This document discusses various types of labeled immunoassays, including fluorescent, radioactive, chemiluminescent, and enzyme labels. It describes the key characteristics and procedures for competitive and noncompetitive assays. Specific enzyme immunoassay techniques are covered in detail, such as competitive and noncompetitive ELISA, capture (sandwich) assays, rapid immunoassays, and homogeneous assays. The advantages of enzyme immunoassays include safety compared to radioimmunoassays and the ability to perform assays without expensive instrumentation. Challenges include potential interference from inhibitors or nonspecific binding.

1. Done by : Ala’a Abdullah
Supervised by : Dr. Hassan Abu-al Ragheb

2. Labeled Immunoassays
Designed for antigen and antibody that may be small in size
or present in very low concentration.
Current techniques include the use of Fluorescent,
radioactive, chemiluminescent and enzyme labels.

3.  The label must not alter the reactivity of the molecule.
 It should remain stable for the shelf life of the reagent.
 For radioimmunoassay, this involves a system for counting radioactivity,
while for other labels such as enzymes, fluorescence, or
chemiluminescence, typically a change in absorbance in a substrate is
measured by spectrophotometry.
 There are two major formats for all labeled assays: competitive and
noncompetitive.
Characteristic of labeled assay

4. Competitive versus Noncompetitive Assays
 In a competitive immunoassay, all the reactants are mixed together
simultaneously, and labeled antigen competes with unlabeled patient
antigen for a limited number of antibody-binding sites. The amount of
bound label is inversely proportional to the concentration of the labeled
antigen. This means that the more label detected, the less there is of patient
antigen.
 In a typical noncompetitive immunoassay, antibody, often called
capture antibody, is first passively absorbed to a solid phase. Unknown
patient antigen is then allowed to react with and be captured by the
antibody. After washing to remove unbound antigen, a second antibody
with a label is added to the reaction. In this case, the amount of label
measured is directly proportional to the amount of patient antigen.

5. Separation Methods
 A partitioning step, or a way of separating reacted from unreacted analyte .
 Most immunoassays use a solid-phase ( polystyrene test tubes, microtiter plates,
glass or polystyrene beads, magnetic beads, and cellulose membranes) for
separation.
 Antigen or antibody is attached by physical adsorption, and when specific binding
takes place, complexes remain attached to the solid phase.
 The bound and unbound fractions are usually separated by physical means,
including decanting, centrifugation, or filtration. This is followed by a washing
step to remove any remaining unbound analyte.

6. ENZYME IMMUNOASSAY
 Enzymes used as labels for immunoassay are typically chosen according to
the number of substrate molecules converted per molecule of enzyme, ease
and speed of detection, stability, availability and cost of enzyme and
substrate.
 Enzyme labels can either be used qualitatively to determine the presence
of an antigen or antibody or quantitatively to determine the actual
concentration of an analyte in an unknown specimen.
 Typical enzymes that have been used as labels in colorimetric reactions
include horseradish peroxidase, glucose-6-phosphate dehydrogenase,
alkaline phosphatase, and β-D-galactosidase.
 Alkaline phosphatase and horseradish peroxidase have the highest
turnover (conversion of substrate) rates, high sensitivity, and are easy to
detect, so they are most often used in such assays.

7. ENZYME IMMUNOASSAY
 Enzyme assays are classified as either heterogeneous or homogeneous on
the basis of whether a separation is necessary:
 Heterogeneous enzyme immunoassays require a step to physically
separate free from bound analyte.
- Competitive EIA
- Noncompetitive EIA
- Capture Assays (Sandwich Immunoassays)
- Rapid Immunoassays
 In homogeneous immunoassays, on the other hand, no separation step
is necessary, because enzyme activity diminishes when binding of antibody
and antigen occurs.

8. Competitive EIA
 EIA is a competitive assays based on the principles of RIA.
 Enzyme-labeled antigen competes with unlabeled patient antigen for a limited
number of binding sites on antibody molecules that are attached to a solid phase.
 After carefully washing to remove any nonspecifically bound antigen, enzyme
activity is determined.
 Enzyme activity is inversely proportional to the concentration of the test
substance, meaning that the more patient antigen is bound, the less enzyme-
labeled antigen can attach.
 This method is typically used for measuring small antigens that are relatively pure,
such as insulin and estrogen.

9. Competitive EIA

11. Noncompetitive EIA
 The tendency in the laboratory today is toward the use of noncompetitive assays,
because they have a higher sensitivity (less than 1 pg/Ml), specificity ,
simplicity, and low cost.
 Noncompetitive assays are often referred to as indirect enzyme-linked
immunosorbent assays (ELISA), because the enzyme-labeled reagent does not
participate in the initial antigen–antibody binding reaction.
 A variety of solid-phase supports are used, including microtiter plates,
nitrocellulose membranes, and magnetic latex beads.
 The amount of enzyme label detected is directly proportional to the amount of
antibody in the specimen.

12. General ELISA Procedure
1. When antigen is bound to solid phase, patient serum with
unknown antibody is added and given time to react.
2. After a wash step, an enzyme-labeled antiglobulin is added.
This second antibody reacts with any patient antibody that is
bound to solid phase.
3. If no patient antibody is bound to the solid phase, the second
labeled antibody will not be bound. After a second wash step,
the enzyme substrate is added.
4. The amount of enzyme label detected is directly
proportional to the amount of antibody in the specimen.

13.  The reaction is allowed to progress for a defined period after which the reaction
is stopped by altering the pH of the system.
 Stop Solution is a used to terminate the enzyme substrate reaction for ELISA
applications after attaining the desired color intensity which is an indication of
analyte level.
 For eg. The TMB substrate reacts with immobilized horseradish peroxidase
(HRP) conjugated antibodies to produce a blue solution. Reaction may be
stopped by 0.2 M sulphuric acid which offers a yellow end product read at 450
nm.
 Alkaline phosphates stop solution (0.5M NaOH) does not change the yellow
color or the absorbance of the chromogen, and so the absorbance is read at 405
nm to 420 nm.
Stop solution

14.  Noncompetitive ELISA. Patient
antibody is incubated with solid-phase
antigen.
After a wash step, enzyme-labeled
antiimmunoglobulin is added. This will
bind to the patient antibody onsolid
phase.
A second wash step is performed to
remove any unbound anti-
immunoglobulin, and substrate for the
enzyme is added.
Color development is directly
proportional to the amount of patient
antibody present.

15. Noncompetitive EIA

16. ELISA Applications
 Used to measure antibody production to infectious agents that are difficult
to isolate in the laboratory and has been used for autoantibody testing.
 Viral infections especially are more easily diagnosed by this method than
by other types of testing.
 This technique remains the preferred screening method for detecting
antibody to HIV, hepatitis A, and hepatitis C.
 ELISA-based tests are also used to identify Epstein-Barr–specific
antibodies produced in infectious mononucleosis.

17. Capture Assays (sandwich immunoassays)
 If antibody is bound to the solid phase, Antigens captured in these assays must
have multiple epitopes.
 Capture assays are best suited to antigens that have multiple determinants, such
as antibodies, polypeptide hormones, proteins, tumor markers, and
microorganisms, especially viruses.
 Rotavirus in stool and respiratory syncytial virus in respiratory tract secretions are
two examples of capture assays.
 It much easier to detect parasites such as Giardia lamblia and Cryptosporidium in
the stool.
 Useful for identifying fungi such as Aspergillus, Candida, and Cryptococcus.

18. To Utilize This Assay:
1 . One antibody (the ‘capture’ antibody) is purified and bound to a solid phase
typically attached to the bottom of a plate well.
2. Antigen is then added and allowed to complex with the bound antibody.
3 . Unbound products are then removed with a wash, and a labeled second
antibody (the ‘detection’ antibody) is allowed to bind to the antigen, thus
completing the “sandwich”.
The second antibody may recognize the same or a different epitope than the solid-
phase antibody
4 . The assay is then quantitated by measuring the amount of labeled second
antibody bound to the matrix, through the use of a colorimetric substrate.

19. Patient Ag
Enzyme-labeled antibody
 Patient antigen is incubated with solid-phase antibody. After washing,
enzyme-labeled immunoglobulin is added, which combines with additional
determinant sites on the bound patient antigen. After a second wash step,
substrate for the enzyme is added. Color development is directly
proportional to the amount of patient antigen present.

21. Another major use of capture assays is in the measurement of immunoglobulins,
especially those of certain classes. instance, the presence of IgM can be specifically
determined, thus indicating an acute infection. Measurement of IgE, including
allergen-specific IgE, which appears in minute quantities in serum, can also be
accomplished with this system.
When capture assays are used to measure immunoglobulins, the specific
immunoglobulin class being detected is actually acting as antigen, and the
antibody is antihuman immunoglobulin.
Indirect ELISA tests are more sensitive than their direct counterparts, because all
patient antigen has a chance to participate in the reaction, there is more manipulation
than in direct tests, because there are two incubations and two wash steps.
Capture Assays (sandwich immunoassays)

22. Rapid Immunoassays
Membrane-Based Cassette Assays
Are a relatively new type of enzyme immunoassay, rapid, easy to perform , and give
reproducible results.
Typically these are designed as single-use, disposable assays in a plastic cartridge.
The membrane is usually nitrocellulose, which is able to easily immobilize proteins
and nucleic acids.
The rapid flow through the membrane and its large surface area enhance the speed and
sensitivity of ELISA reactions.
Either antigen or antibody can be coupled to the membrane, and the reaction is read
by looking for the presence of a colored reaction product.

23. The analyte is applied at one end of the strip and migrates toward distal end,
where there is an absorbent pad .
The labeling and detection zones are set between the two end.
As the sample is loaded, it reconstitutes the labeled antigen or antibody, and
the two form a complex that migrates toward the detection zone.
An antigen or antibody immobilized in the detection zone capture the immune
complex form colored line for positive test.
Excess labeled immunoreactant migrates to the absorbent pad.
This type of test device has been used toIdentify microorganisms such as
Streptococcus pyogenes and Streptococcus agalactiae .Test for pregnancy, for
troponin in a heart attack For hepatitis B surface antigen.
Immunochromatography

25. Homogeneous Enzyme Immunoassay
 Homogeneous assays are generally less sensitive than heterogeneous assays, but
they are rapid, simple to perform, and adapt easily to automation.
 No separation step is necessary, no washing steps are necessary.
 Their chief use has been in the determination of low-molecular-weight analytes
such as hormones, therapeutic drugs, and drugs of abuse in both serum and
urine.
 An example of a homogeneous immunoassay is the enzyme multiplied
immunoassay technique (EMIT) developed by the Syva Corporation.
 Homogeneous assays are based on the principle of change in enzyme activity as
specific antigen–antibody combination occurs.

26.  Reagent antigen is labeled with an enzyme tag.
 When antibody binds to specific determinant sites on the antigen, the active
site on the enzyme is blocked, resulting in a measurable loss of activity.
 Free analyte (antigen) competes with enzyme-labeled analyte for a limited
number of antibody-binding sites, so this is a competitive assay.
 Enzyme activity is directly in proportion to the concentration of patient
antigen or hapten present in the test solution.
 General procedure :

27. The sensitivity of this method determined by:
1. Detectability of enzymatic activity.
2. Change in that activity when antibody bind to antigen.
3. Strength of antibody binding.
4. Susceptibility of the assay to interfernce from cross reacting
antigen ,enzyme inhibitor.

28. Advantages and Disadvantages
of Enzyme Immunoassay

29. I. Enzyme immunoassays have achieved a sensitivity similar to that of RIA without creating
a health hazard or causing disposal problems.
II. The use of non isotopic enzyme labels with high specific activity in noncompetitive assays
increases sensitivity and does so using shorter incubation times than the original RIAs.
III. There is no need for expensive instrumentation, because most assays can be read by
spectrophotometry or by simply noting the presence or absence of color.
IV. Reagents are inexpensive and have a long shelf life.

30. I. Some specimens may contain natural inhibitors.
II. The size of the enzyme label may be a limiting factor in the design of some
assays.
III. Nonspecific protein binding is another difficulty encountered with the use of
enzyme labels.

32. Thank you for being good
listeners