enzyme linked immuno assay

1. ELISA

2. ELISA
ELISA= enzyme linked immunosorbant assay
ELISA or EIA is an immunoassay which uses
enzyme labeled antibodies for the quantitatve
measurement of antigen or antibody mostly in
a solid phase

3. Immunoassay
An immunobiochemical reaction with the help
of which we can measure(both qualitatively or
quantitaively) the levels of an antigen or an
antibody in a test sample.

4. Antigen

5. Hapten

6. Antibody

8. Solid phase
• Agarose gel
• Polyacrylamide
• Plastic beads
• Polystyrene
• Iron oxide coated particles
• Streptavidin
• NeutrAvidin

9. StreptAvidin
• Streptavidin is a tetrameric protein,
which can bind four biotins per one
molecule; each monomer binds one
molecule. (Figure 1)
• Biotin binds to streptavidin with a
very high affinity ( Kaff = 1013 M-1 )
• Streptavidin/biotin complexes are
very stable over a wide range of
temperature and pH
• Streptavidin doesn’t have any
electrical charge in neutral or slightly
basic; it doesn’t cause for example
unspecific electrostatic binding
• Because the binding of compounds on
streptavidin plates is based on the
strong affinity of streptavidin towards
biotin, the compounds must be
biotinylated prior to use

10. NeutrAvidin
• Near-neutral isoelectric point (6.3) and no
glycosylation, unlike avidin
• No RYD recognition sequence like streptavidin
• Generally lower nonspecific binding than
avidin and streptavidin

11. Enzyme labeling
Peroxidase
Horseradish plant
MW= 40,000
Turnover rate = 10,000
Periodate oxidation(Nakane method)
TMB =Tetramethylbenzidine
ABTS =Azino-bis ethylbenzthiazoline
sulfonic acid
HPPA=Hydroxyphenylpropionic Acid ,
Alkaline phosphatase
Bovine intestine
MW=100,000
Turnover rate=250,000
Glutaraldehyde method
pNPP = p-Nitrophenyl phosphate
MUP = 4-Methylumbelliferyl
phosphate
Beta-galactosdase
E. Coli
MW=530,000
Turnover rate=318,000
Dimaleimide method

12. Classification of immunoassays
• Precipitation IA
• Particle IA
• Radioimmunoassay
• Enzyme linked IA
• Fluorescence IA
• Chemiluminescence IA

13. Types of ELISA
Heterogenous
• Direct
• Indirect
• Sandwich
• Competitive
• Multiplex
• Fluorescent Enzyme
Immunoassay
• Chemiluminescent Enzyme
Immunoassay(CL-EIA)
Homogenous
• Mention ahead

14. Direct ELISA

15. Direct ELISA
Advantages
• Quick
• No cross reactivity
Disadvantages
• Reduced immunoreactivity
• Expensive

16. Indirect ELISA

17. Indirect ELISA
Advantages
• More sensitive
• More imunoreactive
primary antibodies
• Versatile as labelled
secondary ab shoudnt
be antigen specific
Disadvantages
• Less specific due to
cross reactivity by sec.
ab.
• Time consuming bcz of
extra incubation step

18. Sandwich ELISA

19. Sandwich ELISA
Advantages
1.Can measure very small
amount of an antigen
2.Antigen purification is
not required
3.Very specific
4. Signal amplification
Disadvantages
1.Multivalent antigens only
2.Match pairing should be
done to avoid hindrance of
binding sites.

20. Competitive ELISA

21. Competitive ELISA
Advantages
• Suitable for complex
samples, since the antigen
does not require
purification prior to
measurement
• Flexibility and sensitivity,
since both direct and
indirect detection methods
can be used
• No hook effect
• No match pairing is required
Disadvantages
• Cross reactivity

22. Standard curve for noncompetitive
ELISA

23. Fish Hook effect
Tumor markers
hCG
Ferretin
CRP
Dilution
Competitive
Beer’s law

24. Cross reactivity
Indirect
competitive

25. The Matrix effect
• Serum or plasma sample is a complex compound of
lipids, proteins, carbohydrates, salt and water. The
sum of interferences of all sample components (with
exception of analytes), which affect the target
analyte to be measured is known as “the matrix
effect”
• Example–Heparin therapy in patients with acute
myocardial infraction (AMI) affects the result of
determination of troponin I concentration

26. The Edge effect
• Temperature differences occur between wells
in the middle and at the edge of the microtiter
plate
• Temperature difference can cause change in
reaction kinetics
• A difference of 2 degree has been noted
• Use incubators

27. Homogenous immunoassay
COMPETETIVE CONJUGATE Modulation manner
EMIT Ag + lysozyme or G6PD Steric hindrance
SLFIA Ag + substrate ‘’
ARIS Ag + prosthetic group ‘’
ENZYME CHANNELING Ag + G6PD or hexokinase Enhancement to proxmity
BIOTIN ENZYME AVIDIN Ag + avidin Steric hindrance
CEDIA Ag + fragments of subunits ‘’
NONCOMPETETIVE
HYBRID ANTIBODY Hybrid ab specific to ag or
to inhibitor
‘’
PROXIMAL LINKAGE Antibody to G6PD &
hexokinase
Substrate cascade by
proxmity
EHIA Ab to amylase Steric hindrance
ENZYME ENHANCEMENT Ab to beta galactosidase Charge effect
AEST Ab to peroxidase stabilization

28. Enzyme Multiplied IT
Rubenstin1972
Enzyme act= free haptens
G6PD
LYSOZYME

29. Substrate Labeled Fluoroscent IA
Burd 1977
Substrate=B-galactosylumbeliferone
Enz=beta galactosidase

30. Apoenzyme Reactivation IS
MORRIS1981
APOGLUCOSE OXIDASE

31. Cloned Enzyme Donor IA
Handerson 1986
Beta-galactosidase genetically engineered

32. Enzyme Inhibitory Homogenous IA
Ashihara1988
HMW antigen=ferretin,AFP
Alfa-amylase,Dextranase
noncompetitive

33. Advantages and Disadvantages of
Enzyme Immunoassay
A. Advantages
1. Sensitive assays can be developed by the amplification effect of
enzymes.
2. Reagents are relatively cheap and can have a long shelf life.
3. Multiple simultaneous assays can be developed.
4. A wide variety of assay configurations can be developed.
5. No radiation hazards occur during labeling or disposal of wastes.
B. Disadvantages
1. Measurement of enzyme activity can be more complex than measureme
of the activity of some types of radioisotopes.
2. Enzyme activity may be affected by plasma constituents.
3. Homogeneous assays at the present time have the sensitivity of 10−9 M
and are not as sensitive as radioimmunoassays.

34. ELISPOT

36. RIA
I-125
I-131
H-3
C-14
P-32

37. Chemiluminescent Immunoassay
• Chemiluminescent immunoassays (CLIAs) use
chemiluminescence generating molecules as
labels, such as luminol derivatives, acridinium
esters, or nitrophenyl oxalate derivatives and
ruthenium tri-bipridyl[Ru(bpy)32+] with
tripropylamine for electrochemiluminescence
• Oxidation-reduction reactions generate
light(OH−/H2O2 as a chemical trigger)

41. Preis Biochemische Analytik, Munich, April 1976.