Antigen antibody reactions
Enzyme linked immunosorbantassay (ELISA)
The method depends on conjugation of an enzyme to eitherAg or Ab, then substrate is added as a quantitative measure ofenzyme activity.
There are 3 ways of ELIZA testing:
1.Indirect method.
2.Direct or Double antibody
technique (sandwich technique).
3.Competitive method
Non competitive
The test is performed in a 8 cm x 12 cm plasticplate which contains 96 wells, each of which areabout 1 cm high and 0.7 cm in diameter.
96-well microtiter plates being used for ELISA
Principle:
Use of enzyme-labeled immunoglobulin to detectantigens or antibodies.
Signals are developed by the action of the hydrolyzingenzyme on chromogenic substrate.
Optical density measured by micro-plate reader.
Examples:
Hepatitis A (Anti-HAV-IgM, anti-HAV IgG)
HIV
Types of ELISA
Direct ELISA sandwich (doubleAb technique):
used for detection of Ags.
Known specific Ab is immobilized byadsorption onto a plastic surface.
Clinical sample is added (if Ag present it willbind to the immobilized Ab)
Enzyme-labelled specific Ab is added
(attach to the fixed Ag if present)
Add the substrate
If Ab specific to Ag
change the colour
If Not specific
No colour change
AbAb
++
Antibody labelledAntibody labelled
with enzymewith enzyme
washwash
++
substratesubstrate
AgAg
E
E
E
E
E
E
Change in colour
Change in colour
Indirect ELISA:
In this test an enzyme- labelled anti-human Ig isused to detect the presence of specific Abs inpatients’ sera.
Known Ag is fixed by adsorption onto a plasticsurface.
The serum sample is added ( if specific Ab ispresent, it will bind the fixed Ag).
Add the enzyme-labelled antihuman Ig.
add the substrate, then quantitatively measurefor the degree of color change.
Competitive ELISA
Titre wells coated with antibodies
Unknown antigen competes with enzyme labeledknown antigen.
Incubate: till antigen antibody reaction is complete
Wash remove unbound antigens
Add substrate.
Enzyme + Substrate Product measure colour
Colour inversely related to antigen in patient sample.
The less color the more Ag.
Radioimmunoassay
Radioimmunoassay (RIA)
This method is used for the quantification of antigens thatcan be radioactively labeled.
History:
The technique was introduced in 1960 by Berson and Yalow asan assay for the concentration of insulin in plasma.
It represented the first time that hormone levels in the bloodcould be detected by an in vitro assay.
The principle
Is that radioactively labeled antigens competes with the nonlabeled antigens in a test sample.
For example 125 Iodine (125I), 14 Carbon (14C) or 3 Hydrogen(3H) are used in RIAs for labeling and are called as tracers.
Method Used:
1.Take an antigen labeled with a radioactive material.
2.Then add primary antibody which complexes with labeled antigen.
3.Then add an unlabeled antigen, which competes with labeledantigen for binding to primary antibody.
4.Then we add secondary antibody to the solution which precipitatesantigen-antibody complexes.
5.Then we measure radioactivity of supernatant that indicates freeantigen.
6.And the radioactivity of precipitate indicates bound antigen.
The differencebetween theradioactivity in thecontrol “A” and theunknown sample “B”is proportional to theamount of unlabeledantigens in theunknown sample
Gamma Counter for calculatingradioactivity
Merits:
Radioimmunoassay is widely-used because of its great sensitivity.
Using antibodies of high affinity, it is possible to detect a fewpicograms (10−12 g) of antigen in the tube.
Demerits:
The main drawbacks to radioimmunoassay are the expense and hazards ofpreparing and handling the radioactive antigen.
125 Iodine (125I), 14 Carbon (14C) and 3 Hydrogen (3H) emit gammaradiation that requires special counting equipment
Uses:
RIA has become a major tool in the clinical laboratory where itis used to assay
Plasma levels of:
Most of our hormones
Digitoxin or digoxin in patients receiving these drugs and
Certain abused drugs
To detect presence of hepatitis B surface antigen (HBsAg) indonated blood;
To detect anti-DNA antibodies in systemic lupus erythematosus(SLE).
Flow cytometry
Flow cytometers are instruments capable of analyzingproperties of single cells from the general population.
The analyzed properties include:
1.Physical: size, volume, refractive index, viscosity.
2.Chemical: DNA, RNA, proteins and enzymes content.
The data generated can be analyzed statistically by flow cytometrysoftware.
Application in microbiology:
1.Rapid detection & counting of bacteria in pure culture.
2.Detection of viruses in tissue culture and clinical samples.
3.Detection of viral Ags on cells of infected person.
Flow Cytometry
In this test, the patient's cells are labeled withmonoclonal antibody, tagged with a fluorescent dye, suchas fluorescein or rhodamine.
Single cells are passed through a laser light beam, cellsthat fluoresce is counted . The data generated can beanayzed statistically by flow cytometry software toreport cellular characteristics such as size , complexity ,phenotype & health.
Instrument Overview
The primary systems of the flowcytometery are :
The fluidic system , which presents samples to the interrogation point& takes away the waste.
The lasers , which are the light source for scatter & fluorescence .
The optics , which gather & collect the light.
The detectors , which receive the light.
The electronics & The peripheral computer system whichconverts the signals from the detectors to the digital data to perform thenecessary analyses .
