Script – Glyphosate Magnetic Particle ELISA, using Abraxis Kit PN 500081

Change log:

When	Who	Comment
2017 06 21	sp17	First version. This represented the first success after five failed sessions in 2014-2015. The first fully successful run was 2017 06 27.
2017 07 06	Sp17	Single tube per sample during derivatization.
2017 07 11	Sp17	Reflects experience in first four kit uses.
2018 07 21	Sp17	New VWR photometer; recipes for spiking using 50 ppb glyphosate solution made from crystals. Singleton samples. Other refinements.
2020 08 26	Sp17	Minor changes to cover diluting samples.
2021 02 19	Sp17	Added Thermo Genesys-20 spectrophotometer.
2021 03 12	Sp17	Adapted into OneNote. This becomes the primary version instead of the MS Word file. Recent runs are using triplicate calibrators and duplicate checks and samples in the assay stage. The derivatization stage continues to use singletons since there is not enough derivatization reagent provided in a kit to duplicate all calibrators and samples.
2023 06 15	Sp17	Convert to Markdown.

See also the two corrected Abraxis flowcharts, attached below as a scanned PDF.

glyphosate-elisa-flowcharts-fixed.pdf

Objective:

Determine concentrations of glyphosate in liquids.

This customizes and elaborates upon the written Abraxis procedure as follows:

Selects the primary derivatization approach, not the alternative. This employs a second set of test tubes in a non-magnetic rack. The tubes used are plastic rather than glass, following USGS Kansas OGRL recommendation.
Repeater pipettor tip choices to match volumes of reagents needed. This is from our particular available tips.
In some cases, the repeater pipettor tip has to be loaded from a temporary reagent container with a wider mouth than the Abraxis container. The temporary container is a new, cut-off Nalgene 250 mL bottle.
Corrects inconsistencies in technique and detail across Abraxis narrative, detailed flow chart, and video.
Aggressive mixing, during derivatization only, with the vortex mixer at high speed (#10) and 9 touches, as distinct from the lower (#3) speed, 1-touch mixing we use for atrazine and metolachlor ELISA. This is from the Abrazis Youtube video demo. The assay part vortexes gently (#3).
The single shot pipettor for transferring samples into derivatization tubes then later into assay tubes is inserted deeply, straight down into the test tube. The repeater pipettor is inserted at an angle and squirts against the tube wall 1/4 to 1/2” below the rim. In both cases squirt gently to limit splashback.

The Abraxis youtube video demonstrating derivatization is:

https://www.youtube.com/watch?v=urSPfmr-E2E

Equipment and reusable supplies needed:

Photometer capable of 450 nm wavelength absorbance measurements.
If using square cuvettes in photometer: Clean square cuvettes matching photometer; 1 mL of sample must rise high enough to shine light through. Foam boxes to hold empty (washed) and full cuvettes. We generally use round tubes.
Eppendorf repeater pipettor.
Single shot pipettor, calibrated and tested. Eppendorf adjustable 100-1000 μL, set for 250 μL then 300 μL.
Mag particle ELISA rack, with magnetic base.
Vortex touch mixer.
Rack to hold up to 41 6-15 mL plastic test tubes (used in derivatization).
Timer, wristwatch, or cell phone to count down the durations of incubation steps from 2 minutes to 30 minutes.
Cleaned square cuvettes with narrowed bottoms, one per assay tube plus 1 for a photometer blank. Two boxes to hold fresh and filled ones.

Expendable supplies needed:

Abraxis Kit PN 500081
Repeater pipettor tips, separate per reagent. Sizes used are 50, 25 and 5 mL, but 50 mL can be used in some cases. [See size details in steps until I transcribe a list here.] The tips may be reused across sessions close in time, for the same reagent, if they are washed with DIW twice between consecutive sessions and allowed to air dry. Store them in a ziplock bag after drying.
Single shot pipettor tips, usable at 250 uL and 300 uL. We use a 100-1000 adjusable pipettor. Lots of them.
7-15 mL plastic test tubes for derivatizing, one per sample plus 6 for calibrators and control.
Lab paper towels for blotting the rims of test tubes and for drying washed cuvettes.
Kimwipes to wipe photometer cuvettes.
Optional: Spiking solution, 50 ug/L glyphosate made from crystals (same as LC-MS LCGY approach except single solute instead of three)
- Make 1000 ppm using 10 mg crystals in 9.99g LC-MS water, or 20 mg/L crystals in 19.98 g water.
- Make “ISM” intermediate dilution, 10 ppm using 250 μL of above 1000 ppm bringing final weight to 25 g using MilliQ or LC-MS water.
- Make “WSM” final spiker at 50 μg/L using 50 uL of ISM bring to 10.00g using MilliQ or LC-MS water.

Steps:

Plan sequence. There should be at least two replicates of calibrators and checks at the assay stage; duplicates or singlteons of samples are acceptable, with some tradeoffs. Early results were mostly consistent (CV <0.15) so we will usually not go beyond two replicates of samples.
1. There is insufficient quantity of derivatization reagent in a kit to replicate at the derivatization stage.
2. Note that singleton samples should only be used for preliminary screening because it is possible to screw up processing of a single tube without discovering that. With duplicates, the difference between the pair is a signal that one was screwed up.
3. In 2021 we are triplicating the calibrators including the zero standard, and duplicating samples. There is enough derivatized calibrator per tube to fill three replicates at the assay stage.
Find and thaw samples. Best to thaw in refrigerator overnight.
Bring samples and kit reagents to room temperature.
Mark the fresh test tubes for both derivatization and ELISA assay with ZS, S1-S4 (75, 200, 750, 4000 ng/L), C and numbers 1..N for the samples. Replicates are next to one another in the magnetic rack; replicates are not used for the derivatization step. (This is because of insufficient reagent provided in the Abraxis kit to do more than about 32 derivatization tubes per batch.) The tubes are in in order of ZS, then S1-S4, C, and samples. We also do another C pair within the sequence, and another in the middle if there are more than 50 tubes in the batch. (Another S2 or S3 pair can be used instead of the C pair.) Tubes in both racks should be laid out identically in row/column order as driven by the mag particle rack structure – six rows of ten, with position 1 in the front left corner. The deriv rack should have one row of tubes per row in the mag rack, i.e. rows of three (when triplicating in assay) to five (when duplicating in assay) tubes. Since the mag rack can hold 60 tubes and you are not using all positions, you may want to leave a gap between the calibrator tubes and the control as a synchronizing aid.
Optional if in the plan for the batch: Spike aliquots of any samples that will be fortified for recovery testing. (Spiking does not require room temperature.) This is done in a temporary 15 mL test tube. Keep the spiked sample in freezer with original sample afterward.
Dilute samples if necessary to bring within range. Dilution is done in 15 mL centrifuge tubes, using diluent (also called zero standard) that comes with the kit. To do 10x dilution, add 900 uL of diluent to 100 uL of sample, 20x add 1900 uL to 100 uL of sample, etc. It is best to keep the sample at 100 uL or above for best precision; you can dilute in stages to conserve diluent. Prepare all dilutions in a rack that allows the bottoms of the tubes to be seen to check for evenness. Arrange the rack in order matching the derivatization tube rack with gaps for standards and non-diluted samples, to make it clear which diluted version matches which derivatization tube. Mark the cent tubes with numbers.
- Note: if you spike a diluted sample, take account of when you spike, before or after dilution.
The practical limit of tubes in one batch is determined by the time needed to read absorbances and to add the most time sensitive reagents (derivatization and mag particle). Also pay attention to remaining kit reagents. There is enough derivatizer to do three batches of 30 derivatization tubes, just enough to fill the assay rack’s 60 positions if duplicating both calibrators and samples. Other reagents may be more limiting.
Turn on the photometer so its lamp warms up before you need to use it. Remove any old tubes from its reading chamber. Set it for 450 nM wavelength (lambda button or similar). Set up a rack near it to hold test tubes or cuvettes after reading them.
If making your own “made concentration” samples or spiking natural samples:
1. Spikes:
  1. Mark one 15 mL centrifuge tube or other small, cappable container with the original sample ID and the spiking level, such as “+0.5 ppb gly”.
  2. It is best to work on a scale sensitive to 0.01g.
  3. Tare the empty container without cap.
  4. Add the spiking volume of WSM to the tube. Repeater pipettor with fresh 1 mL tip.
    1. +0.25 = 10 uL
    2. +0.50 = 20 uL
    3. +1.0 = 40 uL
    4. +2.0 = 80 uL
    5. +5.0 = 200 uL
    6. +10.0 = 400 uL
  5. Bring the final weight of liquid in the tube to 2 g (2 mL total volume) using original sample and single-shot pipettor. Change tip if using a different sample from previous tube.
  6. Cap.
  7. Repeat for each spiked sample.
2. Made concentrations
  1. This is identical to spiking except that instead of a sample you are adding kit diluent to reach 2 g liquid weight.
  2. You can use a repeater pipettor instead of single shot to transfer kit diluent. Different tip than the one used for WSM.
  3. Mark the containers as “0.5 ug/L made gly ELISA” etc with the date made.
Lay out the ZS, S1-S4 calibrators, check, and sample containers in the order of tubes in the racks in a way to eliminate getting the wrong stuff into the wrong tubes.
Derivatize:
1. Dilute the derivatizer reagent. Pipette 3.5 mL of the derivatizer diluent into the container of the derivatizer reagent. Use single shot adjustable pipettor 100-1000 to transfer the diluent into the reagent container. Prime/calibrate the tip by squirting back twice into the diluent container, then load and squirt until 3.5 mL is transferred. Re-cap the derivatizer reagent vial. Touch-mix at least 10 times on the vortexer, allowing the mixture to settle between touches. Do not foam. This will sit for a little while you carry out the next few steps.
2. Note: You are making 1.35 mL of derivatized sample, enough to fill four assay tubes at 300 uL each, with 150 uL left over. Thus one derivatizer tube can supply quadruplicate assay tubes, with great care in filling and priming due to the very small margin.
3. Transfer sample, standards, et al into derivatizer tubes. Pipette 250 uL of each ZS blank, calibrator, standard, check, and sample into the derivatization tubes. Single shot pipettor (100-1000 range) set for 250 uL, blue 1mL tips. Use a fresh tip for each sample/standard/blank etc to avoid cross-contamination. Prime each tip by loading, eject back into source container, load, squirt back to source again, load, squirt gently into derivatizing tube. The two prime steps are to ensure that the volume ejected is precise. Squirt downwards, gently, with tip far into the tube slowly to avoid splashing back. You also do not want any liquid droplets on the tip.
  1. Important: after filling all tubes in the derivatization rack, visually check that your tubes all have essentially the same volumes before proceeding. It is fine to top-up or withdraw liquid at this point, using a fresh tip in a single shot pipettor, probably best using the 10-100 pipettor size.
4. Pipette 1.0 mL of Assay Buffer into each of the derivatizing tubes. After each squirt, mix tube on vortexer at high speed, touching four times briefly. (i.e. it is squirt, mix, squirt, mix – don’t wait to mix.) Use repeater pipettor with 25 mL or 50 mL tip. When loading the tip with reagent, prime this tip by ejecting 1 mL at least twice back into the Assay Buffer container before the first destination squirt. For all reagent ejection, squirt at a small angle so that reagent runs down the side of the tube to minimize splashback.
  1. The order of mixing and squirting are based on the Abraxis demonstration video. This different from the USGS procedure and the written Abraxis instructions. The corrected flow chart is consistent with the video.
  2. If you cannot load the repeater pipettor tip from the reagent container, pour from container into a new cut-off Nalgene 250 mL sample bottle before loading, then pour back into reagent container after the step is completed. You can reuse these temporary containers if they are washed between sessions with deionized water and the same container is always used for the same reagent.
  3. Important: Visually check the tubes for having the same volumes of sample+Assay Buffer. Note any overages in the log. It is OK to top up any shortages, if you re-mix the tube.
5. Pipette 100 uL of diluted derivatizer mixture into each tube. Use repeater pipettor with 5 mL tip. Touch mix the tube 9 times on vortexer. Allow to settle between touches. Do not foam. Kit narrative says to mix repeatedly until there are no swirl lines showing; what are these? Flow chart says to vortex each tube for 15-30 seconds. Vortex mix the tube immediately after the squirt, as with assay buffer.
  - USGS says to add derivatization reagent to a few samples at a time, then wait ten minutes (step 8.f), then transfer into the assay tubes (step 9.b). They believe that the derivatizing is very time sensitive and that it stops when the reacting mixture is transferred into the assay tubes. Then process more samples. This is specifically for use of an entire microtiter plate at once, 96 wells.
  - With the magnetic particle kit, there are fewer than 96 tubes processed in one batch. We are following the Abraxis procedure which processes all tubes in one batch, and we adopt USGS use of extra 0.5 check samples to determine derivatization drift. Based on these extra check samples, we have not seen drift with up to 54 tubes run in one batch.
  - You will not be able to see the difference in volumes between tubes because of the 100 uL added to 1.25 mL already in the tubes. Thus be careful with the repeater pipettor to watch its squirt. We have seen problems from missing squirts, particularly early in the rack and near the end of the rack if the reagent is almost exhausted in the tip. It is OK to squirt a second time if you see no first squirt, before mixing; note any such problems in the log.
6. “Incubate” at room temperature for 10 minutes.
Assay (begin immediately after derivatizing incubation is over)
1. Transfer 300 uL of each ZS, calibrator, check, and sample from derivatizer tubes into assay tubes in ELISA plastic rack, not yet using the magnetic base. Single shot 100-1000 adjustable pipettor set for 300 uL, fresh tip for each derivatizer tube (use same tip for all replicate assay tubes), prime the tip twice in the derivatizer (source) tube before final load/move/eject into the first assay (destination) tube. Insert tip vertically down in destination tube and squirt gently so whole sample is in bottom, no splash onto sides or onto pipettor tip.
2. Put aside the derivatizer tubes in their rack, those are done with.
3. Mix the Magnetic Particle reagent container by hand by rolling it until the brown particles are distributed evenly over the volume. Do not shake, to avoid making bubbles.
4. Transfer 500 uL of mag particle solution into each assay tube. Repeater pipettor 25 mL or 50 mL tip. (50 mL tip needed for over 46 tubes, to avoid needing to reload.) Load pipettor, eject twice back into source container before beginning to squirt in the rack. Dose all tubes before proceeding to mixing step 9.f . Finish dosing in 2 minutes.
5. Vortex each assay tube with the touch mixer on low speed (#3), 1-2 seconds, minimizing foaming. Note that (1) this is a different approach from the multiple touches at high speed used in derivatizing, and that (2) mixing is left until reagent is added to all tubes.
6. Incubate for 30 minutes at room temperature.
7. Mix enzyme conjugate gently, avoiding foaming. Add 250 uL of enzyme conjugate to each assay tube using repeater pipettor and 25 mL tip.
8. Vortex each tube for 1-2 seconds at low speed (#3) minimizing foaming. Again, wait for mixing until reagent is added to all tubes.
9. Incubate for 30 minutes at room temperature.
10. Put the rack into the magnetic base.
11. Wait two minutes.
12. Dump tubes into sink, while holding rack within the magnetic base. Gently blot rims on paper towels laid out next to sink. Keep the rack within the base so the magnet holds onto the magnetic particles in the next steps.
13. Add 1 mL of Washing solution to each tube using repeater pipettor and 50 mL tip. You will need to reload if you have >46 tubes. Take care not to squirt more than once into a given tube – you can look through the side of the rack or remove/reinsert a tube.
14. Wait two minutes.
15. Dump tubes in rack (still holding magnetic base). Gently blot rims on fresh paper towels.
16. Repeat steps m through o two more times (i.e. three washes). Put down fresh paper towels before each dump.
17. Remove the tube rack from the magnetic base.
18. Make a photometer blank by adding 1 mL of wash solution into a square photometer cuvette.
19. Mix the color solution bottle without bubbling it.
20. Transfer 500 uL of color solution into each assay tube. Repeater pipettor 25 mL or 50 mL tip (use 50 mL tip if over 46 tubes to avoid needing to reload).
  1. Inspect rack quickly to ensure that there is essentially the same amount of liquid in each tube. It is OK to top up ones that are short, but be quick about it before mixing.
21. Vortex each tube 1-2 seconds at low speed minimizing foaming. Wait to mix until after all tubes have received color reagent. At this stage, if you note any tubes that are short in volume, add reagent to top up and mix again.
22. Incubate at room temperature for 20 minutes.
  1. This is a good time to read the absorbance of the photometer blank made in 10.r .
  2. This is also a good time to set up your rack of clean photometer cuvettes, a rack for used cuvettes, and a logbook grid page for recording photometer readings.
  3. Toward the end of the 20 minutes the blue color has developed so that your samples with the most glyphosate are the palest and the ones with none detectable are deepest blue.
23. Transfer 500 uL of Stopping Solution into each tube. Use repeater pipettor with 25 mL or 50 mL tip (50 mL if over 46 tubes). This does not require mixing. The color changes to yellow immediately.
Read the absorbances in the photometer. You have 15 minutes after stopping the reaction.
1. VWR spectrophotometer (temporarily using manual recording of absorbances)
  1. You will have warmed up the photometer earlier and put it in the basic mode. Also you will have filled a spare glass tube with wash solution and left that in the photometer’s reference position. Finally you will have put an empty rack to hold the ELISA assay tubes already read, in order of their original rack.
  2. Reading is done by
    1. Transfer next tube to the reading position in the photometer.
    2. Close the lid.
    3. Allow the reading to stabilize.
    4. Write down reading.
    5. Move tube to “done” rack in same position. Move it slowly to avoid ejecting droplets.
  3. Re-read the initial row to assess color drift.
2. Thermo Genesys-20 spectrophotometer [new 2020 02 19]
  1. You will have powered on the photometer earlier and it is set to 450 uM and fully warmed up.
  2. Press A/T/C until A is showing. With the wash solution tube in position, zero the photometer using the 0 abs/100%T button to read the blank.
    - As a reference, one drop of McCormick yellow food dye in 100 mL of water reads at 0.76 absorbance at 450 nm. It reads 1.36 at 427 nm, the peak absorbance for this dye.
  3. Reading is done by
    1. Transfer next tube to the reading position in the photometer.
    2. Close the lid.
    3. Allow the reading to stabilize.
    4. Write down reading.
    5. Move tube to “done” rack in same position. Move it slowly to avoid ejecting droplets.
  4. Re-read the initial row to assess color drift.
Cleanup and supplies reuse:
- We reuse all repeater pipettor tips except the derivatizer one. Rinse the disassembled tips using deionized water three times, forcing the water out of the nozzle at high velocity to maximize cleaning force. Also we reuse the temporary reagent containers.
- We do not reuse derivatization tubes, the derivatizer repeator pipettor tips, assay tubes, or single-shot pipettor tips. Rinse all of these except single shot pipettor tips with cold tap water before disposal or recycling. Note that the assay tubes containing remaining processed samples are moderately acidic from the stop solution, so have cold tap water running before dumping them. (Not much liquid per batch so we do not neutralize.)
Transcribe data from logbook to calibration spreadsheet.
- Again Abraxis needs to copy-edit their kit instructions. The instructions say to use ln/ln data transformation, the manual plotting instructions refer to the vertical B/B0 axis as linear, and the graph paper provided is logit(B/B0) versus log(conc).
- The calibration spreadsheet for atrazine uses logit() versus log() thus can be used here, adjusted for the different calibrator concentrations and one quality assurance check range. The non-detect limit is 0.05 as for atrazine, but there is no quantification limit thus any number down to and including 0.05 may be considered quantified.
- Abraxis provides one quality assurance check: the Control sample which is specified as having 0.5 ppb +/- 0.15. The other QA checks in the spreadsheet such as fit of the calibration are from the Modern Water atrazine kit.
Interpreting data
- Quality assurance results for the batch.
- Individual scatter.