Binding affinity and toxicity of thirteen microcystin congeners
Changing the Paradigm for Water Pollution Monitoring
Oral Presentation
Prepared by , M. Citriglia
Contact Information: bhattacharyyad@neorsd.org; 330-541-7475
ABSTRACT
Cyanobacteria produce several secondary metabolites known as cyanotoxins, that are toxic to humans and animals upon ingestion. The most commonly observed cyanotoxins are microcystin, cylindrospermopsin, anatoxin, and saxitoxin. Microcystins are hepatotoxins that are observed to be most frequently associated with the HAB and its chemical structure consists of a cyclic heptapeptide with more than different congeners characterized. Microcystins is most commonly detected using HPLC coupled with a PDA detector or dual mass spectrophotometer or biochemical techniques such as ELISA or PPIA. Both method types are acceptable for monitoring however there is much controversy on which method is the most accurate and reliable. Data obtained from between the chemical and biochemical methods can vary widely due to matrix effects, competition, method sensitivity, and specificity. Cross-reactivity and competition with the ELISA antibody among the different congeners are key reasons for this discrepancy between the methods. The ADDA-specific antibody is expected to alleviate the large differences in cross-reactivity over the range of the congeners. However, our research indicates large variations in recoveries due to cross-reactivity between 14 different microcystin congeners. Calibration curves were used to evaluate the binding affinity of 14 different microcystin congeners. The Abraxis ELISA kit which use an ADDA-specific polyclonal antibody was used to for all experiments. The calibration curves were compared, evaluated and statistically analyzed using a 4- parameter logistic curve fit and a Log-Logit fit for the same 14 individual congeners. All the congeners observed had individual binding patterns and were different from the MC-LR curve fit. The EC50 of all the congeners were compared to that of the previously published values of few of the congeners. The EC50 values were similar considering the changes in the assay method, reagents during the development and commercialization of the assay procedure. The EC50 values derived from the logistic curve indicate that the MC-RR (EC50 = 0.629 ppb) has the least affinity for the antibody whereas the [D-Asp3] MC-LR (EC50 = 0.275 ppb) has the highest affinity for the ADDA antibody. The data obtained from the research indicates that using a single correction factor to determine cross-reactivity might not be the most accurate method because the cross-reactivity varies at the different points along the calibration curve. The cross-reactivity data generated from this study is being used to compare the results of sample containing a mixture of microcystin congeners that have been analyzed by LC/MS/MS and ELISA to best determine false positives and false negative of the methods and the best method to use when a sample matrix contains more of the other congeners than MC-LR. This presentation will discuss the results of the affinity study and variations between samples analyzed.
Changing the Paradigm for Water Pollution Monitoring
Oral Presentation
Prepared by , M. Citriglia
Contact Information: bhattacharyyad@neorsd.org; 330-541-7475
ABSTRACT
Cyanobacteria produce several secondary metabolites known as cyanotoxins, that are toxic to humans and animals upon ingestion. The most commonly observed cyanotoxins are microcystin, cylindrospermopsin, anatoxin, and saxitoxin. Microcystins are hepatotoxins that are observed to be most frequently associated with the HAB and its chemical structure consists of a cyclic heptapeptide with more than different congeners characterized. Microcystins is most commonly detected using HPLC coupled with a PDA detector or dual mass spectrophotometer or biochemical techniques such as ELISA or PPIA. Both method types are acceptable for monitoring however there is much controversy on which method is the most accurate and reliable. Data obtained from between the chemical and biochemical methods can vary widely due to matrix effects, competition, method sensitivity, and specificity. Cross-reactivity and competition with the ELISA antibody among the different congeners are key reasons for this discrepancy between the methods. The ADDA-specific antibody is expected to alleviate the large differences in cross-reactivity over the range of the congeners. However, our research indicates large variations in recoveries due to cross-reactivity between 14 different microcystin congeners. Calibration curves were used to evaluate the binding affinity of 14 different microcystin congeners. The Abraxis ELISA kit which use an ADDA-specific polyclonal antibody was used to for all experiments. The calibration curves were compared, evaluated and statistically analyzed using a 4- parameter logistic curve fit and a Log-Logit fit for the same 14 individual congeners. All the congeners observed had individual binding patterns and were different from the MC-LR curve fit. The EC50 of all the congeners were compared to that of the previously published values of few of the congeners. The EC50 values were similar considering the changes in the assay method, reagents during the development and commercialization of the assay procedure. The EC50 values derived from the logistic curve indicate that the MC-RR (EC50 = 0.629 ppb) has the least affinity for the antibody whereas the [D-Asp3] MC-LR (EC50 = 0.275 ppb) has the highest affinity for the ADDA antibody. The data obtained from the research indicates that using a single correction factor to determine cross-reactivity might not be the most accurate method because the cross-reactivity varies at the different points along the calibration curve. The cross-reactivity data generated from this study is being used to compare the results of sample containing a mixture of microcystin congeners that have been analyzed by LC/MS/MS and ELISA to best determine false positives and false negative of the methods and the best method to use when a sample matrix contains more of the other congeners than MC-LR. This presentation will discuss the results of the affinity study and variations between samples analyzed.
