1. Labeled Immunoassays Part 2
Enzyme Linked Immunosorbent Assay (ELISA)
Lab. 3

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)
Can be used to detect both antibody and antigen
Very Sensitive (ng & pg/mL),
Relies on Monoclonal Abs
An enzyme is used as an indicator molecule
The enzyme does not provide detection directly but through the break-down of a substrate

4. Enzymes used in ELISA
Enzymes used as labels for immunoassay are chosen according to the following criteria:
Turnover number
Sensitivity
Ease and speed of detection
Stability
Absence of interfering factors in patient samples
Availability and cost of enzyme and substrate
turnover number: the maximum number of molecules of substrate that an enzyme can convert to product per catalytic site per unit of time

5. Enzymes used in ELISA The most commonly used enzymes are:
Horseradish peroxidase (HRP)
Alkaline phosphatase (AP)
Each has a high turnover number (rapid conversion of substrate to a product) resulting in high sensitivity
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

6. Enzyme Substrates
A large selection of substrates is available for performing the ELISA with an HRP or AP conjugate
Substrates for AP and HRP, depending upon the plate-reading equipment available and the level of sensitivity required in the ELISA
An array of, and substrates is available for use with either enzyme
Chromogenic Color spectrophotometer
Fluorogenic Fluoroescence fluorometer
Chemiluminescent light luminometer

7. Enzyme Substrates
Chemiluminescent and chemifluorescent substrates provide a stronger signal than Chromogenic substrates
Steadily gaining in popularity because of their:
sensitivity (less than 1 pg/ml “10-12”),
large linear range for detection
and excellent antibody conservation

8. Enzyme Substrates Chromogenic substrates
A common ELISA substrate for HRP is:
Tetramethylbenzidine (TMB),
TMB is oxidized to yield a blue product that is water-soluble and absorbs light at 650 nm
The reaction can be halted by addition of acid or another stop reagent
Using sulfuric acid turns TMB yellow color which may be read at 450 nm

9. Enzyme Substrates
The most common chromogenic substrate for alkaline phosphatase is:
p-nitrophenyl phosphate (PNPP)
PNPP yields a yellow reaction product that is water-soluble and absorbs light at 405 nm

10. Enzyme Substrates Chemiluminescent substrates
Luminol is one of the most widely used chemiluminescent reagents and its oxidation by peroxidase results in creation of an excited state product called 3-aminophthalate
This product decays to a lower energy state by releasing photons of light
Using chemiluminescence allows multiple exposures to be performed to obtain the best image
Sensitivity: picogram or femtogram level (1.0 × 10-15 grams)

11. Enzyme Substrates Fluorogenic substartes
A Fluorogenic Substrate is a nonfluorescent material that is acted upon by an enzyme to produce a fluorescent compound
Different substartes are also available for AP and HRP enzymes
p-Hydroxphenylpropionic acid (HPPA) is used for HRP enzyme

12. 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
The heterobifunctional cross-linker
Cysteine amino acid

13. Linkage of Enzyme Treatment with periodate
Glycoproteins such as horseradish peroxidase can be activated for conjugation by treatment with periodate
This provides a mild and efficient way of generating reactive aldehyde groups for subsequent conjugation with amine- or hydrazide-containing molecules

14. Linkage of Enzyme The use of biotin- avidin binding
The secondary Ab is labeled with biotin
An enzyme-avidin conjugate is added

15. 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
Hydrophobic interactions describe the relations between water and hydrophobes (low water-soluble molecules). Hydrophobes are nonpolar molecules and usually have a long chain of carbons that do not interact with water molecules.

16. Coating of Microplate
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

17. 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
Analyte = antibody Analyte = antigen
Coated with Antibody when analysing antigen
Coated with Antigen when analysing antibody
Incubate, wash

18. ELISA Protocol Incubate, wash 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 [blocking agent ( μl/well)]
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
Analyte = antibody Analyte = antigen
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.
Incubate, wash

19. ELISA Protocol 3. Add sample. Incubate, Wash
Analyte = antibody Analyte = antigen
4. Add conjugate. Incubate, Wash
Analyte = antibody Analyte = antigen
E E E E

20. ELISA Protocol 6. Incubate, stop, 5. Add substrate
measure color change
5. Add substrate
ENZYME
Colourless OD
Concentration

21. Types of Enzyme Immunoassay
Three main types:
1. Competitive ELISA
2. Indirect ELISA
3. Sandwich ELISA

22. 1- Competitive ELISA such as insulin, and estrogen
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

23. 1- Competitive ELISA
Competition ELISA to detect Antigens (Antibody-coated plate)
1. Anti-analyte
2. Analyte-E
+ sample
Low [analyte] High [analyte]
E E E E E E E
3. Wash
2. Analyte- E
+ sample
1. Anti-analyte
Low [analyte] High [analyte]
4. Substrate
3. Wash
2. Analyte-E
+ sample
1. Analyte
Low [analyte] High [analyte]

24. 2- 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.

25. 2- Indirect ELISA Screening of hybridoma supernatants
Detecting clinically important antibodies (eg. Autoantibodies)
E E E E
3. Anti-(human) Ig-enzyme
2. Sample (human) antibody
1. Antigen
1. Antigen
E E
4. Substrate
3. Anti-(human) Ig-enzyme
2. Sample (human) antibody
1. Antigen
2. Sample (human) antibody
1. Antigen

26. 3- Sandwich ELISA
If the antibody is bound to the solid phase, these assays are called sandwich immunoassays
Antigens captured in these assays must have multiple epitopes
Used for detection of hormones, drugs, tumor antigens, cytokines

27. 3- Sandwich ELISA 3. Anti-analyte-enzyme 2. Sample 1. Anti-analyte
4. Substrate
3. Anti-analyte-enzyme
2. Sample
1. Anti-analyte
2. Sample
1. Anti-analyte

28. Lateral Flow Immunoassay (LFIA)
(Immunochromatography System Assays)
(Membrane based immunodiagnostic assay)

29. Principle
Are based on the migration of nano or micro particles on strips for analytes detection in several areas
In principle, any coloured particle can be used, however latex (blue colour) or nanometer sized particles of gold (red colour) are most commonly used
The technology is based on a series of capillary beds, such as pieces of porous paper
These elements has the capacity to transport fluid (e.g., urine, plasma,..) spontaneously

31. Monoclonal Ab labelled with colored particles
Principle
Immoblized Ab
(Test Line)
support
Sample pad
Monoclonal Ab labelled with colored particles
(Control Line)
While the sample fluid start moving it dissolves particles and in one combined transport action the sample and conjugate mix while flowing through the porous structure

33. Principle
Apply sample solution, upon application of sample biochemicals dissolve
Immobilised
Antibody area
Control area
Negative: no antigen
Positive: antigen present

34. Uses
Human and animal diagnostics Point of Care testing (PoCT)
Forensics
Environmental testing
Advantages
Rapid test (<15 minutes)
Reliable and easy-to-use (no special equipment required nor trained staff)
Semi-quantitative or quantitative results
Non-refrigerated storage

35. Lateral Flow Immunoassay (LFIA)
Detection of HBsAg
Lateral Flow Immunoassay (LFIA)

36. Intended Use
HBsAg One Step Hepatitis B Surface Antigen Test cassette (Serum/Plasma) is a rapid chromatographic immunoassay for the qualitative detection of Hepatitis B Surface Antigen in serum or plasma

37. Summary and explanation
Viral hepatitis is a systemic disease primarily involving the liver, and in most cases is caused by one of three viruses:
Hepatitis A (HAV),
Hepatitis B (HBV)
or Hepatitis C (HCV).
The antigen found on the envelope of HBV is designated Hepatitis B Surface antigen (HBsAg) and its presence in serum or plasma indicates active HBV infection.
In a typical Hepatitis B infection, HBsAg will be detected 2 – 4 weeks before ALT levels become abnormal and 3-5 weeks before symptoms or jaundice develop.

38. Principle
The membrane is precoated with anti-HBsAg antibodies on the test line region of the test
During testing, the serum or plasma specimen reacts with the particle coated with anti-HBsAg antibody
The mixture migrates upward on the membrane chromatographically by capillary action to react with anti-HBsAg antibodies on the membrane and generate a colored line
The presence of this colored line in the test region indicates a positive result, while its absence indicates a negative result

39. Quality Control A procedural control is included in the test
A red line appearing in the control region (C) is the internal procedural control
It confirms sufficient specimen volume and correct procedural technique (membrane wicking has occurred)

40. Limitations
LINEAR HBsAg cannot detect less than 1 ng/mL of HBsAg in specimens
If the test result is negative and clinical symptoms persist, additional follow-up testing using other clinical methods is suggested
A negative result at any time does not preclude the possibility of Hepatitis B infection

41. Procedure
Allow the test strip to equilibrate to room temperature prior to testing
Place the test device on a clean and level surface. Add 100 µl of serum or plasma to the specimen well (S) and then start the timer.
Wait for the red line (s). The result should be read at 15 minutes

42. Interpretation
Control line
Test line
Positive
Negative
Invalid

43. The End