serological methods

1. Antigen - Antibody
interaction

2. Analyte
• The sample being analyzed.
• In immunoassays the analyte is either Antibody or Antigen

3. Points to remember
• Antigen antibody interaction is a
Bimolecular association
• Like an enzyme substrate interaction
• Association involves non-covalent
interaction between epitope and paratope
• Reaction does not lead to irreversible
chemical alteration

4. Features of Ag-Ab interaction
• Reversible
• Non-covalent interaction

5. Affinity and Avidity
Affinity
• Measure of the strength of the binding
• Ease of association or dissociation
Avidity
• Increase in affinity due to multivalent binding
• The summation of multiple affinities

6. Affinity vs
Avidity

8. Factors Affecting Measurement of Ag/Ab
Reactions
1. Affinity and avidity
2. Ag : Ab ratio
3. Physical form of Ag

9. Antigen-Antibody reactions
• Ag –Ab reactions are highly specific
• Antigen will react only with antibodies elicited by itself
• Due to this specificity, Ag-Ab reactions are utilized for identifying one by using
the other
This is the basis of serologic reactions

10. Cross reaction
Ag-Ab reaction are highly specific
But in some cases
Ab elicited by one Ag can cross-react with an unrelated Ag

11. Cross reaction
Such cross-reactivity occurs if two different antigens share an
• Identical or
• Very similar epitope

12. Cross reaction
• The ability of an
individual Ab
combining site to react
with more than one
antigenic determinant.
• The ability of a
population of Ab
molecules to react
with more than one Ag

13. Homogenous immune assay
Immunoassay system in which
• both antigen-antibody reaction and
• the measurement of the degree of immune reaction
are performed in solution without separation of the free and
antibody-bound components.

14. EMIT - Enzyme Multiplied Immunoassay
Technique

15. Heterogenous immunoassay
• Any form of immunoassay that involves physical separation, at
some stage of the procedure, of antibody‐bound antigen from
remaining free antigen.
• Example - ELISA

16. Luminescence
• Luminescence is the emission of light by a substance as a result
of a chemical reaction (chemiluminescence) or an enzymatic
reaction (bioluminescence).

17. Types of luminescence
• Bioluminescence
• Chemiluminescence
• Electroluminescence – Ruthenium, Tris chelate
• Photoluminescence - Inorganic phosphorus
• Thermoluminescence

18. Bioluminescence
• Fungi
• Firefly – Firefly luciferase enzyme oxidize firefly luciferin
• Lanternfish
• Anglerfish
• Squid
• Jellyfish

19. Chemiluminescence and ELISA
• Modified version of ELISA
• Luxogenic substrate instead of chromogenic substrate used here
• Stopping reagent not required
• Incubation period is small

20. Chemiluminescence
• The process of chemiluminescence occurs when energy in the form
of light is released from matter during a chemical reaction.
• Intensity of light produced by Chemiluminescence's is measured by
SPECTROPHOTOMETER

21. Chemiluminescence
• Antibody tagged with enzyme HRP mixed with antigen in sample
• Ag-Ab reacts
• Oxidation of the luminous substrate by H2O2, produces light
Ab-HRP+Ag Ab-HRP-Ag Light
Luminol+H2O2

22. • 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.
• Can be used for heterogeneous or homogeneous assays.
• Can attach label to antigen or antibody.
Chemiluminescence

23. Chemiluminescence
• Heterogeneous assays use competitive and sandwich assay.
• Competitive assays used to measure smaller analytes.
• Sandwich assays are used to measure larger analytes.

24. Molecules capable of chemiluminescence
• Luminol
• Acridium esters
• Ruthenium derivatives
• Nitrophenyl oxalates
Use sodium hydroxide as a catalyst

25. Application
• Chemiluminescence immunoassay
• DNA hybridization detection
• Western blotting
• Forensic science
• Food analysis

26. Monoclonal Ab coated well
Test sample (Serum)
HRP labelled antibody conjugate
Test Ag sandwich between solid
phase Ab and enzyme labelled Ab
Incubate
Remove unbound enzyme labeled
Ab
Chemiluminescence reagent added
Measure light in Relative light unit
with luminometer