Enzyme Diagnostics

Question 1

how are enzymes used as reagents?

Answer 1

as specific bio-reagents
reacting with substrate / analyte
as a means of producing observable signal
as removers of interference
as signal amplifiers ‘labels’ in immunoassay

Question 2

example of an enzyme as a specific bio-reagent?

Answer 2

glucose oxidase
glucose + o2 —- (glucose oxidase) —-> gluconolcatone + H2O2
H2O2 + o-toluidine —-(peroxidase)—-> oxidised o-toludine + H20 coloured
glucose oxidase is very specific for glucose, but peroxidase can react with many reagents

Question 3

what can provide specificity for enzymes as reagents?

Answer 3

a converse arrangement- lack of specificity in the primary enzyme (hexokinase) can be compensated by a high specificity in the secondary enzyme linked to detection of the initial product

Question 4

what is an example of a converse enzyme arrangement?

Answer 4

glucose
glucose + ATP (hexokinase)-> Glucose-6-Pi + ADP
Glucose-6-Pi +NADP (G-6-Pi-DH) -> 6-phosphogluconate + NADPH
the glucose 6-Pi-DH is very specific for its substrate

Question 5

modern analysers and enzymes as reagents

Answer 5

hundreds of analyses per day, no hazardous chemicals, everything takes place at a single controlled temperature and inter-batch CVs are about 2-3%

Question 6

why is a 5th enzyme (ascorbate oxidase) also present for creatinine?

Answer 6

to prevent interference from ascorbic acid

Question 7

creatinine enzymatic assay

Answer 7

creatinine + H20 (creatininase) -> creatine
creatine + H20 (creatinase) -> sarcosine and urea
sarcosine + O2 + H20 (sarcosine oxidase) -> glycine + HCHO + H202
H202 + dye reagent (peroxidase) -> colour

Question 8

the traditional ‘jaffe’ method for creatinine

Answer 8

used alkaline picrate as a colour reagent, long established and cheaper method, but more prone to interferences, particularly from bilirubin. modern analysers allow for both methods on the same instrument and can be programmed only to do an enzymatic creatinine analysis if a high bilirubin is detected

Question 9

end point assays (fixed time measurements)

Answer 9

the reactions are allowed to go to completion. zero order kinetics. can demand relatively high amounts of enzyme reagents and they work best with enzymes with a low km (high affinity) for the substrate which is the subject of the analysis. less sensitive to changes in assay conditions.

Question 10

when is a trapping reagent used

Answer 10

where a reaction equilibrium is unfavourable to the desired direction, a ‘trapping’ reagent may be used to ‘pull’ the equilibrium that way e.g. the analysis of lactate.
lactate + NAD <– (LDH–>) pyruvate and NADH
pyruvate + hydrazine —> hydrazone.
as pyruvate is continually removed from the equilibrium by conversion to its hydrazone, the production of the detection chromophore, NADH, continues until all the lactate is used up

Question 11

kinetic assays

Answer 11

the rate of reaction is used to assess the concentration of the analyte being measured. several measurements required over a period of time. smaller amounts of the primary enzyme may allow a slower process which is then easier to monitor over time. the theory behind enzyme kinetics means that an enzyme with a high km for the substrate being determined may be advantageous as is it desirable to work at substrate/analyte concentrations below 0.2x km

Question 12

substrate concs below 0.2 x km

Answer 12

to achieve situation where the observed reaction velocity is proportional to the substrate conc. first-order kinetics

Question 13

kinetic assays - factors

Answer 13

need to be strictly controlled to give precise results

Question 14

enzyme reagents employed as part of ‘production detection’ (coupled assays)…

Answer 14

must not be rate limiting - use in high concentrations relative to the enzyme reacting with analyte/substrate

Question 15

kinetic assays may be faster than end point assays as

Answer 15

there may be less need to wait for a reaction to achieve an equilibrium

Question 16

example of an kinetic assay

Answer 16

ethanol
ethanol + NAD - (ADH) -> acetaldehyde + NADH
Only requires a single enzyme which reacts with substrate/analyte and also produces an observable product.
kinetic assays can also use multi-enzyme systems to produce a convenient spectrophotometric change if needed

Question 17

enzymes as labels in immunoassay

Answer 17

immunoassay often analyse very low concentrations of analyte. as enzymes can cycle for as long as they have appropriate substrate. they can amplify the signal

Question 18

what enzymes are advantageous for enzymes labelled immunoassays

Answer 18

enzymes with high turn-over numbers (kcat) as they produce more signal per unit time.

Question 19

what are some examples of enzymes used as labels in immunoassay?

Answer 19

peroxidase and ALP

Question 20

what 2 common techniques for ELIA

Answer 20

EMIT- a ‘homogeneous’ immunoassay where no separation step is needed
ELISA - a ‘heterogeneous’ immunoassay method

Question 21

ELISA are usually…

Answer 21

2 stage assays - capture antibody is used to bind analyte in the sample to a solid phase followed by a wash step and then addition of a second antibody for the analyte which is linked to an enzyme label.

Question 22

‘dry’ reagent systems

Answer 22

enzyme reagents can be immobilised onto a solid support e.g. cellulose and used in test ‘strips’. Urine testing for glucose generally uses this method – the glucose oxidase, the peroxidase and dye (potassium iodide chomogen) are incorporated into the reagent pad. used in POCT