L.13 Enzyme Assays & Immunoassays

Question 1

How can enzyme activity be measured?

Answer 1

By monitoring the rate of formation of a product or the rate of disappearance of a substrate

Example: NADH measurement at 340nm

Question 2

What happens to substrate and product during an enzyme reaction?

Answer 2

Substrate decreases and product increases

Question 3

What is Vmax in enzyme kinetics?

Answer 3

Max rate of reaction reached when all enzyme active sites are saturated with substrate

Question 4

What is the lag phase in enzyme reactions?

Answer 4

The reaction may not start immediately

Question 5

What characterizes the linear phase of an enzyme reaction?

Answer 5

The reaction is linear when enzyme is saturated with substrate

Question 6

What occurs during the depletion phase of an enzyme reaction?

Answer 6

The reaction may slow down as substrate is used up

Question 7

What happens in the inhibition phase of an enzyme reaction?

Answer 7

The reaction may stop when enzyme is denatured

Question 8

When is it most accurate to measure enzyme activity?

Answer 8

In the linear phase

Question 9

How does the rate of reaction relate to enzyme concentration?

Answer 9

The rate of reaction is proportional to the enzyme concentration when the substrate concentration is high

Question 10

What are the two methods to determine enzyme activity?

Answer 10

• Fixed time: measure change in absorbance at a fixed time
• Continuous monitoring: measure change in absorbance continuously

Question 11

What is critical when measuring enzyme activity?

Answer 11

To measure enzyme activity in the linear phase

Question 12

What does the rate of ALT activity measurement depend on?

Answer 12

The rate of NADH formation

Question 13

What are coupled enzyme reactions used for?

Answer 13

To measure the activity of enzymes that do not produce easily measurable products

Question 14

How is ALT activity measured using a coupled enzyme reaction?

Answer 14

By coupling the reaction with LDH

Question 15

What is the purpose of the indicator reaction?

Answer 15

The indicator needs to be directly proportional to the enzyme activity from the primary reaction

Question 16

What is a common example of an indicator reaction in clinical chemistry?

Answer 16

• NADH/NADPH formation or removal (340nm) for ALT, AST, LDH, Urea
• p-nitrophenol formation (yellow - 405nm) for ALP

Question 17

What enzyme is used to measure urea?

Answer 17

Urease

Urease catalyzes the hydrolysis of urea into ammonia and carbon dioxide.

Question 18

Which enzyme is used to measure glucose?

Answer 18

Glucose oxidase

Glucose oxidase catalyzes the oxidation of glucose to gluconolactone.

Question 19

What enzyme is used to measure triglycerides?

Answer 19

Lipase

Lipase catalyzes the hydrolysis of triglycerides into glycerol and free fatty acids.

Question 20

How does enzyme concentration affect reaction rate?

Answer 20

Rate of reaction ∝ enzyme concentration

In substrate excess, increasing enzyme concentration increases the reaction rate until the substrate is depleted.

Question 21

What happens to the reaction rate when all substrate is used up?

Answer 21

Plateau

The reaction rate levels off when no substrate is available for the enzyme to act upon.

Question 22

How does substrate concentration affect enzyme activity?

Answer 22

Effect depends on enzyme concentration

The impact of increasing substrate is contingent on how much enzyme is present.

Question 23

What effect does pH have on enzyme activity?

Answer 23

Affects ionisation of substrate or enzyme

Extreme pH levels can lead to enzyme denaturation, affecting its function.

Question 24

What is the effect of temperature on enzyme activity?

Answer 24

Low activity at low temperatures; denaturation at high temperatures

Enzymes have an optimal temperature range for activity.

Question 25

Name two factors that affect enzyme activity.

Answer 25

* Inhibitors * Co-factors ## Footnote Inhibitors can reduce enzyme activity, while co-factors are often necessary for enzyme function.

Question 26

What is a key issue with enzyme assay standardization?

Answer 26

Comparison of results between methods and labs ## Footnote Standardization is crucial for accurate clinical diagnosis and interpretation of results.

Question 27

Why is enzyme preparation used as a standard?

Answer 27

To calibrate the assay ## Footnote A standard allows for consistent measurement across different labs and methods.

Question 28

What is the SI unit for enzyme activity?

Answer 28

Katal (kat) ## Footnote The katal measures the rate of reaction in moles per second.

Question 29

What is the International Unit (IU) of enzyme activity?

Answer 29

Amount of enzyme that catalyzes one micromole of substrate to product per minute ## Footnote The IU is a more practical unit than the katal for laboratory use.

Question 30

Fill in the blank: The amount of enzyme which will catalyze the transformation of one mole of substrate to product per second is defined as _______.

Answer 30

katal ## Footnote The katal is considered too small for practical use in many laboratory settings.

Question 31

What are isoenzymes?

Answer 31

Enzymes that can occur in multiple forms with different subunits or amino acid sequences.

Question 32

Isoenzymes vary with respect to which characteristics?

Answer 32

* Kinetic properties * Electrophoretic mobility * Localisation

Question 33

How can isoenzymes help in clinical settings?

Answer 33

They can help identify the source of enzyme activity in the body.

Question 34

How many isoforms does LDH (lactate dehydrogenase) have?

Answer 34

5 isoforms.

Question 35

How many isoforms does CK (creatine kinase) have?

Answer 35

3 isoforms (CK-MM, CK-MB, CK-BB).

Question 36

What is one advantage of measuring enzymes?

Answer 36

Specificity - each enzyme has a specific substrate.

Question 37

List two advantages of measuring enzymes.

Answer 37

* Sensitivity - can detect small changes in enzyme activity * Versatility - wide array of enzymes available for measurement.

Question 38

What is the reproducibility of enzyme measurement?

Answer 38

Standardised; reaction rate will be the same if conditions are the same.

Question 39

What is a disadvantage of enzyme measurement related to localisation?

Answer 39

Enzyme activity may not be specific to a particular organ.

Question 40

Give an example of an enzyme that is found in multiple organs.

Answer 40

ALT is found in liver, heart, and skeletal muscle.

Question 41

What is the timing of sample collection important for enzyme measurement?

Answer 41

Enzymes rise and fall as they are released and cleared.

Question 42

What is the principle of immunoassays?

Answer 42

Use of antibodies to detect and quantify specific analytes in a sample.

Question 43

What forms when antibodies bind to their target analytes in immunoassays?

Answer 43

Antibody-antigen complex.

Question 44

What is detected in an immunoassay?

Answer 44

The antibody-antigen complex using a signal producing reaction.

Question 45

What is the purpose of immunoassays?

Answer 45

Determination of specific and clinically relevant analytes.

Question 46

How do immunoassays utilize antibodies?

Answer 46

They utilize the specificity and sensitivity of an antibody towards a target analyte to rapidly detect and quantify the analyte of interest.

Question 47

What are common applications of immunoassays?

Answer 47

* Hormone measurement (thyroid, reproductive hormones) * Drug monitoring (TDM and toxicology) * Cardiac markers (Troponins, NT-proBNP) * Tumour markers * Specific plasma proteins * Infectious disease testing * Allergies and autoantibodies ## Footnote These applications highlight the versatility of immunoassays in various medical fields.

Question 48

What is a Direct Immunoassay?

Answer 48

A type of immunoassay used for rapid tests and tissue staining ## Footnote Direct immunoassays involve the direct binding of an antigen to an antibody.

Question 49

What is an Indirect Immunoassay?

Answer 49

A type of immunoassay used for the detection of antibodies, such as HIV and HCV ## Footnote Indirect immunoassays typically involve a secondary antibody that binds to the primary antibody.

Question 50

What is a Sandwich Immunoassay?

Answer 50

A type of immunoassay used for the detection of antigens, such as hormones and tumour markers ## Footnote This method uses two antibodies to capture and detect the target antigen.

Question 51

What is a Competitive Immunoassay?

Answer 51

A type of immunoassay used for drug monitoring, toxicology, and detection of small molecules ## Footnote In competitive immunoassays, the sample competes with a labeled analyte for binding sites on the antibody.

Question 52

What is a Magnetic Particle Immunoassay?

Answer 52

An immunoassay that uses magnetic particles to capture the antibody-antigen complex ## Footnote This type of assay is adaptable to automation and suitable for high throughput testing.

Question 53

What are the advantages of immunoassays?

Answer 53

* High specificity * High sensitivity * Flexible * Automation * Rapid results ## Footnote These advantages make immunoassays a preferred choice in many diagnostic applications.

Question 54

What is a disadvantage of immunoassays related to cross-reactivity?

Answer 54

Antibodies may cross-react with similar molecules ## Footnote This can lead to false-positive results in certain cases.

Question 55

What is a limitation of immunoassays regarding dynamic range?

Answer 55

Limited dynamic range may not allow detection of high and low concentrations ## Footnote Samples may need to be diluted or concentrated to fit within the assay's range.

Question 56

What is a common high-cost factor associated with immunoassays?

Answer 56

High-cost ## Footnote The technology and materials used in immunoassays can be expensive, impacting overall testing costs.

Question 57

Fill in the blank: A __________ immunoassay is used for the detection of antibodies like HIV and HCV.

Answer 57

Indirect Immunoassay ## Footnote Indirect immunoassays are critical for diagnosing viral infections.

Question 58

True or False: Sandwich immunoassays are used to detect small molecules.

Answer 58

False ## Footnote Sandwich immunoassays are primarily used for detecting larger antigens like hormones and tumour markers.