Immunoassay

Question 1

What is the Hooke Effect and when is it clinically significant?

Answer 1

The Hooke Effect occurs in immunoassays.

It is where there is a too high concentration of substrate, so they saturate the enzyme, in this way the enzyme can’t bind (sandwich assay) or fluoresce (competitive assay).

So the sample can appear to have much lower concentrations of analytes than are really present = falsely low result.

This sample would require dilution prior to immunoassay.

Clinically you will know if this is the case if it doesn’t fit with the clinical picture.

Question 2

What are the requirements for immunoassay?

Answer 2

Standards for calibration
Specific antibodies
Labelled antigen or antibody
Separation system to separate complex from free antibodies and antigens
Detection system
Quality control

Question 3

What must be considered for immunoassay standardisation for calibration?

Answer 3

• The standard should contain the analyte in a form identical to that found in the sample or similar to the extent that the forms behave identically in the assay system.
• Must be sure with calibrator that what its measuring represents all the forms, but also that nothing else with a similar structure to any fragments can interfere.
• The standard should be suitable for use in the widest possible range of assay systems (standard should be transferable across platforms), with the same type of concentration units and potency.
• The standard should be available in sufficiently large quantities and be sufficiently stable to permit continuity of use through time and between labs.
• The matrix in which the standard is introduced into the assay system should behave identically to the sample matrix.
• Where possible assay should be calibrated with an International Standard (IS), so it is traceable to any lab that uses that standard, for UKAS must be able to quote where your calibration material comes from.
  IS are defined on the basis of an international collaborative studies.

Question 4

What primary antibody is used?

Answer 4

A primary antibody is an immunoglobulin developed against specific target antigen of interest. IgG is used almost exclusively in immunoassay as it has highest yield, highest affinity for epitope, stable, several functional sites available for chemical coupling with minimal loss of antibody binding.

These days immunoassay use monoclonal antibodies that are specific for one epitope on the antigen, so reduce cross reactivity and increase specificity. These are produced from a single cell line, that can produce antibodies consistently and indefinitely.

Question 5

What is a competitive immunoassay?

Answer 5

If to a solution of solid phase antibody, an equal volume of labelled and sample analyte are added they will compete for antibody binding sites on the tube, with equal affinity. This leads to 50% of each bonding as they are present in the same amount. However, if there is more analyte in the sample (ie it is very concentrated) then it will outcompete the labelled analyte. All unbound is washed away so you only measure what is bound. You then count how much labelled is present. So the more analyte in the sample the less label you see.

Either way, must always include a fixed amount of labelled antigen to indicate the distribution of bound and free analyte and count bound fraction after separation and washing.

Can then form a calibration curve where values are lower at higher concentrations, proportional to a higher sample concentration, therefore a lower concentration of bound labelled analyte.

Eg testosterone and aldosterone.

Question 6

What is a sandwich assay?

Answer 6

A tube is coated with an excess of solid phase antibodies. The antigen is added followed by an excess of labelled antibody. The antigen is complementary to both antibodies so both bind causing a sandwich. The more antigen present in the sample the more sandwich complexes can form and the more label is measured.

In this the calibration curve shows an increase in label activity with increasing concentration.

Eg PTHrp

Question 7

What are the advantages and disadvantages of the sandwich assay?

Answer 7

Advantages:
Increased sensitivity - measure down to picomoles
Increased precision
Better specificity - directing to two different epitope.
Greater assay range - lots of concentrations
Shorter assay times - around 20 minutes, though Trop T take an hour

Disadvantages:
Need for large quantities of pure antibody - must be two sites available for the complex (monoclonal antibodies usually employed)
2 antibody binding sites required (limit range of analysis), can clog the sites so that the complex cant form giving spuriously low results, must add lots of dilution steps.
High dose “hook” effect
Need for multiple washing steps
Non specific interference due to heterophyllic antibodies

Question 8

What are homogenous immunoassays?

Answer 8

Assays that do not require separation are referred to as homogenous immunoassays. No separation step means the assay is simple and easy to automate but it lacks sensitivity.

Examples are:

• Fluorescence polarisation
• Turbidimetry (immu)
• Nephelometry (immu)
• latex agglutination (haemk

Question 9

What is turbidometry?

Answer 9

Measure how much light can reach a parallel detector through the Antibody antigen complex

More complex = cloudier sample

Often used for measuring CRP, analytes that are in higher concentrations

Question 10

What is nephlometry?

Answer 10

Antibody-antigen complex prevents light passing through resulting in light scattering
more scattering = more complex

Question 11

What are heterogenous immunoassays?

Answer 11

Assays that require separation of the bound Ag-Ab complex from the loose unbound antibodies/antigens are referred to as heterogenous immunoassays.

Examples are:
Liquid Phase Separations
	- separation by salts (PEG, ammonium sulphate)
	- Double antibodies 
	- decantation methods
Surface Coated Solid Phases
	- glass and plastic particles
	- magnetizable particles
	- Tubes, wells and micro-titre plates

Question 12

Explain the use of radioactive labels and what are the advantages of using them?

Answer 12

Radioactive labels are more commonly used in research than the clinic.
For example, 125-Iodine, half-life of 60 days (careful not to allow build up when you’re ordering kits in, but also be aware of expiry date so as to not run out).
Detection by gamma-counters, detected as counts per minute
Labelling by direct substitution of 125-I onto the aromatic ring of tyrosine
Or more commonly iodinating a carrier and then coupling the carrier onto the to the antigen

Advantages:
Simple coupling reactions 
Label properties do not alter on coupling
No background signal
Efficient/convenient detection systems
No additional cost to detect signal
Very useful for research assays
BUT THEY CAN’T BE AUTOMATED WHICH IS WHY THEY HAVE SOMEWHAT GONE OUT OF FASHION

Question 13

What are the advantages and disadvantages of non-isotopic labels?

Answer 13

Advantages:
No radioactivity, therefore safety aspects
Easier disposal
Extended life of label - usually a year
Speed of detection - usually a second or milisecond
Ease of automation
Theoretical increase in sensitivity
Possibility of homogeneous assay eg turbidometry

Disadvantages:
Serum/buffer effects
Extra manipulations in detection, if there is an issue with recounting must repeat
Inefficient detection in some cases
No recounting possible in some systems
Limitation of separation system
“Dedicated” instruments to one manufacturer leading to commercial pressures

Question 14

Explain the use of enzymatic assays:

Answer 14

Used to measure Alkaline phosphatase, horse radish peroxidase and β-D-galactosidase

Signal generated can be detected by:

• colour
• fluorescence (Antibody immobilised to magnetic bead and the analyte in the sample. In a competitive immunoassay with a fluorometric end point, the enzyme is measuring, for example, testosterone in the sample. The analyte is testosterone, both enzyme-labelled and not labelled. Both compete for binding sites on the antibody, then wash away those that didn’t bind (heterogeneous assay).Second step involves adding substrate, the enzyme converts the substrate causing it to fluoresce).
• chemiluminescence (Antibody fixed to a magnetised particle, complementary to the analyte in the sample.A second antibody, labelled to acridinium ester, is added. Form antibody antigen particle on the magnetised bead, then wash away excess 2ndary antibody (heterogenous). Add H2O2 and pH adjusting reagent which allow production of luminescent. More analyte more illuminescence).

Question 15

How does the Streptavidin-Biotin Detection System work for competitive assays?

Answer 15

Antibody on solid face. Testosterone and biotin labelled testosterone analytes compete for the antibody. Wash away any excess and add streptavidin enzyme conjugate and the substrate. Strep binds to biotin to make complex due to high affinity. Th substrate can then bind to the enzyme for detection.
Complex, but allows automation to allow faster detection.

Question 16

What does the process of evaluating automated immunoassays involve?

Answer 16

This process of verification must occur every time the manufacturer makes a changes, or when you bring in a new assay. It also must be considered every time you go out for tender.

This involves measuring:
Sensitivity – LoD, LoQ, LofB pmol/L, TSH, PSA
Precision
- lot to lot variation - variability across analysers, tells you if a result has really changed or whether it was just measured on a diff analyser. This can be seen with assays from different lots(some manufacture’s acceptance testing may be up to 10% diff)
- clot detectors (in the pre analysis section)
Linearity (concentration range it can measure up to without dilution and how much you can/should dilute by - will be in the kit inserts - would prefer a large linearity, but this may reduce sensitivity. Often better if the machine can dilute on board, otherwise you must know diluent etc…)
- sometimes difficult to assess
- wide linearity
- diluent, on board dilution
- hook effect
Specificity
- cross reactivity (Eg Cortisol in Cushing’s disease. The drug given blocks the pathway one step above cortisol. The assay can detect the intermediate build up above cortisol as cortisol, so it was reported that the drug wasn’t working)
- interference
- matrix effects (most assays are only calibrated to serum)
Accuracy
- lack of international standards
- calibration, must know where your assay is calibrated to

Question 17

What are the advantages and disadvantages of automated immunoassays?

Answer 17

Advantages:
Improved precision, better pipetting, mixing, timing and separation steps
Greater within lab uniformity, less technique dependable
Greater inter lab uniformity
Decreased turnaround time
Less labour intensive

Disadvantages:
Lack of reagent choice
Total reliance on manufacturer
Lack of range of analytes
Compromises on assay design
Little use for “research” assay
Increased reagent and maintenance costs