June 5, 2018
EPA Method 546Theresa Hattenrath-Lehmann, Ph.D.
& Ellen Braun-Howland, Ph.D.
Environmental Biology Laboratory
American Water Works Association Webinar
June 5, 2018 2
Method 546: Determination of Total Microcystins &
Nodularins in Drinking water & Ambient water by ADDA
ELISA
• >100 microcystin variants
• ELISA ADDA specifichttps://www.youtube.com/watch?v=YOWNA6VSQkA&feature=youtu.be
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Sample
requirement
overview
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Temperature Requirements
• Method 546 states: “Samples must be
confirmed to be at, or below, 10°C when they
are received by the laboratory”
• All samples are measured with a NIST
traceable thermometer
• Samples >10°C will be qualified and may be
rejected
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Chlorine Requirements
• Residual chlorine in drinking water must be
neutralized at the time of collection
• All samples are measured for residual
chlorine using test strips
• Samples containing residual chlorine will be
REJECTED
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pH Requirements
• Sample pH must be between 5-11.
• Adjust sample pH with NaOH or HCl so it falls
within the acceptable range.
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Preparing
samples and QC
samples for an
Analysis Batch
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Preparing samples for analysis
Method 546: Determination of Total Microcystins & Nodularins in
Drinking water & Ambient water by ADDA ELISA
• After passing the temperature & chlorine requirements, samples are aliquoted to 4ml amber vials
• In addition, several quality control samples are prepared:• Laboratory Reagent Blanks• Minimum reporting level (MRL) verification• Laboratory Fortified Blanks• Laboratory Fortified Matrix Samples• Quality Control sample provided with each kit
Prepared using
certified reference material
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Preparing samples for analysis: Method 546
Freeze/thaw process
35°C water bath
Filter with glass fiber syringe filters
Ethanol/dry ice bath
3x
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Quality Control Sample (QCS): Method reference 9.2.6
Method 546 states: “The MC-LR used for the QCS must be obtained from a source independent from the source of the calibration standards. Prepare the QCS in reagent water near the EC50. A QCS
supplied with the ELISA kit may be used if these criteria are met”.
The QCS is assayed in duplicate wells on the ELISA plate
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Low Calibration Verification (Low CV): Method
reference 9.2.3
A low CV must be prepared and assayed with each Analytical Batch. This is prepared at a concentration ≤ the MRL. Do NOT add sodium thiosulfate, do NOT lyse, do NOT filter.
The low CV is assayed in duplicate wells on the ELISA plate
Prepare 1 low CV at 0.3ppb: 1988µl of DI (with NO thiosulfate) + 12µl of 50ppb NRCC spiking stock
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Preparation of Sodium thiosulfate stock
for use in Analysis Batch QC preparation
• Add 200ml of autoclaved DI water to a 250ml media jar. • Add a 20 mg tablet of sodium thiosulfate (e.g., Brim
Technologies)• Invert 10x, let the tablet dissolve completely and mix
well.• This can be used for preparing Laboratory Reagent Blanks
(LRBs) and Laboratory Fortified Blanks (LFBs).
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Laboratory Reagent Blank (LRB):
Method reference 9.2.2
Aliquot 2ml of well mixed sodium thiosulfate stock into 4ml amber vial
Sample undergoes 3 freeze/thaw cycles
LRB is vortexed and then syringe filtered with glass fiber filters into new 4ml amber vial
LRB is assayed in duplicate by placing one pair of well replicates on opposite sides of the ELISA plate (4 wells total).This helps to assess plate drift
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Laboratory Fortified Blanks (LFBs):
Method reference 9.2.4
Samples undergo 3 freeze/thaw cycles
Samples are vortexed and then syringe filtered with glass fiber filters into new 4ml amber vials
Both samples are assayed in duplicate wells on the ELISA plate. These can be placed on opposite sides of the plate.
Prepare 2 LFBs: Aliquot 1970µl of well mixed sodium thiosulfate stock into 4ml amber vial, spike with 30µl of 50ppb spiking stock for a final concentration of 0.75ppb.
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Recreational and Drinking water samplesAfter passing the temperature & chlorine requirements, well mixed samples (2ml) are aliquoted to 4ml amber vials (note that we are requiring all submitted samples have sodium thiosulfate added prior to
shipment)
Samples undergo 3 freeze/thaw cycles
Samples are vortexed and then syringe filtered with glass fiber filters into new 4ml amber vials
Samples are assayed in duplicate wells on the ELISA plate
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Laboratory Fortified Sample Matrix (LFSM) and duplicate
(LFMSD): Method reference 9.2.5
Samples undergo 3 freeze/thaw cycles
Samples are vortexed and then syringe filtered with glass fiber filters into new 4ml amber vials
Both samples are assayed in duplicate wells on the ELISA plate.
Prepare a LFSM & LFSMD: Aliquot 1970µl of sample into 4ml amber vial, spike with 30µl of 50ppb spiking stock for a target concentration of 0.75ppb. Require one set of drinking water and recreational per 20
samples. Use any ‘finished’ drinking water sample in that batch. Use the sample with the least biomass for recreational samples.
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Enzyme-Linked Immunosorbent Assay (ELISA)Indirect competitive principle
substrate
Secondary antibodyconjugate
microcystin
Primary antibody
https://www.slideshare.net/sakeenagilani/enzyme-likned-immunosorbent-assay-elisa-and-its-tyoes-with-emphasis-on-competitive-elisa
mic
rocy
stin
=3hr 10minutes
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Automated vs Manual
Total time investment= 6 - 7 hours
Cyanotoxin Automated Assay System (CAAS)
Manual pipetting and spectrophotometer
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Data analysis: The Standard Curve
Absorbance= 1.00
concentration= 0.71ppb
• Each analytical batch has a standard curve
• 6 standards with a known concentration
• 4 parameter curve fitted to these standards
• Sample absorbance is compared to the standard curve
Ab
sorb
ance
(4
50
nm
)
Microcystin concentration (ppb)
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Analysis cost:
Start-up
Consumables
ELAP certification
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Plate reader (BioTek Epoch) $11,000
Computer to run program for plate reader $500
refrigerator to store samples and kits $2,000
1ml Pipettor $400
5ml Pipettor $400
200ul Pipettor $400
20ul Pipettor $400
reagent basins (case of 100) $90
300ul 8 channel pipettor $600
water bath $1,100
Total $16,890
Method 546 Equipment & consumable costs
Equipment
5ml pipette tips (200pk) $103
1ml pipette tips (5000/case) $235
200ul pipette tips (4800/case) $700
20ul pipette tips (4800/case) $700
sodium thiosulfate (bottle of 500 20mg tablets) $70
Certified Reference Material for spiking QC samples $95
Methanol $60
Parafilm $70
graduated cylinder (250ml) $50
500ml Erlenmeyer (pack of 6) $65
Total $2,148
Consumables
* Current cost per 96 well ELISA kit $440
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Estimated cost per sample
uom cost/uom cost/unit cost/plate #/plate#/sample
cost/sample
Abraxis kit Each (96 wells) $440.00 $4.58 $137.50 30 wells 2 wells/sample $9.17
Amber Glass Sample Bottles 12/cs $57.00 n/a $4.75 1 bottle/sample $4.75
Disposable Pipettes, 5mL 200/cs $103.88 n/a $0.52 1/sample $0.52
Amber Glass Vials (4mL) 144/pk $39.01 n/a $3.25 12 vials 2 vials/sample 0.54
Syringes, 3mL, Luer-Lock 200/pk $19.74 n/a $1.18 12 syringes 1 syringe/sample 0.0987
Syringe Filters 50/pk $105.42 n/a $25.30 12 filters 1 filter/sample 2.10840
Cost $167.76a $17.18b
a cost to run QC samples b cost to run a single sample without considering QC costs
Plate of 12 samples costs $32/sample*
Plate of 30 samples costs $23/sample*
*These costs include consumable supplies only and do not account for employee salary
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THANK YOU FOR YOUR ATTENTION ANY QUESTIONS??