The document describes the enzyme-linked immunosorbent assay (ELISA) technique. ELISA allows for qualitative and quantitative analysis of antigens and antibodies. It is highly specific and sensitive. There are four main types of ELISA - direct, indirect, sandwich, and competitive. ELISA involves an antigen or antibody being immobilized on a plate and subsequent washes and incubations with conjugated enzymes and substrates to produce a detectable color change. ELISA has various applications in fields like quality control, disease diagnosis, and more.

2.  Serological technique
 Qualitative & Quantitative
analysis of:
› Antigen
› Antibodies
 Highly Specific &
Sensitive(1000X more)
 Quality control check in
various industries
 Detection of toxins, allergens
and HBV & HCV markers in
serum
 Screening of HIV in blood
samples, drugs and antibiotics
 Diagnostic tool in medicine &
plant pathology

3.  Antigen bound to ELISA plate
 Specific antibodies bind to antigen
 Antibodies conjugated with an enzyme
 Addition of substrate results in a colored product
 Intensity of color detected by ELISA reader or
spectrophotometer
o Both antigen & antibody can be coated onto polystyrene plates
o Both antigen & antibodies can be conjugated with enzyme

4.  Direct ELISA
 Indirect ELISA
 Sandwich ELISA
 Competitive ELISA

5. Used for
detection
and
enumeration
of an
antigen in
sample.
Plate-Ag + Ab*enzyme +
Substrate  Read

6.  Advantages
o Quick
methodology since
only one antibody
is used
o Elimination of cross
reactivity with
secondary antibody
 Disadvantages
o Expensive- primary
antibody labeled
individually
o Time consuming
o inflexible

7. For
detection
of specific
antibodies
in the serum
sample
Plate-Ag +Ab+AB*enzyme
+Substrate  Read

8.  Advantages
o Increased sensitivity, since
more than one labeled antibody
is bound per primary antibody
o Flexibility, since different
primary detection antibodies
can be used with a single
labeled secondary antibody
o Cost savings, since fewer
labeled antibodies are required
 Disadvantage
o Cross reactivity with
secondary antibodies can occur

9.  Name depicts: antigen ‘sandwiched’ b/w
antibodies
 Plate coated with capture antibody
 Sample added: specific Ag binds to Ab
 Conjugated Detecting antibody added: binds to
Ag (for antigen detection)
 Conjugated secondary antibody binds to
detecting antibody (for antibody detection)
 Substrate added & colored product produced

10. Used for
detection of
antigen
Primary Ab &
Capture Ab
must not be
from same
specie
Plate-Ab + Ag + AB*E +
Substrate  Read

11. Used for detection
of antibodies
Primary Ab &
Capture Ab must
not be from same
specie
Secondary
conjugate
antibody is against
the Primary Ab
Plate-Ab + Ag + AB + anti-
AB*E + Substrate  Read

12.  Advantages:
o High specificity, since two
antibodies are used, the
antigen/analyte is
specifically captured and
detected
o Suitable for complex
samples, since the
antigen does not require
purification prior to
measurement
o Flexibility & sensitivity,
since both direct and
indirect detection
methods can be used
 Disadvantage:
o Two antibody binding
sites must be present on
an antigen

13.  Most complex form of ELISA
 Also called ‘Inhibition ELISA’
 Competition exists b/w Ag & Ab
 Competition can be:
› Direct Antibody Competition
› Direct Antigen Competition
› Indirect Antibody Competition
› Direct Sandwich Competition
› Indirect Sandwich Competition

14.  Passive adsorption of Ag
 Wash
 Incubation of sample serum(Ab)along
conjugate-Ab specific to plate-Ag
 Addition of mixture to plate-Ag &
incubate
 Wash
 Add substrate & read

15. The more antibody in the sample, the less conjugate antibody will be
able to bind to the antigen in the well, hence "competition"
Plate-Ag + Ab + AB*Enzyme + Substrate  Read

16.  Passive adsorption of known antigen
 Wash
 Simultaneous addition & incubation of
sample Ag along conjugate-Ab in the
plate
 Wash
 Substrate added & color produced

17. The more antigen in the sample, the less conjugate antibody will be
able to bind to the antigen in the well, hence "competition."
Plate-Ag + Agsample + Ab*Enzyme + Substrate  Read

18.  Passive adsorption of known antigen
 Simultaneous addition & incubation of
sample/competing cAb along detecting
AB specific to plate-Ag
 Wash, add anti-AB (conjugated), incubate
 Wash, substrate added & color produced

19. cAb & ABnot from same donor
Anti-ABonly against detecting AB
P-Ag + cAb + AB + AB*Enzyme + Substrate  Read

21. Capturing Ab,Sapmle Ab, Detecting Ab & Ab*E should be from different species

22.  Competitive ELISAs yield an inverse curve,
where higher values of antigen/antibody in the
samples or standards yield a lower amount of
color change.
 Competitive ELISA System can be made for all
other ELISA systems (direct, indirect & sandwich)
If sample & conjugates added simlutaneouslycompetition
If sample added first, conjugate laterinhibition

23. o When the antigen to
be detected is in low
quantities
o When the
particles/molecules
have low antigenicity
o No sample processing
required
o Flexibility & sensitivity
o Suitable for complex
samples
o High specificity

24.  Horse Radish Peroxidase
› ortho phenylene diamine (OPD)/H2O2
› 2,2', azino-di-ethylbenzothiazolinesulfonic acid (ABTS)/H2O2
› Tetra methyl benzidine (TMB)/H2O2
› 5-amino salicyclic acid (5AS)/H2O2
 Alkaline Phosphatase
› PNPP (para-Nitrophenyl Phosphate)
 Β-galactosidase
› ortho-nitrophenyl B-galactosidase (ONB)
 Urease
› Bromocresol purple

25. Thank You