Script – Atrazine ELISA (Modern Water)

Priority: low
Updating: when company changes its instructions

Change log:

When	Who	Comment
2016 05 12	Sp17	Last update to contents.
2021 03 18	Sp17	Imported without change to OneNote. This needs updating to incorporate learning during glyphosate ELISA. We also did an extraction from stream sediment before this, perhaps incorporate it here.
2023 06 15	Sp17	Convert to Markdown.

S Pacenka

sp17@cornell.edu BEE S&W Lab

1. Objective:

Determine atrazine concentrations in liquid samples.

This script is for Atrazine kits from ModernWater (formerly Strategic Diagnostics Inc (SDI)). The script differs from 2011-2013 and earlier Atrazine and Metolachlor kit scripts in newer repeating pipettor and different photometer use.

This script is for one round of 50-60 analyses – 20-25 samples + 5 standards

(including QA check), all in duplicate. This is roughly half an ELISA kit. A whole ELISA kit is

rated for 40 samples + 2x5 standards, but can be stretched easily to cover 46 total samples + 2x5 standards, by using six fresh test tubes from an earlier kit.

Learners should practice the following using deionized or tap water before doing this for production:

* Repeating pipettor loading and use.

* Single pipettor loading (without bubbles), squirting, tip eject.

Use the scale to confirm that your pipettor is consistent in delivering liquid, and that your technique excludes air from the loading and ejection.

It is a good idea to do a throwaway run with an expired kit before doing production work. It is a good idea to do a run with an experienced person before doing a solo run.

Changes from 2013 0802 Atrazine script:

1. This version uses a different photometer which requires pouring the samples into cuvettes before absorbance measurement.

2. The switch to Combitips Advanced further complicates repeater pipettor use. At this writing we do not have 10 mL tips in stock which would be better than 5 mL or 25 mL for three of five tips required.

3. Recommended using an extra pair of ZS (zero standard) tubes as the last pair in the rack.

4. 20160517: Minor copy edits related to Combitips.

2. Equipment:

Eppendor single pipettor, 200 uL fixed volume or adjustable volume.
Eppendorf repeater pipettor, recent model using Combitips Advanced. Not compatible with Combitips Plus or Combitips.
Rack for fresh single pipettor tips.
SDI RaPID tube rack, RaPID magnetic base.
Photometer capable of reading absorbance at 450 nanometers.
Waste box or bag for used solo pipette tips and sometimes repeater pipettor tips.
Racks for clean and used cuvettes; we use foam ones, probably as received with new cuvettes.
Timer or wristwatch.
Lab sink with tap water and deionized water.
Lab logbook.
Pen or pencil for writing in logbook.

3. Supplies:

5 Eppendorf Combitips Advanced repeating pipettor tips, 50 mL, 25 mL, and 5mL; fresh, or marked 1-5 during previous uses for atrazine ELISA. These can be reused three or four times for the same type of kit and same reagent each time. If you make new tips, number them with the number being replaced. Tip #3 must be 50 mL size, and tips 1, 2, 4, and 5 are all 5mL. (Tips 2, 4, and 5 could also be 25 or 50 mL since they dispense 500 uL, but the large 25 and 50 mLs tips have too large a diameter to fill from the small reagent bottles. 10 (best) or 25 mL (better than 50) size are usable for 2, 4, and 5.)
ModernWater (formerly Strategic Diagnostics) Atrazine immunoassay kit.
Disposable nitrile gloves. At least one pair per batch. Wear them during prep, execution, and cleanup.
Paper towels.
Single pipettor tips, 200 uL, enough for one per sample and per standard. Up to 30+spares for a whole batch (batch = half of ELISA kit).
Fine point Sharpie to write on test tubes.
Kimwipes.
Cut-off bottoms of Nalgene sample bottles to use for loading reagents when bottle necks are too narrow.
Deionized water for washing combitips.
Print of this script.
Square plastic cuvettes for reading light absorbances with the photometer. These may be reused between sessions if they are cleaned well with deionized water shortly after use. Discard them if they are no longer optically clear.

4. Safety of your samples and you:

There is a moderately strong acid used in the “stop” step (fifth reagent), and the herbicides in the standards are designed to kill life. Wear gloves to protect yourself.
The samples and reagents are in danger from your hands. Wear gloves throughout to protect your samples and the expensive reagents from contamination.

5. Advance prep:

Bring Photometer into lab if not present.
Check that vortex mixer is working. Move it near your work bench. Set for “low speed”.
Warm samples to room temperature. Can accelerate with a fan or immerse bottles in room temperature water. Two hours should be adequate for samples at fridge temperature; will take several hours for frozen samples.
Take ELISA kit reagents, standards, and kit test tubes out of refrigerator at least two hours in advance - all should be at room temperature to use.
Clean the Combitips with deionized water, twice. (Should have been cleaned after previous use; use new ones if carryovers were not cleaned then!). Shake out droplets well, press out with plunging action, leave parts separated to air dry.
Label near tops of tubes from kit with standards and samples codes. Use a fine point Sharpie. Plain numeral for samples, ZS, S1, S2, S3, and C for Standards and Control. Two of everything (disregard kit instruction that says only one of Control). Label the samples in any unique way, such as plain numbers – include these in the logbook. Wear gloves to avoid fingerprints on lower half of tubes.
Also it is a good idea to do a second pair of ZS at the end of the rack. That is the most important analysis of the batch since the absorbance of that pair is used as a denominator for all other absorbances when interpreting results.
In lab logbook, make a list of standards and samples in rack order, replicate pair in the same row.
This will be used to record absorbances.
Set up rack of fresh 200 uL pipettor tips. 30 at a time, plus a couple of spares.
Set up racks for clean cuvettes and full cuvettes.
Set up RaPID rack of marked standard/sample tubes, separate from magnetic base. Insert tubes in rack.
Set up a waste container for used single tips.
Array standards and sample bottles in order of positions in rack. Peel foil tops from standards bottles if they are unopened; I use a pocket knife.
Array reagents in the order to be used: 1. enzyme conjugate, 2. magnetic particles, 3. wash, 4. color reagent, 5. stop solution. Have the respective repeater pipettor tips for these ready; they can be inserted tip-down into the rack of single-shot pipettor tips.

6. Steps

The remainder of the procedure requires close attention to time and careful focus for 2-3 hours. It does not allow for breaks except during two incubations of 30 and 20 minutes. Use the rest room before you start or during these two breaks.

Turn on photometer; toggle switch on rear. Must warm up for at least 30 minutes. Set wavelength to 450 nanometers. [needs instructions how to set wavelength]
Using single-shot pipettor, add 200 uL of standards and samples to tubes, in pairs.

Shake to mix the sample or standard bottle before drawing liquid into pipettor.
Insert pipettor/tip vertically into destination test tube, and eject gently so as not to splash sides or bottom.
Two uses per tip (i.e. both replicates), into consecutive tubes. Eject tip into waste container.

Swirl the “enzyme conjugate” reagent bottle gently to mix; this stuff tends to bubble which is not good. Using repeating pipettor with Combitip 1 (5 mL size), add 250 uL (dial=2.5) of conjugate to each tube.

Note: Just after loading the repeating pipettor from the reagent bottle, eject one squirt back into the bottle. This is a “calibration” step. The pipettor’s display will flash to help you remember to do this.
Note: All uses of repeating pipettor should gently eject liquid onto the inner wall of the test tube. Aim a little down into the tube so there is no splash upwards.
Note: All uses of repeating pipettor should proceed from position 1 in the rack to the highest numbered position. Front row, left to right. Second row, left to right. Etc. This is to give the closest possible reaction durations for every tube.
Note: If a repeating pipettor combitip leaks through its tip, that is a sign that it is worn out. Discard it after you have finished using it. (Don’t break your rhythm to change tips in the middle of injecting a reagent into tubes.)
Note: A 5 mL pipettor tip is good for dispensing to 20 tubes 250 uL each if you are perfect in filling it; it is a better idea to reload after 15 tubes in case you were not perfect in filling.

4. Swirl mag particle bottle to mix. Using repeating pipettor with Combitip 2 (5 mL size, or 25 mL size), add 500 uL (dial=5.0 or 1.0) of magnetic particle solution to each tube. Use no more than 2 minutes to inject into all tubes. Reload after each 8 or 9 tubes when using 5 mL tip, after 40 using 25 mL tip.

5. Move rack with tubes to near vortex mixer. Mix each tube for 1-2 seconds. Do in order, to maintain similar reaction times.

6. Wait 15 minutes.

Write down the start of this interval in the lab log or on a copy of this script.

This is a good time to put all samples back into fridge or freezer; note, however, that if you wish to rerun a sample in a later ELISA session on the same day that it will need to be at room temperature. You can also wash the repeater pipettor tips #1 and #2.

7. Put RaPID tube rack into magnetic base. Make sure that rack fits properly into base, press down on all tubes to get bottoms fully into magnetic zone.

8. Wait 2 minutes. While waiting, set up paper towels at rim of sink, at least two layers deep.

9. Invert tube+rack+base assembly into sink. Careful to keep base + rack + tubes together so magnets are in full play. Keeping assembly inverted, tap gently on paper towels to remove more droplets from tube rims. Do not bang the tubes on the paper towels, press gently.

10. Swirl wash solution bottle to mix. Using repeating pipettor with Combitip 3 (50 mL size), add 1 mL (dial=1) of wash solution into each tube. Let sit 2 minutes after you finish the last tube. While waiting, add more paper towels near sink, at least 2 layers.

11. Invert tube+rack+base assembly into sink. While inverted, press gently on paper towels to remove more droplets.

12. (2nd round of washing) Repeat 10 including the 2 minute wait.

13. Repeat 11.

14. Remove tubes+rack from magnetic base.

15. Swirl color reagent to mix. Using repeating pipettor with Combitip 4 (5 mL or 25 mL size), add 500 uL (dial=5 or 1) of color reagent to each tube. Reload each 8 or 9 tubes for 5 mL tip, after 40 for 25 mL.

16. Vortex-mix each tube for 1-2 seconds, at low speed.

17. Wait 20 minutes.

Write down the time at the start of this interval in your lab log or

While waiting, make up the photometer blank in one clean square cuvette 1 mL (repeating pipettor, Combitip 3, dial=1) of wash solution. Wipe the tube with a kimwipe. Insert cuvette into photometer chamber, close door, zero it using a keyboard function [needs clarification]. Remove and dump cuvette in sink; do not reuse in this session. We are now ready for reading standard and sample absorbances.

Ready the lab logbook and pen/pencil near the photometer, for recording photometer reatings in step 19.

18. Swirl stop solution container to mix. Using repeating pipettor with Combitip 5 (5 or 25 mL size), Add 500 uL (dial=5 or 1) of stop solution to each tube.

19. Read each tube photometrically as follows, in no more than 15 minutes for all. Time will be tight because of having to pour from tubes into cuvettes. Work from rack position 1 to highest. Array cleaned cuvettes in a rack (long before this time!) so that they are in rows of 10 like the test tubes. Have an empty cuvette rack ready to receive cuvettes after they are read so that some can be reread. Per test tube: - Pour the entire contents of test tube into a cleaned cuvette. - Wipe the bottom of the cuvette with a Kimwipe to maximize optical clarity of the container. - Discard the emptied test tube. - Insert cuvette into photometer chamber in the proper orientation. Close door to exclude ambient light. - Wait for absorbance to stabilize. Write it down. Remove the cuvette and set it in the correct position

It is a good idea to reread the beginning cuvettes after reading the last cuvette, to check for drift. This is most important when working solo.

When two people are available, one person reads absorbances and writes them down, the second person pours from tubes into cuvettes and wipes the cuvettes, handing them to the reading-person when that person is ready.

When one person is working solo, it is workable to prepare four cuvettes together via pouring from consecutive test tubes, then switch to reading/recording those four. Be careful not to mix these up, i.e. always fill them from left to right.

20. Turn off photometer power.

21. We’re done with time sensitive activity in the lab.

22. Clean up: - Complete washing of five combitips thoroughly, with deionized water. Allow to air dry. Return to storage ziplock bag. - Dump cuvettes into sink. Rinse with deionized water and dry on paper towels in a clean area for reuse. - Rinse test tubes with tap water and put into trash. - Discard paper towels (to trash), any worn out combitips (to recycle bag), test tubes (to trash). - Put kit back into fridge if there are any reagents left.

23. Transcribe absorbances from logbook to calibration spreadsheet. The spreadsheet plots the standards, and applies several quality assurance tests.

7. Wastes:

Test tubes, pipettor tips, gloves, paper towels. Liquid from cuvettes. Reagent containers. Reagents (dumped in sink during sample processing).

8. Potential improvements:

10 mL Combitips tips, to dispense 250 uL and 500 uL.
Make an adapter or substitute part for the photometer to read absorbances in the circular test tubes instead of requiring that the liquid be poured into a square cuvette. (This is not simple with the current photometer since the circular tube will not fit into the square cuvette holder, unlike in the prior photometers that could fit the circular tubes directly.)