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??