ELMA kit : S&P chemical 10/08/2010

A user’s guide…

Some relevant background information advertising the benefits of the kit:

Quantification of the amount of glucose is essential to assess the current

health conditions of those suffering from high or low blood glucose levels

(hyperglycaemia and hypoglycaemia respectively).

Glucose plays the lead role as the energy reservoir for every cell in our body,

attained from the food we consume on a regular basis. The glucose is further

attended to by regulatory hormones, insulin and glucagon which serve to regulate

it’s presence and utilize it effectively.

A considerable portion of a given population will be subject to an irregularity

of either of these hormones and thus interrupting optimal glucose utilization, a

consequence evident from the glucose concentration in the bloodstream.

Our ELMA assay kit achieves this purpose via Enzyme-Linked Metabolism

with easy to follow instructions that ensures accurate quantification of any glucose

sample. However, if any nasty surprises do occur as a result, a trouble-shooting

guide is provided quickly get back on track.

NOTE: this kit is designed ONLY for Laboratory purposes

 What are the general principles of this assay kit?

General principles of the assay in simple terms

Glycation begins as a reaction between the aldehyde group on glucose with

the amino groups on proteins forming a reversible schiff’s base. A further

rearragnement results in a stable aminoketone modification of the protein

known as Amador rearrangement. This modification in hemoglobin Hb A1c is

measured in diabetics to monitor the management of blood glucose.

Assay involves two steps: first oxidizes D-glucose to D-glucono-1,5-lactone.

This reaction is catalysed by glucose oxidase (GOD). GOD removes a pair of

hydrogen from glucose. In doing so, the redox cofactor employed by GOD,…

In short this means oxidation of glucose.

Second step involves the enzyme Peroxidase (POD) which cleaces the

hydrogen peroxide produced from the first reaction. An oxidsable substrate

is required for this reaction to take place and in this case is phenol. 4-

aminoantipyrine (4-AAP) reacts with oxidized phenol to yield a red

quinoeimine adduct.

So essentially, the more glucose present in the sample, consumed by the

first enzymatic reaction, the more quinoeimine adduct and red colour,

produced by second reaction.

Some information about meaning of different blood [glucose] results.

Time required

This kit has been equipped with pre-measured reagents that will be used in

the assay, which in all will take about 15-20mins to setup and 45-50 mins to

run. Further on in the test, the acquired absorbance values will be plotted

via a spreadsheet that is also provided for your convenience.

Intended use: the types of samples the kit is designed for

Reagent provided (consider enzymes to be included unmixed)

Material required but not provided; both equipment and reagents

Warnings, precautions and storage instructions

 10/08/2010

Abstract
The extensive use of commercial kits in molecular biology and

biochemistry has prompted us to design a series of practical sessions to help

students become familiar with the uses and limitations of pre-packaged

assay systems. To facilitate an understanding of these assay systems and to

promote reflection on their appropriate use, students manufacture their own

kit for one of four enzyme-linked metabolite assays. To do this they must

investigate the role of each of the components in the assay, optimize the

conditions where possible, check for cross reactivity, and then work this

assay up for a few related “real” samples. Students make up all the buffers,

the standard solution, instructions, and even the packaging. The kit is

checked for accuracy by the producers, “marketed” to other students, and

evaluated by their peers. By going through this process, students learn the

benefits and pitfalls of commercial kits as well as reinforcing the basic

principles of metabolite measurement and gaining experience with assay

design, troubleshooting, and problem solving.

ELMA kit : S&P chemical 10/08/2010

Many of the techniques used in molecular biology and biochemistry

rely extensively on the use of commercial kits. These offer an economical

alternative for small-scale experiments and for methods that are carried out

infrequently in the laboratory, saving valuable time working up methods and

troubleshooting. The success of the technique is usually assured, positive

controls are supplied, and extensive quality assurance procedures are in

place to ensure that the product leaves the manufacturer in a functional

state. Kits are, however, not without limitations, and caution must be

applied when drawing conclusions from results obtained by kits in some

circumstances. The decision to use a commercial kit or to “do-it-yourself”

requires an understanding of the principles behind the reactions employed

by the kit, an appreciation of its limitations in certain circumstances, and

experience at using kits. It is these skills that we wish to foster in students

studying practical courses in molecular biology and biochemistry. However,

the cost to purchase commercial kits for >500 students would be prohibitive

and the educational value of simply following instructions would be minimal.

By giving students the task of designing their own kits, there is also the

opportunity to indulge in genuine troubleshooting, rather than that done by

the course coordinator before introducing the practical to the course. With
ELMA kit : S&P chemical 10/08/2010

this in mind, a series of practical sessions has been designed in which

students produce and market their own kit.

Strategy
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In the first session, students are provided with a working protocol for

one of four enzyme-linked metabolite assays (ELMAs)1 : glucose/sucrose,

lactate, alcohol, and monosodium glutamate (MSG). These metabolite

assays were chosen as they are reliable, robust in student hands, and

relatively cheap to assay with a spectrophotometer. In addition, these

metabolites are found in measurable quantities in many foods and

beverages, which makes possible the use of these kits on real samples. After

familiarizing themselves with the assay by obtaining a standard curve with

the protocol, students then investigate the role of each component in the

assay by performing the protocol with a limiting amount of each component

in turn. With this knowledge, the function of each component in the assay

can be determined and the consequences of its omission explored. Students

are divided into groups for this activity, and class results are collated at the

end of the practical session. This section provides a knowledge base for the

troubleshooting exercise in the next session.

ELMA kit : S&P chemical 10/08/2010

In the second session, with the basic optimized protocol in hand,

students now proceed to try their assay on “real” samples. Samples of

various foods and drinks are provided (see Table I) for this session (and

students often choose to provide their own samples). Students are

encouraged to “theme” their kit around related products. For example, if

glucose is to be measured, students could design kits to compare the

carbohydrate content of soft beverages. This session involves much trial-

and-error and extrapolative calculation, as there is often no indication of the

concentration of the metabolite in many of the samples. Many of the

samples require some sort of preparation first, such as centrifuging or

precipitation of proteins. Strategies for sample handling are discussed with

the tutor before the measurements begin. By the end of this session,

students have worked up a reliable protocol that can be used to measure

their assigned metabolite in real samples. A more extensive range of

samples could be explored if time permitted.

ELMA kit : S&P chemical 10/08/2010

A working kit is actually produced by the students in the third session.

The students are responsible for assembling all the components from

standard laboratory reagents (i.e. they make up all their own buffers and

stock solutions). For most of their laboratory classes at university students

have all the necessary solutions provided for them, so this session is a

valuable step in the transition from laboratory class to workplace situation.

Students are shown how to make up buffers and to weigh out and accurately

make up standards and other stock reagents. Like any good manufacturer,

after students have made up their solutions, they perform some quality

control before the kit is “shipped out.” They perform a standard curve using

their own standards and buffers and check that this result is both linear and

achieves the correct response. When they are satisfied with the

measurements, the kit is packaged and stored for the final session.

Documentation
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ELMA kit : S&P chemical 10/08/2010

Before coming to the final session, students produce instructions and

specifications sheets to accompany their kits, both in hard copy and

electronic format. As well as the instructions on how to prepare the samples

and carry out the assay, this material covers topics such as limits of

detection, troubleshooting, principles behind the assay, and cross-reactivity.

Disks are often included with spreadsheet templates for calculating the final

concentrations. In our classes, students have even set up websites for their

product!

The Final Check

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In the final session, the kits are distributed to peers for evaluation.

Where possible, the students evaluating the kit will not have produced a kit

for the same metabolite. Several criteria are considered in the evaluation

process: the consistency and accuracy of the results obtained for both

standards and samples and the user-friendliness of the instructions and

calculations. A proportion of the marks is allocated for the information

sheets and for creative presentation. In our scheme, the final mark is made

up of student evaluation and a mark by the tutor.

THE ASSAYS
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All assays are spectrophotometric, but there is no reason why the

principles cannot be applied to any system.

Sucrose/Glucose
Glucose is measured by the oxidation by glucose oxidase (GOD) [1] to

gluconic acid with the concomitant production of H 2O 2 in equimolar

proportions. Two moles of H 2O 2 then react, under the action of peroxidase

(POD), with 1 mol of 4-amino-antipyrine (4-AAP) and 1 mol of phenol to

produce a red complex that absorbs strongly at 500 nm. To increase the

versatility of this assay, sucrase (or invertase) [2] can be added, enabling an

estimate of the level of sucrose in samples. The standard curve produced by

this reaction is shown in Fig. 1a.

Lactate
The enzymic oxidation of lactate to pyruvate by lactate dehydrogenase

(LDH) [3], with the concomitant reduction of NAD +

to NADH, forms the basis

of this assay. This reaction can be monitored spectrophotometrically as

NADH, and not NAD +, absorbs strongly at 340 nm. Hydrazine, which reacts

with the pyruvate, is added to drive the reaction to completion. The standard

curve obtained with this reaction is shown in Fig. 1b.

ELMA kit : S&P chemical 10/08/2010

Glutamate
Glutamate is measured using glutamate dehydrogenase (GlDH) [4]

under similar experimental conditions to the lactate assay above. This

reaction can be used to measure the level of MSG in samples. Fig. 1c shows

the standard curve produced by this assay.

Ethanol
Ethanol is measured under similar experimental conditions to the

lactate assay except the LDH is replaced with alcohol dehydrogenase (ADH)

[5]. The standard curve resulting from this assay is shown in Fig. 1d.

SESSION DETAILS
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Students can work in pairs for all four sessions with advantage.

Session 1 (2∼4 h): Introduction to Reactions

ELMA kit : S&P chemical 10/08/2010

Students begin the first session by determining the reaction time

course in their assigned assay using different metabolite concentrations.

From these results, a standard curve is constructed and the optimal

incubation time for the assay is determined (Fig. 2). This enables students to

become familiar with the assay and with following instructions/protocols as

the end user. After completing the initial standard curve, examples of less-

than-ideal instructions could be given and the elements that make up a good

protocol could be discussed.

Investigating the Experimental Conditions

After students have obtained a standard curve for their assigned

metabolite, they work, as a group, to investigate the role of each of the

components in the assay. This is done by performing the assays for the

standard curve with a limiting amount of each assay component. Both the

time course and the resultant standard curve are compared with the fully

optimized data.

Adding a limiting amount of enzyme produces a time

course that fails to plateau within the time frame. Students will appreciate

from this exercise that, although enzymes do not “run out,” there may not

be enough enzyme to complete the reaction in a realistic time frame.

ELMA kit : S&P chemical 10/08/2010

If there is an insufficient concentration of one of the

reactants or cofactors involved in the reaction then this reactant becomes

the limiting component of the assay, rather than the metabolite of interest.

The reaction slows or stalls at higher metabolite concentrations and a

“bunched up” time course results, producing a nonlinear standard curve.

Many enzyme-linked assays must be coupled to a second

reaction to drive them to completion. Typically dehydrogenase reactions,

where the production of NADH is monitored, require such a coupled reaction.

In our examples, hydrazine is used to convert the ketone product to a

hydrazone derivative and thus remove it from the equilibrium. If the

hydrazine is omitted, the reaction does not go to completion but rather

reaches an equilibrium and variable results are obtained. While a linear

standard curve is produced, the results are unreliable.

To test for substrate specificity, the response of a number

of possible substrates of the various enzymes used is examined by student

groups. Possible substrates are added to the relevant reaction under the

same assay conditions, and the absorbances for equivalent concentrations

are compared. All the reactions used in this study have proved to be very

specific. The enzymes were even found to distinguish optical isomers, e.g.d

and l lactate.
ELMA kit : S&P chemical 10/08/2010

Each of these studies illustrates the results if a particular solution is

not at the correct concentration in the reaction. Practically, this may arise

from errors in the delivery of the solution or a denatured, deteriorated, or

improperly prepared solution. From these results, an extensive

troubleshooting section is compiled for the kit specification sheets.

The Second Session (∼5 h)

By now, students are confident with their assay using standard

solutions. It is time to apply what they have discovered to real samples. This

session is more demanding for tutors and students alike. Students have

moved from the comfortable zone of predictable standard solutions to real

unknowns. The can of soft drink does not quote quantitative information on

the components of the drink; many of these are proprietary secrets. Some

sports drinks are more informative, but generally some initial guesswork is

needed to establish a working range. Students are encouraged to assay a

series of wide-ranging dilutions and to obtain a narrow, measurable range of

dilutions, which can be then assayed in a second series of reactions. Typical

samples assayed for each ELMA are shown in Table I.

Discussion Arising from this Session

What Happens When the Results Indicate the Sample

Contains None of the Metabolite?—

ELMA kit : S&P chemical 10/08/2010

Is this because there is actually none of the metabolite in the sample

or is there something in the sample that interferes with the assay; perhaps

an inhibitor of the enzyme? To test for this, students are encouraged, after

some discussion, to “spike” the sample with a known amount of the standard

metabolite solution. If the spiked assay returns the predicted result, then it

can be concluded that the sample contained none of the metabolite. If the

response for the spiked sample is lower than predicted, there may be some

interference from the sample.

How to Prepare the Sample?—

If the sample is a liquid, as in the case of soft drinks, the sample only

needs diluting appropriately. If, however, students wish to assay powders

and solid samples (e.g. flavor sachets, cheeses, chips), there is considerable

discussion with the tutor before proceeding. A strategy needs to be devised,

based on the solubility of the metabolite and the nature of the sample.

Commercial kit protocols are sometimes consulted, merely as a guide [6].

The Third Session (∼3 h)

ELMA kit : S&P chemical 10/08/2010

In this session, students make up and check the buffers and solutions

for their kit. The session begins with a discussion on how many assays their

kit should accommodate, including the standard curve (6–12 assays). To

ensure the end user will have enough of each solution to carry out their

analyses, kits are prepared with enough reagents for at least 30 assays.

After the solutions are made up, students are strongly advised to test them

with their own standard. A linear standard curve achieving the desired

absorbance is required. The kit is evaluated, in part, on the standard curve

obtained by the evaluating students. In the event of poor results from the

end user, the manufacturer will be given the benefit of the doubt if the kit

was shipped out in a functional state, i.e. consistent and accurate standard

curve.

Key Learning Objectives from this Session

Making Up and Adjusting the pH of a Buffer—
For most students, this is their first experience of actually making up

the buffer. Use of pH meters, how to adjust the pH then make up to volume,

and a refresher on basic moles calculations are all covered.

Problems with Unstable Compounds—

ELMA kit : S&P chemical 10/08/2010

The hydrazine and the enzymes can be weighed out in advance, but

the liquid must be added to the powders just before use. Developing

strategies to overcome these problems while keeping the protocol simple

and user-friendly has proved to be quite a challenge.

Before Next Session

Students produce information and specifications sheets for their kit.

Topics covered in these sheets include: limits of detection, general assay

principles, cross-reactivity, sample preparation, troubleshooting, assay

procedure, warnings, hazards, and cost. This material is a summary of all

their work over the past 3 weeks. The troubleshooting section in particular

directs students back to the first week's experiments where the role of each

component in the assay was investigated. By this stage, students are aware

of the steps that are crucial and must be performed with extra care, they

have worked out all the tricks to handling their real samples, and they have

experience as the end user with protocol instructions.

The kit presentation can be as creative as students like. Students are

given access to their kits before the final session to add labels and be

creative with packaging. Commercial kit instructions and specification sheets

are available for ideas and as a reference for accepted commercial practice.

Catalogues are provided and kits are costed as well.

ELMA kit : S&P chemical 10/08/2010

The Fourth Session (∼4 h)

Kits are exchanged, usually between pairs who have developed kits for

different metabolites. An evaluation sheet is handed out as a guide and

students are asked to produce a standard curve and analyze the samples

provided and then to make constructive criticisms as they progress. This

session allows students to comment on their peers' work from the

perspective of an end user. In their appraisal, students must consider what

elements go into making a good set of instructions and how it could be

improved. They must critically analyze the consistency and accuracy of their

results and, if necessary, troubleshoot the assay. These are the very

discernment skills we hoped to foster in our students from the outset.

CONCLUSION
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ELMA kit : S&P chemical 10/08/2010

The ELMA kit is the culmination of the semester's practical work for

students in our course. It brings together both the generic problem-solving,

design and interpretation skills, and biochemistry-specific techniques learned

throughout the term. Students enjoy the chance to present their work

creatively, in a different format from the usual report, poster, or

presentation. The effort put into the final kits, both to the accuracy and the

presentation, is very rewarding for the student.

This series of practical sessions gives the students an opportunity to

become “experts” in a technique, rather than having to jump from one

method to the next, which is often the case in laboratory courses. In an

effort to expose students to as many techniques as possible, there is the

danger of “overloading” them with a surface coverage of many methods

without allowing them to understand any of them in depth. Feedback from

end-of-semester student surveys is overwhelmingly positive toward the

ELMA, and this is borne out also in the performance in end-of-semester

theory of practical exams.

ELMA kit : S&P chemical 10/08/2010

Through this series of practical sessions, students are encouraged to

develop the discernment skills necessary to use kits and interpret the results

obtained from kits wisely. The general troubleshooting skills and sample

preparation strategies learned in the four sessions will be invaluable when

using kits for any technique involving molecular biology or biochemistry. The

ability to write a clear set of instructions to carry out a technique will also be

of great help for technical supervision in the workplace.

Extending the Concept

Molecular biology abounds in the use of kits. The student-designed kit

production and evaluation concept could be developed further for inclusion in

molecular biology courses. Methods for common molecular biology

procedures, for example DNA and RNA isolation procedures, could easily be

adapted to facilitate this approach. One of the most common kits used in

molecular biology is the alkaline lysis method for the isolation of plasmid

DNA. Rather than just presenting the instructions in a recipe-style format,

students could design kits for this procedure from basic principles.
ELMA kit : S&P chemical 10/08/2010

Figure FIGURE 1.. a, glucose levels (0–250 μm) were measured in a

0.1 m sodium phosphate buffer, pH 7.4, containing 1 mm phenol and 2.5

mm 4-AAP with the addition of GOD (10 U/ml) and POD (10 U/ml). The

reaction was monitored by absorbance at 500 nm over a 40- to 45-min time

period. b, lactate levels (0–250 μm) were measured in a 0.1 m glycine

buffer, pH 9.5, containing 3 mm NAD, 1.6 m hydrazine, and 2.8 mm EDTA

with the addition of LDH (10 U/ml). The reaction was monitored by

absorbance at 340 nm over a 70-min time period. An estimate of the

percentage completion of the reaction was obtained using the extinction

coefficient ϵ NADH at 340 nm of 6.2 mm−1cm −1

. c, glutamate levels (0–250 μm)

were measured under the same conditions as lactate (see b) except that

GlDH was substituted for LDH and 0.1 mm ADP was also included in the

assay. d, ethanol levels (0–250 μm) were measured under the same

experimental conditions as b except that ADH was added instead of LDH.

ELMA kit : S&P chemical 10/08/2010

Figure FIGURE 2.. The construction of the Standard Curve. A

series of reactions is set up using one of the procedures described in Fig. 1,

in this case glucose (Fig. 1a). Absorbances are monitored at 5-min time

intervals, and from these values a time course is produced for each

substrate concentration. Using the end point absorbances after 40 min at

each concentration, a standard curve is then constructed.

Table Table I. Samples analysed by ELMA kits

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