Last Update: 6 December 2017 Part - I

M-73
RIA & ELISA
Q. Add a short notes on RIA and ELISA (+5)
A. RIA (Radio Immunoassay)
Definition : RIA is a sensitive technique for detecting the concentration of Ag. This technique was first developed by two
endocrinologist, S.A. Berson and R. Yalow in 1960 to determine the level of Insulin. In 1977 the significance of this
technique was acknowledged, by the award of Nobel prize to yellow.
Principle : The principle of RIA involves competitive binding of radio labelled Ag & unlabeled Ag to a high affinity Ab.
The labelled Ag is mixed with Ab at a concentration that saturates the Ag is mixed with Ab at a concentration that
saturates the Ag binding sites of the Ab molecule. They increasing the amount of test sample containing unlabeled Ag of
unknown concentration are added. The Ab does not distinguish labelled from unlabeled Ag. So, the two kind of Ag
compete for available binding site on the Ab with increasing concentration of unlabel Ag more label Ag will be displaced
from the binding site.
Ag* + Ab + Ag Ag*Ab + Ag Ab + Ag* + Ag
Procedure :
1) Radio active Ag specific Abs are added to a series of tube
(Eppendorf tube) which are first bound to the tubes. Radio activity
in each of these standard tubes is then measured.
2) The Ag (Insulin) from a patient is allowed to bind to
another tube. Then radio labelled Ag (Insulin*) & are
added & measured the radio activity by Gamma Counter.
3) To determine the unknown concentration of Ag in a
patients sample, percentage radio activity of a sample solution
that contain unknown concentration of Ag is plotted in the
standard curve.
Application : This technique are used to detect
i) the concentration of antigen (Ag).
ii) the concentration of antibody (Ab).
iii) the concentration of peptide hormone.
iv) the concentration of Steroid hormone.
v) No. of receptors in the plasma membrane.
Advantage : The advantages of RIA as follows.
i) This assay permits great sensitivity often in Pgm (Pico gram) level.
ii) RIA methods are vary prcised.
iii) Ab of very high affinity & great specificity can often be obtained.
Disadvantage :
i) This assay system is expensive.
ii) It generates a considerable amount of radio active waste that harmful for life.
iii) RIA is specific immunologic reaction not biologic.

B. ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) :

Definition : The ELISA is a immunoassay, which employs an enzyme linked anti-immunoglobulin to detect Ags or Abs.
Principle : ELISA is similar in principle to RIA but depends on an enzyme rather than a radio active label. An enzyme
conjugated with an Ab react with a colourless substrate to generate a coloured reaction product. Such a substrate is called
a chromogenic substrate. A number of enzymes have been employed for ELISA, including alkaline phosphates, horse
radish peroxidase (HRP) and -galactosidase.
Ag + Ab1 + Ab2 - HRP Ag Ab1 Ab2 HRP
H2O2
O.D. ELISA reader Colour solution
(1)
A number of variations of ELISA have been develop to detect quantitatively & qualitatively either Ag or Ab.
Types of ELISA :
There are three types of ELISA these are as
follows
i) Indirect
ii) Sandwich
iii) Competitive.
i) Indirect : Ab can be detected either
qualitatively or quantitatively with an indirect
ELISA (Fig : 1). Serum or some other sample
containing primary Ab (Ab1) is added to an
microlitre plate containing Ag and allowed to react

with the Ag attached to the well. After any free Ab1 is washed
away, the presence of Ab1 bound to the Ag is detected by adding
an enzyme conjugated secondary Ab (Ab2). Any tree Ab2 then is
washed and a substrate for enzyme is added. The amount of
colour reaction product that form is measured by specialized
spectrophotometric plate reader (ELISA reader).

Indirect ELISA is the method of choice to detect the

serum Ab against HIV, the Ag which causes AIDS.
ii) Sandwich ELISA :
Ag can be detected or measured by a sandwich ELISA
(Fig : 2). In this technique, the Ab is immobilized on a microtiter
well. A sample conteining Ag is added and allowed to react with
the immobilized Ab. After the well is washed, a second Ab
specific for a different epitope of Ag is added to react with
bound Ag. Any free second Ab is removed by washing &
subsequently added a substrate and colon reaction product is
measured by ELISA reader.

iii) Competitive ELISA :

Ag can be detected or measured by compitative ELISA
(Fig : 3). In this method Ab is first incubated in solution with a
sample conteining Ag. Ag-Ab mixture is then added to an Ag
coated microtiter well. Addition of an Ag. Conjugated second
Ab (Ab2) can be used to determined the amount of primary Ab as
well as Ag. In this assay, the higher the concentration of Ag in
the original sample, lower the reaction product.

(2)