Immunoassay techniques RIA ELISA

1. Immunoassay
An immunoassay is a test that uses antibody and antigen
complexes
An antibody: antigen complex is also known as an immune-
complex
"Immuno" refers to an immune response that causes the body
to generate antibodies
"assay" refers to
atest
immunoassay is a
test that utilizes immuno complexing when
antibodies and antigens are brought together

2. Antibodies,AntigensandAnalytes
An analyteis anything measured by a laboratory test.
In immunoassay testing, the analyte may be either an
antibody, or an antigen.
Immunoassays utilize one or more selected antibodies to detect
analytes of interest.
The analytes being measured maybe:-
1. That are naturally present in the body (such as a thyroid
hormone)
The body produces but are not typically present (such as a
cancer antigen)
3 Do not naturally occur in the body (such as an abused drug)
2.

3. 1959, RoasalynYalowand SolomenBerson developed this techniques for measuringthe
plasma Insulin level.
They received Nobel prize for the same in 1977.

4. Radioimmunoassay (RIA)
This technique is used to determine
the concentration of antigen present
in blood/serum of the patient

5. Principle
The principle of RIA is based on the
competitive binding between the radio
labeled antigen (HOT antigen) and unlabelled
antigen (COLD antigen) with selected antibody
and the radioactivity is determined.

6. What is HOT/COLD antigen?
Hot antigen The antigen which are labeled with
radioactive isotopes
Cold antigen = The antigen which needs to be
determined (present in your sample)

7. RIA could be used for qualitative
and quantitative assay
Qualitative =Only presence or absence
of particular antigen/analyte
Quantitative =the exact amount of
antigen/analyte could be determined
Let's understand this...

8. Radioimmunoassay (RIA)
Very sensitive: can detect material
present at concentrations of <0.001
micrograms/ml.
Cpm Generate standard curve with known
amounts of unlabeled antigen
Measure unknown using standard curve.
O Labeled
antigen
Amount unlabeled Antigen Unlabeled
antigen O
O
YYYY L
L

9. Infccted serum 251] HBsAg 251] HBsAg Uninfectcd
serum
Unlabeled
HBsAg
Anti-HBsAg-
251 bound TI251 bound
(b)
RIA to detect
hepatitis
pproximately linear part of curve
detects hepatitis virus in 1 microliter
of blood.
Used for screening blood for
transfusions.
Concentration of unlabclkd HBsAg. ng/ml

10. Enzyme-linked /mmune sorbant
assay (ELISA)
Capture
antigen using
plate-bound
antibody
Add second specific
antibody-enzyme
conjugate
Colorometric
assay
YYYY r i i
Detection by secondary antibodies conjugated to enzymes (alkaline
phosphatase, horse radish peroxidase. B-galactosidase). Break-
down of substrate by enzyme produces a visible color.

12. (a) Indirect ELISA Variations of the ELISA method
wash wash Wash
Add enzyme
conjugated
secondary
antibody
Add substrate (S)
Antigen
coated well
Add specific
antibody to be and measure
measured color
(b) Sandwich ELISA
wash wash wash
Y Add substrate
Add enzyme
conjugated
secondary antibody
Antibody- Add antigen
to be measured and measure
Co3red we
color
(C) Competitive ELISA
wash Wash
Incubate
antibody with
Add Ag-Ab Add enzyme
conjugated
econdary
antibody
antigen to be Add substrate
measured mixture to and measure
antigen-coated well color

13. Common Enzymes and substrate used in ELISA
Enzyme Substrate Color
Blue
Tetra methyl
peroxidase (HRP) Banzedine
(TMB)
Horseradish
Para Nitrophenyl
phosphatase (AP) phosphate(PNPP)
Alkaline Yellow
B-galactosidase Ortho- Yellow
Nitrophenyl beta
galastoside