1. Labeled Immunoassays

2. Labeled Immunoassays
Some antigen/antibody reactions not detected by precipitation or agglutination.
Looking for very small amounts.
Measured indirectly using a labeled reactant.
Referred to as receptor-ligand assays.

3. Terminology Ligand is the substance to be measured.
Defined as a molecule that binds to another molecule of a complementary configuration.
Usually binds to the substance the test is trying to detect.
The receptor is what binds the specific target molecule.

4. Terminology Reactions may be competitive or non-competitive
Competitive – labeled known and patient unknown are added to reaction and “compete” for the target.
For example, looking for an antibody.
Add labeled reagent antibody of known specificity, patient sample and known antigen.
Patient antibody competes with reagent antibody for the target antigen.
Concentration is inversely proportional to results.

5. Terminology Non-competitive
Add patient sample, for example looking for antibody, to known reagent antigen.
Reaction occurs and the concentration is directly related to the amount of antibody in patient sample.

6. Terminology Heterogeneous or homogeneous
Heterogeneous assays called separation assays
Require multiple steps
Careful washing of surface to remove unbound reagents and samples.
Homogeneous assays do NOT require a separation step.
Mix reagents and patient sample.
Measure the labeled product.

7. Homogeneous VS Heterogeneous Methods

8. Standards or Calibrators
Substance of exact known concentration.
Usually run for each new lot number
Based on results create standard curve.
Standard curve used to “read” results or built into machine to provide results.

9. Labels Used to detect reaction which has occurred. Most common are:
Radioactive
Enzymes
Fluorescent
Chemiluminescent

10. Radioimmunassay (RIA)
Competitive binding
Sensitive technique used to measure small concentrations of antigens.
Known quantity of antigen is made radioactive, usually with Iodine 125.
Known labeled antigen and patient sample added to the reagent antibody.
Known antigen will compete with the unknown patient antigen for sites on the antibody.
Radioactive label competes with patient for sites
High radioactivity, small amount of patient substance
Low radioactivity high amount of patient substance.

11. Radioimmunoassay
Thus, the results are inversely related to the label detected.
Standards are run and results read off of standard curve.
The bound antigens are separated from the unbound ones.
Can measure the radioactivity of labeled free antigen in the supernatant solution.
Can measure radioactivity of fixed labeled antigen to the well.

12. Radioimmunoassay

13. Radioimmunassay

14. Immunoradiometric Assay (IRMA)
Labeled antibody plus patient antigen
Solid phase antigen added to bind excess antibody.
Labeled antibody binds to both patient antigen, if present, and bound antigen.
Spin down to separate
Labeled antibody/antigen remain in solution.
Measure radioactivity.

15. IRMA
In IRMA, the antibodies are labeled with radioisotopes which are used to bind antigens present in the specimen.
When a positive sample is added to the tubes, radioactively labeled (labeled with I125 or I131 radioisotopes) antibodies bind to the free epitopes of antigens and form an antigen- antibody complex.
Unbound labeled antibodies are removed by a second reaction with a solid phase antigen.
The amount of radioactive remaining in the solution is directly proportional to the antigen concentration.

16. Advantages/Disadvantages
Extremely sensitive and precise
Detects trace amounts of analytes small in size.
Disadvantages
Health hazard
Disposal problems
Short shelf life
Expensive equipment necessary
Enzyme immunoassays have largely replaced radioimmunoassay.

17. Enzyme Immunoassay
Enzymes occur naturally and catalyze biochemical reactions.
Enzymes are
Cheap
Readily available
Have a long shelf life
Easily adaptable to automation.
Automation relatively inexpensive.
Techniques pose no health hazards.
Little reagent enzyme necessary.
Can be used for qualitative or quantitative assays.

18. Enzyme Immunoassay Enzymes selected according to Enzymes used include:
Substrate molecules converted per molecule of enzyme.
Ease and speed of detection.
Stability.
Availability and cost
Enzymes used include:
Horseradish peroxidase
Glucose-6-phosphate dehydrogenase
Alkaline phosphatase
Β-D-galactosidase
Horseradish peroxidase and alkaline phosphatase are the most popular.
Highest turnover
High sensitivity
Easy to detect

19. Types of Enzyme Immunoassay
Heterogeneous enzyme immunoassay
Competitive ELISA
Non competitive ELISA
Immunoenzymometric assay (Indirect)
Sandwich or capture ELISA
Homogenous enzyme immunoassay

20. Heterogenous EIA Competitive
Enzyme labeled antigen competes with unlabeled patient antigen for antibody sites.
Wash to remove unbound reactants.
Add substrate which causes color change.
Results are inversely proportional to concentration.
More patient antigen bound, less color.
If little or no patient antigen bound, dark color.
Used to measure small antigens such as insulin and estrogen.
Enzyme produces color reaction

21. Heterogenous EIA Noncompetitive Noncompetitive are very popular.
Often referred to as enzyme linked immunosorbent assay – ELISA
Enzyme labeled reagent DOES NOT participate in the initial antigen-antibody reaction.

22. Noncompetitive EIA Variety of solid support Microtiter plates
Nitrocellulose membranes
Magnetic beads
Advantages
High sensitivity and specificity.
Relatively simple to perform.
Low cost.

23. Immunoenzymometric assay (Indirect)
Antigen bound to solid phase
Add patient sample, antibody will bind if present
Wash
Add known enzyme labeled antibody (anti Fc portion)
Add substrate
Measure enzyme label

24. Sandwich or Capture Assays
Antibody bound to solid phase.
If looking for antigen must have multiple epitopes, bound antibody specific for one epitope, second labeled antibody added specific for a different epitope.
Antigens detected can be
Antibodies
Hormones
Proteins
Tumor markers
Microorganisms especially viruses
Enzyme label used to detect reaction

25. Sandwich or Capture Assays
Add patient sample with antigen.
Antigen will bind to antibody bound to solid phase.
Add enzyme labeled antibody directed against a different epitope on the antigen.
Add substrate, measure intensity of color.

27. Homogenous immunoassay
Immunoassays those that do not require separation step.
An antigen is labelled by an enzyme.
When antibody reacts with the labelled antigen the enzyme is rendered inactive.
Free antigen completes labelled antigen for the antibody binding site preventing inhibition of enzyme activity.
Enzyme activity is thus directly proportional to the concentration of the free antigen

28. Homogenous immunoassay
Less sensitive BUT rapid, easy to perform and automate.
Chief use is to detect low molecular weight analytes such as:
Hormones
Therapeutic drugs
Drugs of abuse
Can use serum or urine.

29. Chemiluminescent Immunoassays
The process of chemiluminescence occurs when energy in the form of light is released from matter during a chemical reaction. 

30. Chemiluminescent Immunoassays
Large number of molecules capable of chemiluminescence
Luminol
Acridium esters
Ruthenium derivatives
Nitrophenyl oxalates
Use sodium hydroxide as a catalyst
Light emission ranges from quick burst or flash to light which remains for a longer time.
Different types of instruments are required based on emission.

31. Chemiluminescent Immunoassays
Can be used for heterogeneous or homogeneous assays.
Can attach label to antigen or antibody.
Heterogeneous assays use competitive and sandwich assay.
Competitive assays used to measure smaller analytes.
Sandwich assays are used to measure larger analytes.
Many applications.
Can measure antigen or antibody.
Add chemiluminescently tagged analyte.
Measure light which is emitted which is directly related to concentration although competitive binding assays are available.

32. Chemiluminescent Immunoassay

33. Fluorescent Immunoassay
will be discussed in the following lecture