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8/13/2019 ASSINMENTY

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RADIOMMUNOASSAY

Radioimmunoassay is a highly sensitive technique that can

detect antigen or antibody at concentrations less than 0.001 g/ml.

A method of detecting or quantifying antigens or antibodies

using radiolabelled substances.

The technique of radioimmunoassay has revolutionized

research and clinical practice in many areas, e.g.,

blood baning

diagnosis of allergies

endocrinology

The technique !as first developed by t!o endocrinologists,

".A #erson and Rosalyn $alo!, in 1%&0 as an assay for the

concentration of insulin in plasma. 't represented the first time that

hormone levels in the bloodcould be detected by an in vitro assay.

All modern immunochemical methods of quantitation are based upon

having a simple and accurate method for measuring the quantity of

indicator molecules that bind to a solid phase surface.

Radioimmunoassay are commonly used to measure the

levels of hormones in blood and tissue fluids,

The principle of R'A involves competitive binding of radio

labeled antigen and unlabelled antigen to high affinity antibody. The

antigen is generally labeled !ith a gamma(emitting isotope such as

1)*'. +ence R'A is said to be a competitive binding type assay.

SCHOOL OF TECHNOLOGY 1


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METHOD

1. A miture is prepared of

a- Radioactive antigen

#ecause of the ease !ith !hich iodine atoms

can be introduced into tyrosine residues in a protein, the radioactive

isotopes 1)*' or 11' are often used.

b- Antibodies against that antigen.

). no!n amounts of unlabeled cold- antigen are added to samples

of the miture. These compete for the binding sites of the antibodies.

. At increasing concentrations of unlabeled antigen, an increasing

amount of radioactive antigen is displaced from the antibody

molecules.

2. he antibody(bound antigen is separated from the free antigen in the

supernatant fluid, and*. The radioactivity of each is measured. 3rom these data, a standard

binding curve, lie this one sho!n, can be dra!n.

Fig- A standard curve is obtained by adding increasing concentration on

unabeed H!sAg to a "i#ed $uantity o" %&'(I) H!sAg and s*eci"ic antibody+



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&. The samples to be assayed the unno!ns- are run in parallel.

4. After determining the ratio of bound to free antigen in each

unno!n, the antigen concentrations can be read directly from the

standard curve as sho!n above-.



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EN,YME IN.ED IMMUNOSOR!ENT ASSAY /EISA01

56'"A is so named because the technique involves the use

of an immunosorbent an absorbing material specific for one of the

components of the reactions, the antigen all antibody. 56'"A is

similar in principle to R'A but depends on an enzyme rather than a

radioactive label.

56'"A is usually done using %&( !ell microtitre plates

suitable for automation. An enzyme con7ugated to an antibody reacts

!ith a colorless substrate to generate a colored reaction product.

A number of enzymes have been employed for 56'"A,

including alaline phosphatase, horseradish proidase and p(nitro

phenyl phosphatase. 8hen mied !ith suitable substrate, each of

these enzymes generates a colored reaction product.

A number of variations of 56'"A have been developed,

allo!ing detection and quantitation of either antigen or antibody.

These are as follo!s9

1- 'ndirect 56'"A

)- :ompetitive 56'"A.

- "and!ich 56'"A

5ach type of 56'"A can be used qualitatively to detect the

presence of antibody or antigen.

Alternatively a standard curve based on no!n concentration

of antibody or antigen is prepared from !hich the unno!n

concentration of a sample can be determined.



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&0 Indirect EISA1

An indirect 56'"A is used to detect or quantitate antibody.

"erum or some other sample containing primary antibody Ab1- is

added to an antigen(coated micro titer !ell and allo!ed to react !ith

the bound antigen. After any free Ab1is !ashed a!ay the presence

of antibody bound to the antigen is detected by adding an enzyme(

con7ugated secondary anti(isotype antibody Ab)- !hich binds to the

primary antibody. Any free Ab)then is !ashed a!ay and a substrate

for the enzyme is added. The colored reaction product that forms is

measured by specialized spectrorphoteometric plate readers, !hich

can measure the absorbance of a %&(!ell plate in less than a minute.

Fig - Indirect EISA

An indirect 56'"A has been the method of choice to detect

the presence of serum antibodies against human immunodeficiency

virus +';-, the causative agent of A'


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=enerally, serum antibodies to +'; can be detected by indirect

56'"A !ithin & !ees of infection.

'0 2o3*etitive EISA

'n this technique antibody is first incubated in solution !ith a

sample containing antigen.

The antigen(antibody miture is then added to an antigen(coated

micro titer !ell. The more antigens present in the sample, the less

free antibody !ill be available to bind to the antigen coated !ell.

Addition of an enzyme con7ugated secondary antibody Ab)- specific

for the isotope of the primary antibody can be used to quantitate the

amount of primary antibody bound to the !ell as in an indirect

56'"A. 'n the competitive assay, ho!ever, the higher the

concentration of antigen, in the original sample, the lo!er the

absorbance.

Fig - 2o3*etitive EISA

40 Sand5ic6 EISA1

The technique of "and!ich 56'"A allo!s detection or

quantitation of antigen. 'n this case the antibody is immobilized on a

micro titer !ell. A sample containing antigen is added and allo!ed to

react !ith the bound antibody. After the !ell is !ashed, a record



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enzyme(lined antibody specific for a different epitope on the antigen

is added and allo!ed to react !ith the bound antigen. After any free

second antibody is removed by !ashing substrate is added and the

adored reaction product is measured.

METHOD:

An 56'"A is generally performed in !ells of micro plates.

1- 8ells of the plates are coated !ith unlabelled monoclonal

antihuman 'g= subclass(specific antibody and !ashed figure A->

)- Test samples, standard(and control sera are introduced in the

respective !ells and incubated> The 'g= subclass to be

determined !ill bind to the solid phase and non(bound 'g= is

removed by !ashing figure #->

- 5nzyme(labelled anti(human 'g= antibodies are added to each

!ell and non(bound con7ugate is removed by !ashing figure :->

2- ?lates are incubated !ith substrate solution>

*- After incubation, the colored reaction product is measured

photometrically figure

&- The concentration of the 'g= subclasses in the test samples is

calculated relative to the values of the calibration curve.



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Advantages o" EISA1- 't is economic.

)- 't is ;ersatile, sensitive and simple.

- Absence of radiation hazard.

2- 't is safer and less costly.

*- Availability of test its and facility for automation have

added to their ?opularity.



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IMMUNO7RE2I7ITATION

'mmunoprecipitation is one of the most !idely used

immunochemical techniques. 'mmunoprecipitation involves the

interaction bet!een a protein and its specific antibody, the separation

of these immune complees !ith ?rotein = or ?rotein A, This

technique provides a rapid and simple means to separate a specific

protein from !hole cell lysates or culture supernatants.

'mmunoprecipitation follo!ed by "


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). ?reclearing

. Antibody incubation/formation of antibody(antigen complees

2. ?recipitation

*. Analysis by "


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&. "pin cell lysates at 10,000g for 1* minutes at 2C:.

4. :arefully collect supernatant, !ithout disturbing the pellet

and transfer to a clean tube. The cell lysates can be frozen at t

term storage at minus D0C:.


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. Add *0ul of !ashed ?rotein = slurry in prechilled 6ysis

#uffer prepared as instructed in ?reclearing "tep 1 above-.

2. 'ncubate for 1 hour at 2C: on a rocing platform or a rotator.

*. "pin the eppendorf tube at1G.FFFg for 0 seconds at 2C:.

&. :arefully remove supernatant completely and !ash the

beads (* times !ith *00ul of 6ysis #uffer. To minimize bacgr

be given to remove the supernatant completely in these

!ashes.

4. After the last !ash, aspirate supernatant and add *0ul of 1H

6aemmli sample buffer to bead pellet. vorte and heat to =

minutes.

D. "pin at 10,000g for * minutes, collect supernatant and load

onto the gel. "upernatant samples can be collected and I

point if the gel is to be run later.

%. 3ollo! manufacturerJs instructions for "


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7rotease In6ibitor 2oc:tai /&99;01

( ?L"3, *mg *0ug/ml-

( Aprotinin, 100ug 1ug/ml-

( 6eupeptin, 100ug 1ug/ml-

( ?epstatin, 100ug 1ug/ml-

Radia I33unodi""usion /Mancini0 /singe di""usion in t5o

di3ensions0 /Met6od0

The relative concentrations of an antigen can be

determined by simple quantitative assay in !hich an antigen sample

is placed in a !ell and allo!ed to diffuse into agar containing a

suitable dilution of an antiserum as the antigen diffuses into the agar,

the region of equivalence is established and ring of precipitation

forms around the !ell. The area of the precipitin ring is proportional

to the concentration of antigen. #y comparing the area of theprecipitin ring !ith a standard carves, the concentration of the

antigen sample can be determined.

Fig - Diagra33atic re*resentation o" radia i33unodi""usion in a ge



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Doube I33unodi""usion /Ouc6terony /Doube di""usion in t5o

di3ensions Met6od0

'n the Fuchterlony method both antigen and antibody

diffuse radially from !ell to!ards each other, thereby establishing a

concentration gradient. As equivalence is reached a visible line of

precipitation forms. This simple technique is an effective qualitative

tool for determining the relationship bet!een antigens and the

number of different Ag(Ab systems present. The pattern of the

precipitin lines that form !hen t!o different antigen preparations are

placed in ad7acent !ell indicate !hether or not they share epitopes

Fig - Diagra33atic re*resentation o" doube i33unodi""usion

Singe di""usion in one di3ension /Oudin *rocedure0

The antibody is incorporated in agar gel in a test tube

and the antigen solution is layered over it. The antigen diffuses

do!n!ards through the agar gel, forming a line of precipitation that

appears to more do!n!ards. This is due to the precipitation form at

due to the precipitation formed at the advancing front of the antigen

and is dissolved as the concentration of antigen at the site increases



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due to diffusion. The number of bands indicates the number of

different antigens present.

Fig -Singe di""usion in one di3ension /Oudin0

Doube di""usion in one di3ension /


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IMMUNOEE2TRO7HORESIS

Aternative Na3es

'5? ( serum> 'mmunoglobulin electrophoresis ( serum>

=amma globulin electrophoresis> "erum immunoglobulin

electrophoresis

De"inition

"erum immunoelectrophoresis is a test that tells !hether or

not you have immunoglobulins in the blood. 'mmunoglobulins are

proteins that mae antibodies. The proteins can be abnormal. There

are various types of immunoglobulins. 'f you do have these proteins,

this test can also help identify !hat specific type they are.

Ho5 t6e Test is *er"or3ed

#lood is dra!n from a vein, usually from the inside of the

elbo! or the bac of the hand. The puncture site is cleaned !ith

antiseptic. An elastic band is placed around the upper arm to apply

pressure and cause the vein to s!ell !ith blood.

A needle is inserted into the vein, and the blood is collected in an air(

tight viaN or a syringe.


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test strip, or into a small container. A bandage may be applied to the

puncture site if there is any bleeding.

Ho5 t6e test 5i "ee

8hen the needle is inserted to dra! blood, you may feel

moderate pain, or only a pric or stinging sensation. After!ard, there

may be some throbbing.

8hy the Test is performed this test is performed to assess the

clonality monoclonal or polyclonal- of immunoglobulin. @o

monoclonal antibodies are detected.

=6at Abnor3a Resuts Mean

'n some malignant disorders that is, multiple myeloma,

chrome lymphocytic leuemia- a single clone of lymphocytes

produces one type of protein ( a monoclonal immunoglobulin. This isidentifiable as monoclonal all the same type- by

immunoelectrophoresis. "ome people have monoclonal

immunoglobulins, but do not have a malignant disorder.

Ris:s

O 5cessive bleeding

O 3ainting or feeling lightheaded

O +ematoma blood accumulating under the sin-

O 'nfection a slight ris any time the sin is broen-

;eins and arteries vary in size from one patient to another and from

one side of the body to the other. Fbtaining a blood sample from

some people may be more difficult than from others.



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The resolving po!er of immunodiffusion !as greatly

enhanced !hen =raybar and 8illiams devised the techniques of

immunoelectrophoresis. This involves the electrophoresis separation

of a composite antigen "uch as seam- into its constituent proteins,

follo!ed by immunodiffusion against its antiserum resulting, in

separate precipitin lines, indicating reaction bet!een each individual

protein !ith its antibody, thus enables identification and approimate

quantitation of the various proteins present in the serum. The

technique is preformed on other agar or agarose gel on a slide, !ell

and antigen !ell and an antibody through out it.

The test serum is place in the antigen !ell and

electrophoresed for about an hour. Antibody against human serum is

then placed in the trough and diffusion allo!ed to proceed for 1D()2

hours. The resulting precipitin lines can be photographed and the

slides dried, stained and preserved for record.


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Fig - Seru3 se*arated by eectro*6oresis

Fig - Anti seru3 t6roug6 "ied 5it6 antiseru3 to 5e 6u3an seru3+

Fig - Seru3 and antiseru3 ao5ed di""using into agar

Fig - 7reci*itin ine "or3 "or individua seru3 *rotein+

Eectroi33unodi""usion1

The development of precipitin lines can be speeded up

by electrically driving the antigen and antibody9 ;arious methods

have been described combining electrophoresis !ith diffusion of

these one(dimensional double 5lectroimmunodiffusion



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:ounterimmunoelctrophoresis- and one dimensional single

electroimmunodiffuison rocet electrophoresis- are used frequently

in the clinical laboratory.

&0 2ounteri33unoectro*6oresis

This involves simultaneous electrophoresis of the antigen P

antibody in gel in opposite directions resulting in precipitation at a

point bet!een them.

This method produces visible precipitation lines !ithin

thirty minutes and is ten times more sensitive than the standard

double diffusion techniques. The clinical applications are for

detecting various antigens such as alpha(fetoprotein in serum and

specific antigens of :ryptococcus and meningococcal in the

cerebrospinal fluid.

Fig - 2ounter i33unoeectro*6oresis /2IE0+ Antigen and Antibody are

driven toget6er by an eectric current and a *reci*itin ine "or3s+

'0 One di3ensiona singe eectroi33unodi""usion /roc:eteectro*6oresis0

The main application of this technique is for quantitative

estimation of antigens. The antiserum to the antigen to be quantities

is incorporated in agarose and gelled and the glass slide.

The antigen, in increasing concentrations, is placed in !ells punched

in the set gel. The antigen is then electrophoreses into the antibody



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containing agarose. The pattern of immunoprecipitaion resembles a

rocet and hence the name.

Fig - Roc:et eectro*6oresis

Antigen is driven in to gel containing antibody

1. Antibody in agarose gel.

). ?recipitin area

. Antigen !ells

2. 'ncreasing antigen concentration.

40 aure>s t5o di3ensiona eectro*6oresis

'n this technique, the antigen miture is first

electrophoretically separated in a direction perpendicular to that of

the final socet stage. #y this method one can quantitate each of

several antigens in a miture.

Fig - aure>s variant o" roc:et eectro*6oresis /t5o di3ensiona i33unoeectro*6oresis0



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REFEREN2ES

1. Qenis uby, immunology 1th edition, !.s. freeman P

company publication, page no( 1% to 12%.

2. Abbas A.., 6acman A.., ?ober Q.#., 'mmunology *th

edition, "ingapoor high court press P company

publication, page no( *% to &%.

3. Anand L.R., tet boo of microbiology 2thedition, orident

lodgment 6td. ?ublication, page no( DD to 10%.

2. http9//!!!.antibodybeyond.com/application/ia.htm

5. http9//!!!.antibodybeyond.com/elisa/radioimmunoassay

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