EPA-OW: 546: "Total” microcystins (MC) and nodularins (NOD) in water using enzyme-linked immunosorbent assay (ELISA)
|
Official Method Name
|
Method 546: Determination of Total Microcystins and Nodularins in Drinking Water and Ambient Water by Adda Enzyme-Linked Immunosorbent Assay |
|---|---|
|
Current Revision
| 2016 |
|
Media
|
WATER |
|
Instrumentation
|
Immunoassay |
|
Method Subcategory
|
Biotoxin |
|
Method Source
|
|
|
Citation
|
|
|
Brief Method Summary
|
This method is based on the 96-well microtiter plate format. In these wells, microcystins and nodularins in the samples and a microcystin-protein analogue immobilized in the wells compete for the binding sites of a primary detection antibody in solution. After a wash step, an enzyme-conjugate is added to the wells and binds to the primary antibody in an inverse relationship to the original concentration of microcystins and nodularins in the sample. After a second wash step, tetramethylbenzidine substrate is added to develop color via an enzyme-mediated reaction. After a set period, an acidic solution is added to each well to stop color generation. Finally, the absorbance of each well is measured using a plate reader. The concentration of microcystins and nodularins is calculated using a four-parameter logistic calibration curve. |
|
Scope and Application
|
Method 546 is a procedure for the determination of “total” microcystins (MC) and nodularins (NOD) in finished drinking water and in ambient water using enzyme-linked immunosorbent assay (ELISA). |
|
Applicable Concentration Range
|
Approximately 0.3 ug/L to at least 2.2 ug/L. |
|
Interferences
|
(A) Assay Drift: Laboratories must assay five LRBs distributed across the plate and each Analysis Batch must include two LRBs placed on opposite sides of the plate. (B) Sample Matrix Effects in Ambient Water and in Drinking Water: Check and note if present. (C) Cross Contamination: Avoid by segregating glass syringes used to filter ambient water, which may contain high levels of microcystins, from those used to filter drinking water. Alternately, use plastic, disposable syringes. Thoroughly clean glass sample containers if they are reused. Do not reuse septa from bottles containing ambient water samples. |
|
Quality Control Requirements
|
Laboratory Fortified Blank (LFB), Laboratory Fortified Sample Matrix (LFSM), Laboratory Fortified Sample Matrix Duplicate (LFSMD), Laboratory Reagent Blank (LRB), Low-Range Calibration Verification (Low-CV), Primary Dilution Standard (PDS), Quality Control Sample (QCS), Reagent Water, Stock Standard Solution, Calibration Standards, Quality Control Sample (QCS). |
|
Sample Handling
|
Prior to shipment to the field, add sodium thiosulfate to each sample bottle ( 100 mg/L final concentration). For drinking water, open the tap and allow the system to flush for approximately 5 minutes. Fill each bottle, taking care not to flush out the sodium thiosulfate, and invert several times to mix the sample with the reducing agent. The addition of sodium thiosulfate is not required for ambient water samples, but may be added if the laboratory chooses to prepare only one type of sample container. Samples must be chilled during shipment and must not exceed 10 °C during the first 48 hours after collection. Freeze samples upon arrival at the laboratory. |
|
Maximum Holding Time
|
14 days |
|
Relative Cost
|
|
|
Sample Preparation Methods
|
Three freeze-thaw cycles (including any initial freezing). Filter 1 to 2 mL (glass-fiber). |
