The Ethanol ELMA

The Scenario
The lab that we met in the Research Data Management assignment have been out on a team bonding event. As might be expected from such a dysfunctional bunch, there was a lot of kerfuffle ‐with declarations of love (unrequited), punch ups, excessive drinking and embarrassing dancing. At the end of the evening they all went their separate ways in their own cars. On a tip off from a snitch, they have all been pulled over by the police and blood samples taken for alcohol testing.
You are the police biochemist in charge of ethanol testing. You have access to two separate labs that can do the assays for you, but you need to tell them what to do. Specifically, you need to design the set‐up of ONE 96‐well plate on which the plasma from TEN separate lab members will be tested. Once you have designed the plate set up, you will submit that to the labs, and they will do any necessary pipetting, incubations, and plate‐reader set up. They will then send you the absorbance readings from the plate and you will use the values to determine whether each of the 10 lab members that you were allocated were driving legally or not.
In addition to designing the plate and analyzing the results, there are some decisions that you MAY want to make regarding your interpretation of the data from the two labs. Each lab has a particular way of doing things. One employs technicians that are very careful and patient, the other lab has more of a party atmosphere and the staff are a little more cavalier. The mistakes the latter make are quite varied too; some often get distracted and pipette different samples into the same wells, or forget to add the master‐mix, etc. More on this later. For now, let’s think about the assay for ethanol.
The Ethanol ELMA
The enzyme alcohol oxidase catalyses the oxidation of ethanol, converting into acetaldehyde and, in the process, producing hydrogen peroxide.
CH3CH2OH + O2 CH3CHO + H2O2
By substituting alcohol oxidase for glucose oxidase in the glucose ELMA, we can turn the latter into a test for ethanol. Ethanol is oxidised by alcohol oxidase to give hydrogen peroxide which reacts with phenol and 4‐aminoantipyrine (4‐AAP) to produce phenyl‐ aminoantipyrine, a red coloured compound whose appearance can be monitored by its absorbance at 500 nm. This latter reaction is catalysed by a peroxidase (POD) and is exactly the same as the one used in the glucose ELMA.
Phenol+4‐AAP+2H2O2 phenyl‐aminoantipyrinecomplex(red)+4H2O
In the system we will use, reactions are carried out in 100 mM sodium potassium phosphate pH 7.5, using 2.5 mM of 4‐AAP, 1 mM Phenol, 10 units/ml of peroxidase and 6 units/ml of alcohol oxidase. The samples are incubated at 30°C for 30 minutes.
In order to be able to assay many samples in the most convenient, time‐efficient and cost‐ effective way, high‐throughput testing uses plate reader‐based spectrophotometry rather than cuvettes. This allows 96‐incubations to be measured at once, using volumes of 200 μL rather than 1 mL.
In our assay, each well simply consists of 50 μL of a ‘master‐mix’ containing 4X the concentration of the reagents mentioned above, plus water and plasma sample or 10 mM ethanol standard to a total volume of 200 μL. Plasma is simply blood that has been centrifuged to remove red blood cells and give a clear supernatant.
The Law: Intel on range of blood ethanol concentrations to expect
To drive a motor vehicle, the legal limit for blood alcohol concentration (BAC) is 0.05 % ‐ which corresponds to 0.05 g per 100 ml of blood. Since the molecular weight of ethanol is 46, a BAC of 0.05% equates to an ethanol concentration of about 10 mM. This is monstrously high when you think that glucose is at 5 mM and it is certainly thousands of times higher than the pharmacologically active concentration of most drugs.
The assay must obviously be able to distinguish between samples that are above or below 0.05 % (w/v). You could probably argue that it is irrelevant whether a subject’s BAC is 0.01 or 0.02 % (w/v) as both are well under, so sensitivity at the bottom of the range is perhaps not so important. However, it is vitally important to differentiate between a BAC that is just below the legal limit and a BAC that is just above the limit. Similarly, determining the exact level above the limit is likely to dictate the severity of the punishment in the case of a conviction for drink driving.
Given that unconsciousness (and often death) occurs when BAC goes above 0.25 % (w/v), the maximum concentration of BAC likely to be encountered in a driver’s serum is about 0.3 % (w/v). However, next time you are on Google, find the Australian and/or world record BAC – it’s quite sobering. Some more information on alcohol levels and driving can be found on the NSW Health and Transport (RMS) web sites.Results Submission Form – SAMPLE
Unikey of Police Biochemist: abcd1234 Results
Pokemon Ethanol (% w/v) Decision Confidence High/Low
Sample specific Comments/Insights/Smarts
Machamp 0.080 % Illegal High A duplicate outlier ignored
Pikachu 0.048 % Borderline Under Low Too close to call. The duplicates agreed but only to within 10% of each other
Bulbasaur 0.055 % Borderline Over High Duplicates identical. Very confident. GUILTY!!!
Raichu 1.480 % World Record High Absorbances off scale of machine
Ekans 0.480 % Australian Record High Absorbances off scale of standard curve.
Flareon 0.000 % None Low All absorbance values zero. Was sample really pipetted into these wells?
Arbok 0.021 % Well Under High Duplicates identical, nicely on bottom end of standard curve.
Charmander 0.230 % Well Over High Only one reading but nicely on top end of standard curve.
Charmeleon 0.005 % Probably none High Readings very low but look like sample has been added.
Pidgeotto 0.035% Under High Duplicates identical, nicely on middle of standard curve.
General Comments/Insights/Smarts
Very confident with all these results except Pikachu and Flareon which I think should be reanalysed. Dolly Rush is a great technician, but they slipped up a bit with Pikachu (bad duplicates) and I think they forgot to pipette in one of Charmander’s duplicates. I’m suspicious of Flareon’s readings but what can you do?
I was pleased to design the assay with no need for dilutions or GIANT spreadsheet analysis.
I chose not to look at the output from to dodgy company because I thought their data would just confuse and could not offer any insight.SUPPORTING INFORMATION Technician characteristics
Code Property Premium Company Budget Company
LHS Liquid handing skills. Using pipette to dispense solutions Super accurate. Always within 2% of expected volume Bit sloppy. Within 10%
CAL Pipette calibration Regularly calibrated. Always within 2% of expected volume Rarely calibrated. Each pipette consistent within itself but always 20% over or under the ‘real’ volume.
ATT Attention to flow Rarely distracted Will occasionally miss wells, or pipette samples into the wrong wells
NTE Notekeeping Keeps accurate notes of anything that they did that worries them about what they did Doesn’t keep notes
PLR Plate Reader Regularly serviced. Has correct wavelength filter. Reads at wavelength which is different to the lamda max for the coloured product
ENZ Enzyme activity Uses fresh enzyme Enzyme a bit out of date and not as active as it should be.
Marking Rubric
Total value is 25 points which becomes 10% of the entire assessment.
5 points for the results analysis spreadsheet
This includes 1 point for good organisation of data in logical rows and columns, meaningful column headings, and clear formatting. 2 points for the standard curve, which must have appropriately labelled axes and a clear trendline and R^2 value. 2 points for the use of formulae and functions to arrive at the final ethanol % (w/v) values for each subject.
5 points for “Accuracy of Decision” for the 10 subjects
Simply the closeness of your estimates to the actual concentrations and your correct interpretation of the number. This is Columns 2 and 3 of the results form.
5 points for reflection on each individual sample (confidence and insights)
Reflections on the validity of each determination are made in Columns 4 and 5 of the form.
5 points for the overall reflections on the assay and plate design
This is contained in the section at the end of the results form
5 points for inter‐company comparisons
Specific reference to any insights obtained by the comparison of the data from the two companies. Again, in the section at the end of the form
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