Thursday, June 21, 2012

Crticism of the ELISA Technique and Cry1Ab Detection

In ‘Maternal and fetal exposure to pesticides associated to genetically modified Foods in Eastern Townships of Quebec, Canada’ the authors claim to have identified the toxin Cry1Ab in the blood of pregnant women.  Cry1Ab is a protein produced by the bacteria Bacillus thuringiensis (Bt) that is toxic to certain insect pests. Cry1Ab is just one version (event) of this Bt toxin.  Bt toxins have been used extensively by organic farmers and biotechnology has enabled seed companies to develop corn plants that express Cry1Ab proteins giving them a built in defense mechanism against insects susceptible to the toxin, while preserving the biodiversity of friendly insects.  Bt genetics have also been incorporated into cotton. The economic, environmental, safety, and health benefits have made this a very popular  tool used by the majority of family farmers. 
One of the major criticisms of the article was the use of the test used to identify the Cry1Ab protein. In the article the authors state:

‘Cry1Ab protein levels were determined in blood using a commercially available double antibody sandwich(DAS)enzyme-linked immune sorbent assay.’

 There have been many criticisms of this article. Basic statistical techniques show that the ELISA test is one of the most unreliable methods for detecting Cry1Ab toxins.  Dr. David Tribe and Dr. Cami Ryan have done a great job discussing the underlying science and peer review of this article as well.  Digging into Dr. Tribe's commentary you can find a very well written peer review based criticism of this paper:

 Alain de Weck *
* Emeritus Professor of Immunology, Institute of Immunology (University of Bern,
Switzerland), Department of Allergology (University of Navarra, Spain)
Translation from:

"In fact, a second category of doubts and questions arise in terms of immunological technique. Indeed, the only basis for the results presented is a double sandwich ELISA commercial test, decribed to be specifically for Cry1Ab (Agdia, Elkhart, IN, USA) (27). Many immunologists warn that such tests can yield not specifc results , especially in the presence of blood or serum proteins.  Various ELISA tests are unusable in serum, due to non-specific binding, [? and variability within samples?] and from one individual to another (28 and unpublished results). These results and signals from non-specific enzymatic variables give exactly the same type of results as those reported by Aris and Leblanc (1). In addition, peroxidase type enzyme conjugates , such as that used in the Agdiatest , are particularly sensitive to this type of non-specific effect, generating false positive  measurements(29). It has been made clear made clear by two users at least theAgdia test does not give reliable results in blood (16 33). Comparisons carried out by various authors between commercial sandwich ELISA (27,30,31) and various laboratory tests using anti-Cry1Ab polyclonal and monoclonal antibodies (32-36) demonstrate that the environmental tests of sandwich ELISA Cry1Ab to vary greatly in terms of sensitivity and specificity. Tests of this kind are particularly likely to yield non-specific false positive findings, especially in the presence of serum (37)."

(27) Agdia Bt-Cry1Ab/1Ac ELISA Kit -ELISA for the detection of Bt-Cry1Ab/1Ac proteins Catalog number: PSP 06200
(28) Furukawa K, Tengler R, de Weck AL, Maly FE. Simplified sulfidoleukotriene ELISA using LTD4-conjugated phosphatase for the study of allergen-induced leukotriene generation by isolated mononuclear cells and diluted whole blood. J Investig Allergol Clin Immunol. 1994; 4:110-5.

(29) Pino RM. Binding of Fab-horseradish peroxidase conjugates by charge and not by immunospecificity. J Histochem Cytochem. 1985 Jan;33(1):55-8.
(16) Chowdhury EH, Kuribara H, Hino A, Sultana P, Mikami O, Shimada N, Guruge KS, Saito M, Nakajima Y. Detection of corn intrinsic and recombinant DNA fragments and Cry1Ab protein in the gastrointestinal contents of pigs fed genetically modified corn Bt11. J Anim Sci. 2003; 81: 2546-51
(30) Envirologix.QualiPlate™ Combo Kit for Cry1Ab & Cry3Bb1 -Catalog Number: AP 039.

(31) Quantitative ELISA for Bt-Cry1Ab. Immunoassay for quantitative detection of Cry1Ab and Cry1Ac proteins in transgenic crops.

(32) Walschus U, Witt S, Wittmann C. Development of Monoclonal Antibodies Against Cry1Ab Protein from Bacillus thuringiensis and Their Application in an ELISA for Detection of Transgenic Bt-Maize . Food and Agricultural Immunology , 2002; 14 : 231-230

(33) Paul V, Steinke K, Meyer HD. Development and validation of a sensitive enzyme immunoassay for surveillance of Cry1Ab toxin in bovine blood plasma of cows fed Btmaize(MON810). Analytica Chimica Acta, 2008; 607 : 106-113

(34) Icoz I, Andow D, Zwahlen C, Stotzky G. Is the Cry1Ab protein from Bacillus thuringiensis (Bt) taken up by plants from soils previously planted with Bt corn and by carrot from hydroponic culture? Bull Environ Contam Toxicol. 2009; 83:48-58.

(35) Crespo LB , Spencer ZA, Nekl E, Pusztai-Carey M, Moar WJ, Blair D, Siegfried W. Comparison and Validation of Methods To Quantify Cry1Ab Toxin from Bacillus thuringiensis for Standardization of Insect Bioassays. Applied Environmental Microbiology , 2008; 74 :130–135

(36) Zhu X, Chen L, Shen P, Jia J, Zhang D, Yang L. High Sensitive Detection of Cry1Ab Protein Using a Quantum Dot-Based Fluorescence-Linked Immunosorbent Assay. J Agric Food Chemistry. 2011; 59 : 2184-9..

(37) Case JT, Ardans AA. Nonspecific reactions in an enzyme-l inked immunosorbent assay caused by binding of immunoglobulins in situ to egg-propagated infectious bronchitis virus. Avian Dis. 1986; 30: 149-53.

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