1. Labeled Immunoassays Part 2
Enzyme Linked Immunosorbent Assay (ELISA)

2. Labeled Immunoassays
The basic underlying principles of indicator labeled immunoassays are the same.
There are differences with respect to the detail of the protocols.
The designation given to each test differs according to the label used to detect the antigen/ antibody complexes.

3. Enzyme Linked Immunosorbent Assay (ELISA)
One of many assays collectively called enzyme immunoassays (EIA).
The enzyme used an indicator molecule.
The enzyme does not provide detection directly.
The action of the enzyme on a substrate that results in a change in the properties of the solution.
The substrate is brokendown by the enzyme to produce chromogenic, fluorogenic or luminescent products.

4. ELISA Can Be Used To Detect Both Antibody and Antigen
Very Sensitive, ng & pg/mL
Relies on Monoclonal Abs

5. Enzymes used in ELISA
Enzymes used as labels for immunoassay are chosen according to the following criteria:
number of substrate molecules converted per molecule of enzyme
purity
sensitivity
ease and speed of detection
stability
absence of interfering factors in patient samples
in addition, availability and cost of enzyme and substrate.

6. Enzymes used in ELISA The most commonly used enzymes are:
horseradish peroxidase (HRP)
and alkaline phosphatase (AP).
Each has a high turnover number (rapid conversion of chromogenic substrate to coloured product) resulting in high sensitivity.
An array of chromogenic, fluorogenic and chemiluminescent substrates is available for use with either enzyme.
Other enzymes have been used as well, but they have not gained widespread acceptance because of limited substrate options.
These include beta-galactosidase, acetylcholinesterase and catalase.

7. Enzyme Substrates
A large selection of substrates is available for performing the ELISA with an HRP or AP conjugate.
A common ELISA substrate for HRP:
tetramethylbenzidine (TMB),
TMB yields a blue reaction product that is water-soluble and absorbs light at 450 nm.
The most common ELISA substrate for alkaline phosphatase:
the chromogen p-nitrophenyl phosphate (PNPP)
PNPP yields a yellow reaction product that is water-soluble and absorbs light at 405 nm.

8. Linkage of Enzyme
The enzyme label is linked to antibody or analyte by several means.
Glutaraldehyde is often used as a crosslinker to join amino groups of the enzyme and the molecule to be labeled.
Maleimide derivatives are also used to attach the enzyme label.
Cysteine amino acid

9. Coating of microplate
A key feature of the solid-phase ELISA is that antigens or antibodies can be attached to surfaces easily by passive adsorption.
This process is commonly called coating.
Most proteins adsorb to plastic surfaces, probably as a result of hydrophobic interactions between nonpolar protein substructures and the plastic matrix.
Since most of proteins' hydrophilic residues are at the outside and most of the hydrophobic residues orientated towards the inside.
Partial denaturation of some proteins results in exposure of hydrophobic regions and ensures firmer interaction with the plastic.
This can be achieved by exposing proteins to low pH or mild detergent

10. ELISA Protocol
1- Coating antibody or antigen onto the microplate
Dilute the protein to be coated in a buffer such as PBS or Carbonate-Bicarbonate and add 100 μl of this solution per well.
Incubate for hours at room temperature or 4°C.
Block unoccupied sites with a blocking agent ( μl/well) Store plate at 4°C with a desiccant for future use.

11. ELISA Protocol 1- Coat solid phase with
antigen when analysing antibody
antibody when analysing antigen
Analyte = antibody Analyte = antigen
Incubate, wash

12. (eg. AP + Blocking Buffer in TBS, PBS interferes with AP)
ELISA Protocol
2- Blockage of free binding sites
Block the unoccupied sites on the surface of the well to reduce the amount of nonspecific binding of proteins
A variety of blocking buffers ranging from nonfat milk to highly purified proteins have been used to block unreacted sites.
The blocking buffer should improve the sensitivity of the assay by reducing the background interference.
An individual blocking buffer will not be compatible with every system; a variety of blockers in both Tris buffered saline (TBS) and phosphate buffered saline (PBS) are available.
The proper choice of blocker for a given assay depends on:
the antigen itself
and on the type of enzyme conjugate to be used.
(eg. AP + Blocking Buffer in TBS, PBS interferes with AP)
For example, with applications using an alkaline phosphatase conjugate, a blocking buffer in TBS should be selected because PBS interferes with alkaline phosphatase. The ideal blocking buffer will bind to all potential sites of nonspecific interaction, eliminating background altogether, without altering or obscuring the epitope for antibody binding.

13. ELISA Protocol 2- Block free binding sites. Incubate. Wash.
Analyte = antibody Analyte = antigen

14. ELISA Protocol 3. Add sample. Incubate. Wash
Analyte = antibody Analyte = antigen

15. ELISA Protocol 4. Add conjugate. Incubate. Wash. E E E E
Analyte = antibody Analyte = antigen

16. 6. Incubate, stop, measure colour change
5. Add substrate
6. Incubate, stop, measure colour change
ENZYME
Colourless OD
CONCENTRATION

17. Types of Enzyme Immunoassay
A- Heterogenous Enzyme Immunoassay
Three main methods:
1. Competitive ELISA
2. Indirect ELISA
3. Sandwich ELISA

18. 1- Competitive ELISA
Enzyme activity is inversely proportional to the concentration of the test substance.
A sensitivity of nanograms (10-9 g)/ml can be achieved.
This method can be used for measurement of small molecules that are relatively pure,
such as insulin
and estrogen.

19. 1- Competitive ELISA COMPETITION ELISA TO DETECT ANTIGENS
(antibody-coated plate)
1. Anti-analyte

20. 1- Competitive ELISA Low [analyte] High [analyte] 2. Analyte-E
+ sample
1. Anti-analyte

21. 1- Competitive ELISA Low [analyte] High [analyte] 3. Wash 2. Analyte-E
+ sample
1. Anti-analyte
E E E E E E E

22. 1- Competitive ELISA Low [analyte] High [analyte] 4. Substrate 3. Wash
2. Analyte-E
+ sample
1. Analyte

23. 2- Noncompetitive ELISA or Indirect ELISA
Indirect ELISA is more sensitive than the competitive.
Much assays are capable of detecting concentrations of less than 1 pg/ml (10-12 g)/ml.

24. 2- Indirect ELISA screening hybridoma supernatants
detecting clinically important antibodies
- autoantibodies
- anti-pathogens
- anti-allergens
1. Antigen

25. 2- Indirect ELISA
2. Sample (human) antibody
1. Antigen

26. 2- Indirect ELISA 3. Anti-(human) Ig-enzyme 2. Sample (human) antibody
1. Antigen E E E
E E E

27. 2- Indirect ELISA 4. Substrate E E E 3. Anti-(human) Ig-enzyme
2. Sample (human) antibody
1. Antigen
E E E

28. 3- Sandwich ELISA If the antibody is bound to the solid phase,
These assays are called sandwich immunoassays or capture assays
Antigens captured in these assays must have multiple epitopes.

29. 3- Sandwich ELISA eg. hormones drugs tumour antigens cytokines
1. Anti-analyte

30. 3- Sandwich ELISA
2. Sample
1. Anti-analyte

31. 3- Sandwich ELISA E E
3. Anti-analyte-enzyme
2. Sample
1. Anti-analyte

32. 3- Sandwich ELISA E S 3. Or: anti-analyte-biotin followed by
streptavidin-enzyme E S
E-S B S-E
E-S B S-E
2. Sample
1. Anti-analyte

33. ANTIBODY SANDWICH ELISA TO DETECT ANTIGENS
4. Substrate
3. Or:
anti-analyte-biotin
followed by
streptavidin-enzyme
2. Sample
1. Anti-analyte

34. Membrane based cassette assays
This type of ELISA is
rapid,
easy to perform
and give reproducible results.
Designated primarily for primary care clinics and home testing
Many of these have been modified for increased sensitivity and can be made semiquantitative for use in a clinical laboratory by reading the color reaction with a densitometer.

35. Membrane based cassette assays(Dipsticks)
Apply sample solution, upon application of sample biochemicals dissolve
Positive: no antigen
Immobilised
Antibody area
Control area
Negative: antigen present
Immunochromatography
Biochemical components are separated across an absorbent membrane into discrete distinct regions.

36. QUALITATIVE TEST Control support Sample pad Immoblized Ab + substrate
Monoclonal Ab labeled
with enzyme
Immoblized Ab
+ substrate
support
Sample pad

38. B- Homogenous Enzyme Immunoassay
Homogeneous methods have been generally applied to the measurement of small analytes such as abused and therapeutic drugs.
Since homogeneous methods do not require the separation of the bound Ab-Ag* from the free Ag*, they are generally much easier and faster to perform.
Homogenous methods are generally less sensitive than heterogeneous assays.
They are based on the principle of change in enzyme activity as specific antigen-antibody combination occurs.
Enzymatic activity can be altered by steric exclusion of the substrate, or there may be changes in the conformation structure of the enzyme.
Enzymes of this type include lysozyme, amaylase, trypsin and others.

39. 1- Enzyme-multiplied immunoassay
It is based on the principle of change in enzyme activity as specific antigen-antibody combination occurs. 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. A physical separation of bound and free analyte is thus not necessary.

40. Advantages & Disadvantages
Advantages of ELISA
High sensitivity
No need for expensive instruments
Reagents are inexpensive and have a long shelf time
No disposal problems, or health hazard of radiation
Disadvantages of ELISA
Some specimens may contain natural inhibitors
Size of enzyme may be a limiting factor in the design of some assays
Nonspecific binding and sensitivity of enzymes to temperature

41. Toxoplasma gondii
Toxoplasma gondii is a small intracellular parasite, whose life cycle has a sexual and an asexual phase.
Sexual development is restricted to the intestinal cells of (probably exclusively) cats; the oocysts formed are excreted and due to their resistant cell walls they may be infectious under advantageous circumstances for at least 1 year.
Animals and man are intermediate hosts for the asexual proliferation of T. gondii: the ingested parasites will proliferate explosively within the host cells lysing them eventually.
They disseminate throughout the body via circulation and lymphatic system and may infect any cell type. Cysts are virtually impossible to be killed in the intermediate host.

42. Toxoplasma gondii
Most T. gondii infections are asymptomatic or present with a transient
febrile illness, often with lymphadenopathy.
Primary infections acquired during pregnancy can cause
stillbirth or congenital abnormalities,
including blindness,
encephalomyelitis,
impaired brain functions
and convulsions in the newborn.
Infections can also be more severe in individuals who are infected with HIV or who are otherwise immunocompromised.
Toxoplasmosis can be treated with antibiotics but early detection of the disease is crucial for effective therapy.

43. Test Principle Microtiter wells are coated with antigens.
This constitutes the solid phase.
Patient's sample is added to the wells and any antibodies specific for the antigen present will bind to the solid phase.
After removal of unbound material, anti-human IgG or IgM conjugated to an enzyme (alkaline phosphatase) is allowed to react with the immune complex.
After removal of excess conjugate by washing, an appropriate substrate (paranitrophenylphosphate) is added, with which the conjugated enzyme reacts producing a coloured derivative of the substrate.
The colour intensity is proportional to the level of specific antibody bound and can be quantified photometrically.

44. Test procedure
Place the required number of cavities in the frame and prepare a protocol sheet.
Prepare the diluted washing solution
Wash microplate strips once with working washing solution. Remove remaining liquid by tapping on absorbent paper
Dilute test sera by adding 10 µl of sample to 1 ml of diluent
Calibrators 0, 6, 60 and 240 IU/ml are diluted in the same manner
Add each 200 μl of diluted sample or calibrators into the appropriate wells
well A1 0 IU/ml
well B1 6 IU/ml
well C1 60 IU/ml
well D1 240 IU/ml
well E1, F1, G1, etc µl of diluted sample

45. Test procedure
Sample incubation for 60 minutes (+/- 5 min) at 37°C (+/- 1°C).
Prepare working conjugate 15 minutes before the end of first incubation period
After incubation wash all wells with washing solution (by automated washer or manually):
aspirate or shake out the incubation solution
fill each well with 300 μl washing solution
aspirate or shake out the washing buffer
repeat the washing procedure 2 times (altogether 3 times!)
dry by tapping the microtest plate on a paper towel

46. Test procedure Addition of conjugate Addition of substrate
Add 200 μl of working-conjugate to the appropriate well
Conjugate incubation for 60 minutes (+/- 1 min) * at 37°C (+/- 1°C)
After incubation wash all wells 4 times with washing solution (see above)
Addition of substrate
Add 200 μl substrate solution to each well
Substrate incubation for 30 minutes (+/- 1 min) * at 37°C (+/- 1°C) in the dark

47. Test procedure Stopping of the reaction Read optical density
Add 100 μl stopping solution to each well, shake microtest plate gently to mix.
Read optical density
Read OD within 30 minutes at 450 nm against substrate blank

48. Results
The presence and quantity of IgG antibodies to T. gondii in the test sample is determined by comparing the optical density (OD) of the test sample to a standard range.
For validation of the test the following criteria must be met:
OD values
OD R3 ≤ 0.200
OD R4c ≥ 1.000
Ratio:
OD R4a / OD R3 ≥ 2

49. Results
After drawing the curve from the concentration of standards on the x-axis with absorbance of standards on the y-axis, find the value corresponding to the OD reading of the test sample on the y-axis and draw a horizontal line to the standard curve. At the point of intersection with the standard curve draw a vertical line to the x-axis. Read the concentration in IU/ml at the point of intersection with the x-axis.
Interpretation of results
Negative result: titer < 6 IU/ml
Positive result: > 9 IU/ml