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 ban ing 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 blood could 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.

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METHOD 1. A mi,ture 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 1.1' are often used. b- Antibodies against that antigen. ). /no!n amounts of unlabeled 01cold1- antigen are added to samples of the mi,ture. 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, li e this one sho!n, can be dra!n.

Fig- A standard curve is obtained by adding increasing concentration on un abe ed 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 0the un no!ns- are run in parallel. 4. After determining the ratio of bound to free antigen in each un no!n, the antigen concentrations can be read directly from the standard curve 0as sho!n above-.

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EN,YME -IN.ED IMMUNOSOR!ENT ASSAY /E-ISA01

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 al aline phosphatase, horseradish pro,idase and p(nitro phenyl phosphatase. 8hen mi,ed !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 un no!n concentration of a sample can be determined.

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&0 Indirect E-ISA1 An indirect 56'"A is used to detect or quantitate antibody. "erum or some other sample containing primary antibody 0Ab1- is added to an antigen(coated micro titer !ell and allo!ed to react !ith the bound antigen. After any free Ab1 is !ashed a!ay the presence of antibody bound to the antigen is detected by adding an enzyme( con7ugated secondary anti(isotype antibody 0Ab)- !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 E-ISA

An indirect 56'"A has been the method of choice to detect the presence of serum antibodies against human immunodeficiency virus 0+';-, the causative agent of A'<". 'n this assay recombinant envelope and core proteins of +'; are adsorbed as solid phase antigens to micro titer !ells. 'ndividuals infected !ith +'; !ill produce serum antibodies to epitopes on these viral proteins.

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=enerally, serum antibodies to +'; can be detected by indirect 56'"A !ithin & !ee s of infection. '0 2o3*etitive E-ISA 'n this technique antibody is first incubated in solution !ith a sample containing antigen. The antigen(antibody mi,ture 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 0Ab)- 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 E-ISA

40 Sand5ic6 E-ISA1 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(lin ed 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 0figure 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 0figure #-> .- 5nzyme(labelled anti(human 'g= antibodies are added to each !ell and non(bound con7ugate is removed by !ashing 0figure :-> 2- ?lates are incubated !ith substrate solution> *- After incubation, the colored reaction product is measured photometrically 0figure <-> &- 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" E-ISA 1- '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 involves used the

immunochemical techniques.

'mmunoprecipitation

interaction bet!een a protein and its specific antibody, the separation of these immune comple,es !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 "<"(?A=5 and immunoblotting, is routinely used in a variety of applications9 to determine the molecular !eights of protein antigens, to study protein/protein interactions, to determine specific enzymatic activity, to monitor protein post( translational modifications and to determine the presence and quantity of proteins. The '? technique also enables the detection of rare proteins !hich other!ise !ould be difficult to detect since they can be concentrated up to 10,000(fold by immunoprecipitation. 'n the 'mmunoprecipitation method, the protein from the cell or tissue homogenate is precipitated in an appropriate lysis buffer by means of an immune comple, !hich includes the antigen 0protein-, primary antibody and ?rotein A(, =(, or 6(agarose con7ugate or a secondary antibody(agarose con7ugate. The choice of agarose con7ugate depends on the species origin and isotype of the primary antibody. The methods described are comparable and the choice of method depends on the specific antigen(antibody system. The procedure can be divided into 1. "ample preparation

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). .. 2. *.

?reclearing Antibody incubation/formation of antibody(antigen comple,es ?recipitation Analysis by "<"(?A=5

METHOD Ste* I1 2e -ysate 7re*aration 1. +arvest appro,imately 104 cells. @ote9 The total number of cells per ml and the cell equivalent loaded per lane of gel sh specifically for each protein and antibody. ). 8ash cells !ith A10 ml of ?#" in a conical tube and spin at 200,g for 10 minutes. .. <iscard supernatant and repeat step ). @ote9 'f using tissue culture dish adherent monolayer cells, !ash cells in the flat room temperature ?#" and aspirate !ithout disturbing the monolayer. 2. After the second !ash, remove supernatant completely and resuspend the cell pellet in 1 ml of cold 6ysis #uffer contains 'nhibitor :oc tail 0final concentration of 104 cells/ml-. =ently vorte, the tube. @ote9 To prevent protease action effectively should be pre(chilled and all the follo!ing steps should be ept in the cold. The ?rotease 'nhibitor :oc tail should be required volume of the cold 6ysis #uffer 7ust before use. 3or adherent monolayerBs, add 1ml of cold 6ysis #uffer contain 'nhibitor :oc tail per 100mm culture dish. *. ?lace the tube or the dish on ice for .0 minutes, !ith occasional mi,ing.

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&. "pin cell lysates at 10,000,g 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:. <iscard pellet. Ste* II1 2e -ysate 7rec earing 1. Transfer *0E7l of the ?rotein = beads slurry 0commercially available from several vendors- to an eppendorf tube and adc #uffer. "pin at 10,000,g for .0 seconds and remove the 6ysis #uffer. 8ash one more time !ith *00ul of cold 6ysis #uff the beads in *0 ul of cold 6ysis #uffer. ). Add this *0ul of prepared ?rotein = slurry and *00ul of :ell 6ysate to an eppendorf tube and incubate on ice for .0(&0 t .. "pin at 10,000,g for 10 minutes at 2C: and transfer the supernatant to a fresh eppendorf. 'f any bead has been transfer and carefully transfer the supernatant to another fresh eppendorf tube. Ste* III1 I33uno*reci*itation 1. Add *(1 Fug of antibody to the eppendorf tube containing the cold precleared lysates. @ote9 This concentration of monocle suggested as a starting point. 5ach investigator may desire to titrate the concentration of antibody and volume of cell lysate, e,periments to e,tensively determine the optimal conditions. ). 'ncubate at 2C: for 1 hour.

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.. Add *0ul of !ashed ?rotein = slurry in prechilled 6ysis #uffer 0prepared as instructed in ?reclearing "tep 1 above-. 2. 'ncubate for 1 hour at 2C: on a roc ing platform or a rotator. *. "pin the eppendorf tube at1G.FFF,g for .0 seconds at 2C:. &. :arefully remove supernatant completely and !ash the beads .(* times !ith *00ul of 6ysis #uffer. To minimize bac gr 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 10,000,g for * minutes, collect supernatant and load onto the gel. "upernatant samples can be collected and point if the gel is to be run later. %. 3ollo! manufacturerJs instructions for "<"(?A=5. "tain the gel for visual analysis of the immunoprecipitated protein. 'f blot after this step, follo! the accompanying 'mmunoblotting 08#?rotocol. True#lotK is recommended for use as a se 8estern blot. 1?referably, the antibody used for the immunoprecipitation portion is not the same antibody used for the portion. A different clone !ith the same specificity is recommended. I33uno*reci*itation !u""ers N7-89 2e -ysis !u""er1
(

*0mL Tris(+:' p+ D.0 , ( *0mL@a:'

( 1M @?(20

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7rotease In6ibitor 2oc:tai /&99;01 ( ?L"3, *mg 0*0ug/ml( Aprotinin, 100ug 01ug/ml( 6eupeptin, 100ug 01ug/ml( ?epstatin, 100ug 01ug/mlRadia I33unodi""usion /Mancini0 /sing e di""usion in t5o 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 the precipitin ring !ith a standard carves, the concentration of the antigen sample can be determined.

di3ensions0 /Met6od0

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

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Doub e I33unodi""usion /Ouc6ter ony /Doub e 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" doub e i33unodi""usion

Sing e 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 -Sing e di""usion in one di3ension /Oudin0

Doub e di""usion in one di3ension /<u: ey-Fu t6ore 7rocedure0 +ere the antibody is incorporated in gel, above !hich is placed a column of plain agar. The antigen is layered on top of this. The antigen and antibody more to!ards each other through the intervening column of plain agar and form a band of precipitate !here they meet at optimum proportion.

Fig - Doub e di""usion in one di3ension /Oa: ey-Fu t6or*e0

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IMMUNOE-E2TRO7HORESIS
A ternative 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 ma e 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. <uring the procedure, the band is removed to restore circulation. Fnce the blood has been collected, the needle is removed, and the puncture site is covered to stop any bleeding. 'n infants or young children, the area is cleansed !ith antiseptic and punctured !ith a sharp needle or a lancet. The blood may be collected in a pipette 0small glass tube-, on a slide, onto a

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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 clonality 0monoclonal or this test is performed to assess the polyclonalof immunoglobulin. @o

monoclonal antibodies are detected. =6at Abnor3a Resu ts Mean 'n some malignant disorders 0that is, multiple myeloma, chrome lymphocytic leu emia- a single clone of lymphocytes produces one type of protein ( a monoclonal immunoglobulin. This is identifiable as monoclonal "ome 0all the same have typeby immunoelectrophoresis. people monoclonal

immunoglobulins, but do not have a malignant disorder. Ris:s O 5,cessive bleeding O 3ainting or feeling lightheaded O +ematoma 0blood accumulating under the s inO 'nfection 0a slight ris any time the s in is bro en;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 0"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 appro,imate 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. <ue .0 different proteins can be identified by this method in human serum. This is useful for testing for normal and abnormal proteins in serum and urine.

Fig - Se3iso id agar ayered on t6e g ass s ide

Fig - Antigen 5e "i ed 5it6 6u3an seru3

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Fig - Seru3 se*arated by e ectro*6oresis

Fig - Anti seru3 t6roug6 "i ed 5it6 antiseru3 to 5e

6u3an seru3+

Fig - Seru3 and antiseru3 a o5ed di""using into agar

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

E ectroi33unodi""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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0:ounterimmunoelctrophoresisin the clinical laboratory.

and

one

dimensional

single

electroimmunodiffuison 0roc et electrophoresis- are used frequently

&0 2ounteri33unoe ctro*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 i33unoe ectro*6oresis /2IE0+ Antigen and Antibody are driven toget6er by an e ectric current and a *reci*itin ine "or3s+

'0 One di3ensiona e ectro*6oresis0

sing e e ectroi33unodi""usion /roc:et

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 roc et and hence the name.

Fig - Roc:et e ectro*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 e ectro*6oresis 'n this technique, the antigen mi,ture is first electrophoretically separated in a direction perpendicular to that of the final soc et stage. #y this method one can quantitate each of several antigens in a mi,ture.

Fig - -aure >s variant o" roc:et e ectro*6oresis /t5o di3ensiona i33unoe ectro*6oresis0

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REFEREN2ES
1. Qenis

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

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

2. Abbas A./., 6ac man A./., ?ober Q.#., 'mmunology *th

edition,
3.

"ingapoor

high

court

press

company

publication, page no( *% to &%. Anand L.R., te,t boo of microbiology 2th edition, 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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