For type A toxin a “double-sandwich” ELISA detected 50–100 mouse LD 50 of type A and
<100 mouse i.p. LD 50 of type E; and a “double-sandwich” ELISA with alkaline phosphatase
and polystyrene plates was shown capable of detecting 1 mouse i.p. median lethal dose of type
G toxin.
125
E. coli enterotoxins
1. A monoclonal antibody specific for enterohemorrhagic strains of E. coli (EHEC) was shown to
be highly specific when used in an ELISA to detect EHEC strains.
154
2. Two “sandwich” ELISAs were developed based on toxin-specific murine monoclonal capture
antibodies and rabbit polyclonal second antibodies specific for the Stx1 and Stx2 genes of E. coli.
The Stx1 ELISA could detect 200 pg of purified Stx1 toxin, whereas the Stx2 could detect 75
pg of Stx2 toxin.
47
Gel Diffusion
Gel diffusion methods have been widely used for the detection and quantitation of bacterial toxins
and enterotoxins. The four most widely used methods are the single-diffusion tube (Oudin), microslide
double diffusion, micro-Ouchterlony slide, and electroimmunodiffusion. They have been employed to
measure enterotoxins of staphylococci and C. perfringens; and the toxins of C. botulinum. The relative
sensitivity of the various methods is presented in Table 11–1. Although they should be usable for any
soluble protein of which an antibody can be made, they require that the antigen be in precipitable form.
Perhaps the most widely used is the Crowle modification of the Ouchterlony slide test as modified
by Casman and Bennett
28 and Bennett and McClure.
15 The micro-Ouchterlony method can detect
0.1–0.01 µg of staphylococcal enterotoxin, which is the same limit for the Oudin test. The doublediffusion tube test can detect levels as low as 0.1 µg/ml, but the incubation period required for such
low levels is 3–6 days. This immunodiffusion method requires that extracts from a 100-g sample be
concentrated to 0.2 ml. Although other methods such as RIA and RPH (reverse passive hemagglutination) are more sensitive and rapid than the gel diffusion methods, the latter has been more widely
used. Their reliability within their range of sensitivity is unquestioned. Recent studies suggest that
results can be obtained in <8 hours when slides are incubated at 45
◦ C.
Immunomagnetic Separation
This method employs paramagnetic beads (about 2–3 µm in size, about 10
6 –10
8 /ml) that are surface
activated and can be coated with antibody by incubating in the refrigerator for varying periods of time
up to 24 hours. The unabsorbed antibody is removed by washing. When properly treated, the coated
beads are added to a food slurry that contains the homologous antigen (toxin or whole cells in the case
of Gram-negative bacteria), thoroughly mixed, and allowed to incubate from a few minutes to several
hours to allow for reaction of antigen with antibody-coated beads. The latter complex is collected by a
magnet followed by elution of antigen or measurement on beads. The concentrated antigen is assayed