Serological tests (Antigen antibody interactions)

Classification of antigen-antibody interactions:

1. Primary serological tests: (Marker techniq es) e.g. a. Enzyme linked immuono sorben assay (ELISA) b. Immuno flurescent antibody technique (IFAT) c. adio immuno assay ( IA) !. Secondary serological tests: e.g. a. A!!lutination tests b. "om#lement fi$ation tests ("FT) c. %reci#itation tests d. Serum neutralization tests (S&T) e. To$in'antito$in test ". #ertiary serological test: e.g. a. (etermination of the #rotecti)e )alue of an anti serum in an animal.

A. Aggl tination tests:

1. Aggl tination$%emaggl tination *hen the anti!en is #articulate+ the reaction of an antibody ,ith the anti!en can be detected by a!!lutination (clum#in!) of the anti!en. The !eneral term a!!lutinin is used to describe antibodies that a!!lutinate #articulate anti!ens. *hen the anti!en is an erythrocyte the term heamagglutination is used. All antibodies can theoretically a!!lutinate #articulate anti!ens but I!-+ due to its hi!h )alence+ is #articularly !ood a!!lutinin and one sometimes infers that an antibody may be of the I!- class if it is a !ood a!!lutinatin! antibody. a. & alitati'e aggl tination test A!!lutination tests can be used in a qualitati)e manner to assay for the #resence of an anti!en or an antibody. The antibody is mi$ed ,ith the #articulate anti!en and a #ositi)e test is indicated by the a!!lutination of the #articulate anti!en For e$am#le+ a #atient.s red blood cells can be mi$ed ,ith antibody to a blood !rou# anti!en to determine a #erson.s blood ty#e. In a second e$am#le+ a #atient.s serum is mi$ed ,ith red blood cells of a kno,n blood ty#e to assay for the #resence of antibodies to that blood ty#e in the #atient.s serum .

b. & antitati'e aggl tination test A!!lutination tests can also be used to measure the le)el of antibodies to #articulate anti!ens. In this test+ serial dilutions are made of a sam#le to be tested for antibody and then a fi$ed number of red blood cells or bacteria or other such #articulate anti!en is added. Then the ma$imum dilution that !i)es a!!lutination is determined. The ma$imum dilution that !i)es )isible a!!lutination is called the titer. The results are re#orted as the reci#rocal of the ma$imal dilution that !i)es )isible a!!lutination. Pro(one effect - )ccasionally+ it is obser)ed that ,hen the concentration of antibody is hi!h (i.e. lo,er dilutions)+ there is no a!!lutination and then+ as the sam#le is diluted+ a!!lutination occurs. The lack of a!!lutination at hi!h concentrations of antibodies is called the prozone effect. Lack of a!!lutination in the #rozone is due to antibody e$cess resultin! in )ery small com#le$es that do not clum# to form )isible a!!lutination. c. A**lications of aggl tination tests 1. (etermination of blood ty#es or antibodies to blood !rou# anti!ens. !. To assess bacterial infections

e.g. A rise in titer of an antibody to a particular bacterium indicates an infection with that bacterial type. N.B. a fourfold rise in titer is generally taken as a significant rise in antibody titer.

!-Passi'e hemaggl tination: The a!!lutination test only ,orks ,ith #articulate anti!ens. /o,e)er+ it is #ossible to coat erythrocytes ,ith a soluble anti!en (e.!. )iral anti!en+ a #olysaccharide or a ha#ten) and use the coated red blood cells in an a!!lutination test for antibody to the soluble anti!en. This is called #assi)e hema!!lutination. The test is #erformed 0ust like the a!!lutination test. A##lications include detection of antibodies to soluble anti!ens and detection of antibodies to )iral anti!ens. "-Coomb+s #est (Antiglob lin #est): a. ,irect Coomb+s #est *hen antibodies bind to erythrocytes+ they do not al,ays result in a!!lutination. This can result from the anti!en1antibody ratio bein! in anti!en e$cess or antibody e$cess or in some cases electrical char!es on the red blood cells #re)entin! the effecti)e cross linkin! of the cells. These antibodies that bind to but do not cause a!!lutination of red blood cells are sometimes referred to as incom#lete antibodies. In no ,ay is this meant to indicate that the antibodies are different in their structure+ althou!h this ,as once thou!ht to be the case. ather+ it is a functional definition only. In order to detect the #resence of non'a!!lutinatin! antibodies on red blood cells+ one sim#ly adds a second antibody directed a!ainst the immuno!lobulin (antibody) coatin! the red cells. This anti'immuno!lobulin can no, cross link the red blood cells and result in a!!lutination. b. -ndirect Coomb+s #est If it is necessary to kno, ,hether a serum sam#le has antibodies directed

a!ainst a #articular red blood cell and you ,ant to be sure that you also detect #otential non' a!!lutinatin! antibodies in the sam#le+ an Indirect "oomb.s test is #erformed. This test is done by incubatin! the red blood cells ,ith the serum sam#le+ ,ashin! out any unbound antibodies and then addin! a second anti'immuno!lobulin rea!ent to cross link the cells. c. A**lications These include detection of anti'rhesus factor ( h) antibodies. Antibodies to the h factor !enerally do not a!!lutinate red blood cells. Thus+ red cells from h2 children born to h' mothers+ ,ho ha)e anti' h antibodies+ may be coated ,ith these antibodies. To check for this+ a direct "oombs test is #erformed. To see if the mother has anti' h antibodies in her serum an Indirect "oombs test is #erformed. .-%emaggl tination-nhibition The a!!lutination test can be modified to be used for the measurement of soluble anti!ens. This test is called hema!!lutination inhibition. It is called hema!!lutination inhibition because one measures the ability of soluble anti!en to inhibit the a!!lutination of anti!en'coated red blood cells by antibodies. In this test+ a fi$ed amount of antibodies to the anti!en in question is mi$ed ,ith a fi$ed amount of red blood cells coated ,ith the anti!en. Also included in the mi$ture are different amounts of the sam#le to be analyzed for the #resence of the anti!en. If the sam#le contains the anti!en+ the soluble anti!en ,ill com#ete ,ith the anti!en coated on the red blood cells for bindin! to the antibodies+ thereby inhibitin! the a!!lutination of the red blood cells.

/. Preci*itation tests:
1-0adial-mm nodiff sion(Mancini) In radial immunodiffusion antibody is incor#orated into the a!ar !el as it is #oured and different dilutions of the anti!en are #laced in holes #unched into the a!ar. As the anti!en diffuses into the !el+ it reacts ,ith the antibody and ,hen the equi)alence #oint is reached a rin! of #reci#itation is formed. !--mm noelectro*horesis: In immunoelectro#horesis+ a com#le$ mi$ture of anti!ens is #laced in a ,ell #unched out of an a!ar !el and the anti!ens are electro#horesed so that the anti!en are se#arated accordin! to their char!e. After electro#horesis+ a trou!h is cut in the !el and antibodies are added. As the antibodies diffuse into the a!ar+ #reci#itin lines are #roduced in the equi)alence zone ,hen an anti!en1antibody reaction occurs. This tests is used for the qualitati)e analysis of com#le$ mi$tures of anti!ens+ althou!h a crude measure of quantity (thickness of the line) can be obtained. This test is commonly used for the analysis of com#onents in a #atient. serum. Serum is #laced in the ,ell and antibody to ,hole serum in the trou!h. 3y com#arisons to normal serum+ one can determine ,hether there are deficiencies on one or more serum com#onents or ,hether there is an o)erabundance of some serum com#onent (thickness of the line). This test can also be used to e)aluate #urity of isolated serum #roteins.

"- Co nterc rrent electro*horesis: In this test the anti!en and antibody are #laced in ,ells #unched out of an a!ar !el and the anti!en and antibody are electro#horesed into each other ,here they form a #reci#itation line. This test only ,orks if conditions can be found ,here the anti!en and antibody ha)e o##osite char!es. This test is #rimarily qualitati)e+ althou!h from the thickness of the band you can !et some measure of quantity. Its ma0or ad)anta!e is its s#eed.

C. Com*lement fi1ation test:

4 The com*lement fi1ation test is an immunological medical test lookin! for e)idence of infection. It tests for the #resence of either s#ecific antibody or s#ecific antigen in a #atient.s serum. It uses shee# red blood cells (s 3")+ anti's 3" antibody and com#lement+ #lus s#ecific anti!en (if lookin! for antibody in serum) or s#ecific antibody (if lookin! for anti!en in serum). 4 If either the antibody or anti!en is #resent in the #atient.s serum+ then the com#lement is com#letely utilized+ so the s 3"s are not lysed. 3ut if the antibody (or anti!en) is not #resent+ then the com#lement is not used u#+ so it binds anti's 3" antibody+ and the s 3"s are lysed. 4 The Wassermann test is one form of com#lement fi$ation test.

,. 2n(yme-3inked -mm noSorbent Assay (23-SA):

4 2n(yme-3inked -mm noSorbent Assay+ or 23-SA+ is a biochemical technique used mainly in immunolo!y to detect the #resence of an antibody or an anti!en in a sam#le. The ELISA has been used as a dia!nostic tool in medicine and #lant #atholo!y+ as ,ell as a quality control check in )arious industries. In sim#le terms+ in ELISA an unkno,n amount of anti!en is affi$ed to a surface+ and then a s#ecific antibody is ,ashed o)er the surface so that it can bind the anti!en. This antibody is linked to an enzyme+ and in the final ste# a substance is added that the enzyme can con)ert to some detectable si!nal. Thus in the case of fluorescence ELISA+ ,hen li!ht is shone u#on the sam#le+ any anti!en1antibody com#le$es ,ill fluoresce so that the amount of anti!en in the sam#le can be measured. 1.-ndirect 23-SA: 4 The ste#s of the !eneral+ 5indirect+5 ELISA for determinin! serum antibody concentrations are6 7. A##ly a sam#le of kno,n anti!en of kno,n concentration to a surface+ often the ,ell of a microtiter #late. The anti!en is fi$ed to the surface to render it immobile. Sim#le adsor#tion of the #rotein to the #lastic surface is usually sufficient. These sam#les of kno,n anti!en concentrations ,ill constitute a standard cur)e used to calculate

anti!en concentrations of unkno,n sam#les. &ote that the anti!en itself may be an antibody. 8. The #late ,ells or other surface are then coated ,ith serum sam#les of unkno,n anti!en concentration+ diluted into the same buffer used for the anti!en standards. Since anti!en immobilization in this ste# is due to non's#ecific adsor#tion+ it is im#ortant for the total #rotein concentration to be similar to that of the anti!en standards. 9. A concentrated solution of non'interactin! #rotein+ such as 3o)ine Serum Albumin (3SA) or casein+ is added to all #late ,ells. This ste# is kno,n as blockin!+ because the serum #roteins block non' s#ecific adsor#tion of other #roteins to the #late. :. The #late is ,ashed+ and a detection antibody s#ecific to the anti!en of interest is a##lied to all #late ,ells. This antibody ,ill only bind to immobilized anti!en on the ,ell surface+ not to other serum #roteins or the blockin! #roteins. ;. The #late is ,ashed to remo)e any unbound detection antibody. After this ,ash+ only the antibody'anti!en com#le$es remain attached to the ,ell. <. Secondary antibodies+ ,hich ,ill bind to any remainin! detection antibodies+ are added to the ,ells. These secondary antibodies are con0u!ated to the substrate's#ecific enzyme. This ste# may be ski##ed if the detection antibody is con0u!ated to an enzyme. =. *ash the #late+ so that e$cess unbound enzyme'antibody con0u!ates are remo)ed. >. A##ly a substrate ,hich is con)erted by the enzyme to elicit a chromo!enic or fluoro!enic or electrochemical si!nal. ?. @ie,1quantify the result usin! a s#ectro#hotometer+ s#ectrofluorometer+ or other o#tical1electrochemical de)ice. !.Sand4ich 23-SA 6 A sand4ich ELISA: %late is coated ,ith a ca#ture antibody sam#le is added+ and any anti!en #resent binds to ca#ture antibody detectin! antibody is added+ and binds to anti!en enzyme'linked secondary antibody is added+ and binds to detectin! antibody substrate is added+ and is con)erted by enzyme to detectable form. A less'common )ariant of this technique+ called 5sand,ich5 ELISA+ is used to detect sam#le anti!en. The ste#s are as follo,s6 7. %re#are a surface to ,hich a kno,n quantity of ca#ture antibody is bound. 8. 3lock any non s#ecific bindin! sites on the surface. 9. A##ly the anti!en'containin! sam#le to the #late. :. *ash the #late+ so that unbound anti!en is remo)ed. ;. A##ly #rimary antibodies that bind s#ecfically to the anti!en. <. A##ly enzyme'linked secondary antibodies ,hich are s#ecific to the #rimary antibodies. 4

=. *ash the #late+ so that the unbound antibody'enzyme con0u!ates are remo)ed. >. A##ly a chemical ,hich is con)erted by the enzyme into a color or fluorescent or electrochemical si!nal. ?. -easure the absorbance or fluorescence or electrochemical si!nal (e.!.+ current) of the #late ,ells to determine the #resence and quantity of anti!en. ".Com*etiti'e 23-SA6 4 A third use of ELISA is throu!h com#etiti)e bindin!. The ste#s for this ELISA are some,hat different than the first t,o e$am#les6 7. Anlabeled antibody is incubated in the #resence of its anti!en. 8. These bound antibody1anti!en com#le$es are then added to an anti!en coated ,ell. 9. The #late is ,ashed+ so that unbound antibody is remo)ed. (The more anti!en in the sam#le+ the less antibody ,ill be able to bind to the anti!en in the ,ell+ hence 5com#etition.5) :. The secondary antibody+ s#ecific to the #rimary antibody is added. This second antibody is cou#led to the enzyme. ;. A substrate is added+ and remainin! enzymes elicit a chromo!enic or fluorescent si!nal. 4 For com#etiti)e ELISA+ the hi!her the ori!inal anti!en concentration+ the ,eaker the e)entual si!nal. ..A**lications: 4 3ecause the ELISA can be #erformed to e)aluate either the #resence of anti!en or the #resence of antibody in a sam#le+ it is a useful tool both for determinin! serum antibody concentrations (such as ,ith the /I@ testB7C or *est &ile @irus) and also for detectin! the #resence of anti!en. It has also found a##lications in the food industry in detectin! #otential food aller!ens such as milk +#eanuts +,alnuts +almonds + and e!!s B8CThe ELISA test+ or the enzyme immunoassay (EIA)+ ,as the first screenin! test commonly em#loyed for /I@. It has a hi!h sensiti)ity. In an ELISA test+ a #erson.s serum is diluted :DD'fold and a##lied to a #late to ,hich /I@ anti!ens ha)e been attached. If antibodies to /I@ are #resent in the serum+ they may bind to these /I@ anti!ens. The #late is then ,ashed to remo)e all other com#onents of the serum. A s#ecially #re#ared 5secondary antibody5 E an antibody that binds to human antibodies E is then a##lied to the #late+ follo,ed by another ,ash. This secondary antibody is chemically linked in ad)ance to an enzyme. Thus the #late ,ill contain enzyme in #ro#ortion to the amount of secondary antibody bound to the #late. A substrate for the enzyme is a##lied+ and catalysis by the enzyme leads to a chan!e in color or fluorescence. ELISA results are re#orted as a numberF the most contro)ersial as#ect of this test is determinin! the 5cut'off5 #oint bet,een a #ositi)e and ne!ati)e result.