7 - Immunoassays Principles and Assay Design

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Immunoassays:principles and assaydesignM. A. CherneskyJ. B. MahonyImmunoassays may be constructed as l iquidphase (LPIA) or sol id-p has e (SPIA) types. LPIAwere popularized in the f ield of chemistry buthave not been extensively explored for micro-biology. Most LPIA are compet i t ive betweenassayed sample and labeled sample. Af terdetermination of the fraction of labeledsample bound by ant igen or ant ibody theconcentrat ion of sample can be calculated bycompar ison wi th resul ts for appropr iatestandards. In the absence of a sol id phase toseparate labeled and unlabeled samples the

procedure depends upon a change in thespeci f ic enzyme act ivi ty when ant ibody-ant igen complexes form. SPIA for the detec-t ion of ant igens or ant ibodies use as react ionindicators: a radioactive label for the radio-immun oassay (RIA), an enzyme which wi llreact wi th a substrate in an enzyme immuno-assay (EIA), or a f luoresce nt dye used in af luorescent immunoassay (FIA) . The sub-strate to be acted on by an enzyme may bef luorescent , radioact ive, chemi luminescent orchromogenic.

Virology Methods ManualISBN 0-12-465330-8 Copyright 9 1996 Academic Press LtdAll rights of reproduction in any form reserved


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V i r o lo g y m e t h o d s m a n u a l

Antigen detectionMost SPIAs for the detection of antigenemploy one of three methods: (1) competi-t ive; (2) direct (double-antibody sandwichmethod); or (3) indirect (double-antibody sand-wich-antiglobulin method). In competit iveassays, labeled antigen is mixed with the testsample that may contain antigen and theycompete for a l imited amount of antibodyattached to the solid phase. A negative con-trol sample containing only labeled antigen isincluded. Unbound antigen is washed awayand the difference in indicator activitybetween the specimen and control is mea-sured. A direct SPIA for detection of antigeninvolves adding the cl inical specimen to acapture antibod y ( CA ) attached to the solidphase. Unbound antigen is washed awaybefore the addit ion of a labeled detector anti-body (DA). The DA is measured and the morelabeled substrate detected, the more antigenis present in the sample. The indirect test issimilar to the direct assay employing a CA andDA, but the DA is not labeled. Instead, a thirdindicator a ntibod y (IA) that is anti-species toDA is labeled; the remainder of the assay issimilar to the direct procedure. This approachhas become the most popular because of theavailabil i ty of IA conjugates from commercialsources. The indirect test provides som e ampli-f ication of the binding reactions. They maycreate some problems, however, as they arevery sensit ive and anti-species antisera maycross-react nonspecif ically.

Competitive assaySpecif ic antigen present in a test specimeninhibits the binding of a predetermined amountof labeled antibody to antigen immunobil izedon a solid phase. The detec tor antibod y can belabeled with enzym e, 1251 or biotin and the solidphase can be beads, plates or tubes.

The fol lowing protocol describes the detec-tion of rat rotavirus using biotinylated antibody

and peroxidase labeled avidin-biotin complex(Vonderfecht et al 1985):

1. Coat wells of a polystyrene microtiterplate ( immulon 2; DynatechLaboratories Inc., Alexandria, VA) with100 I~1 of ei the r rat rota virus ant igen orcontrol antigen diluted optimally incarbonate buffer, pH 9.6 overnight at4~ The optimal conce ntrations ofviral antigen and biotin-labeledantibody are determined bycheckerboard t i tration.

2. Mix an equal v olum e (50 i~1) of the tes tspecimen, di luted 1 : 5 in fetal bovineserum (FBS), wi th biot in-c0njugatedantirotavirus antibody diluted inphosphate buffered saline (PBS)containing 0.05% Tween 20 and 0.5%gelatin and incubate for 1 h at 37~then place into precoated microtiterplate wells. Incubate for 1 h at 37~then wash wells three t imes with PBS.

3. Add 0.1 ml of preformed peroxidase-avidin-biotin complex (VectorLaboratories, Burlingame, CA) andincubate for 1 h at 37~4. Wash the wells, add the substrate(0.4 mg OPD and 0.4 ~1 of 30% H202per ml of 0.01 M citrate buffer, pH 5)and read at 450 nm. Subtract the ODof wells with control antigen from theOD of wells with rotavirus antigen todetermine specif ic activity anddetermine percent inhibit ion as 100 x(1 - net specif ic activity divided bynegative control). The specif icity of theinhibition reaction can be verified byblocking with specif ic immune serum.

C o m m e n tDespite the relative ease of performingthe competit ive assay and its excellent

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Antigen detectionsensit ivity this assay has not been widelyadopted in cl inical laboratories.

Sandwich assayThe assay can be either a direct (using anenzyme-labeled detector antibody as the indi-cator) or an indirect (using a second enzyme-labeled anti species antibody fol lowing thedetector) assay and employ either polyclonalor monoclonal antibody reagents. Assays withthe combination of polyclonal capture andmonoclonal detector antibodies usually pro-vide the best performance. Sandwich assayshave been developed for a number of viruses(rotavirus, influenza, R SV ) and several areavailable as commercial kits. The fol lowingprotocol describes the detection of rotavirusantigen in stool specimens using 96-wellmicrotiter plates (Yolken et al 1980).

and incubate for 30 min at roomtemperature.5. Calculate specif ic reactivity by

subtracting the mean activity of thespecimen in wells coated with non-immune serum from the mean activityof wells coated with antirotavirusantiserum. A specimen is consideredpositive if its mean reactivity is greaterthan 2 standard deviations above themean of the negative controls.

Co m m e n tThe direct assay has the advantage ofrequiring only a single detectionantibody. The indirect assay may be moreconvenient i f the laboratory is doing anumber of different EIAs and wants touse a single enzyme-labeled indicatorantibody. The indirect assay is generallymore sensit ive than the direct EIA.

1. Coat alternate rows of microtiterplates with goat antirotavirusant iserum and non- immune goatserum (0.1 ml) diluted 1:10,000 in PBS(0.5 t~g ml-1) and incubate overnightat 4~ Wash plate five time s withPBST.2. Ad d 5 0 !~1 dilut ed sto ol filtra te an d50 !~1 N-acetylcysteine (to decreasebinding of stool material to non-immune globul in) in dupl icate to theantirotavirus antibody and controlantibody coated wells, and incubatefor 2 h at 37~ Wash the plate fivetimes with PBS.

3. A dd 100 #1 enz ym e-lab eledantirotavirus antibody optimallydiluted (approximately 1:2000)in PBScontaining 2% fetal bovine serum andincub ate for 1 h at 37~ Wash fivetimes.

4. Add freshly prepared substrate (p-nitrophenyl phosphate for alkalinephosphatase or OPD for peroxidase)

Immunodot assayThe imm uno dot assay (IDA) for antigen is simi-lar to Western blott ing for antibody as both usenitrocellulose membranes. In IDA viral antigenin a cl inical specimen is detected by dott ingthe specimen onto nitrocellulose and reactingthe strip with antiviral antibody in either adirect or indirect format. IDA has been usedto detect HIV fol lowing amplif ication of virusin H9 cell culture (Blumberg et al 1987) and todetect rotavirus antigen in stool specimens. Amodif ication of IDA called immune complexdot assay ( ICDA), whe rean t igen is al lowed toreact with specif ic antibody and the antigen-antibody complex is spotted onto nitro-cellulose and detected with colloidal gold-labeled anti species antibody, has beendescribed (Wu et al 1990). ICDA was moresensit ive than IDA for detecting rotavirusantigen in stool specimens.

The fol lowing protocol describes the detec-tion of rotavirus antigen by IDA:

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V i r o lo g y m e t h o d s m a n u a l1. S pot 1 ~1 of stool s pecim en (10%stool f i l trate in dH20) togetherwith positive and negative controlsonto nitrocellulose strips with aDrummond microdispenser and

allow to dry for 15 min at roomtemperature.2. Block the strips with 2 ml of 1% B SA -0.5% gelatin in PBS for 20 min at37~3. React the strips with rabbit anti-rotavirus antiserum diluted 1:1000 inblocking solution for 90 min at 37~

then wash strips with PBS twice for5 min each.4. React the strips with gold labeled-an tirabbit IgG antiserum (GAR G15,Janssen Chimica) diluted 1:10 for 2 hat room temperature on an orbitalshaker (50 rpm). Wash twice with PBSand once with dH20.5. Place the strips into silverenhancement solution (JanssenChimica) for 20 min then wash withtap water and air dry.

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A n t i b o d y d e t e c t i o nAntibody detectionCompetitivemeasurementin a compet i t ive assay format human ant ibodyagainst a viral antigen(s) can be measured bycombining the sample wi th a predeterminedamount of conjugated ant ibody di rectedagainst the same viral antigen(s) and incubat-ing the mixture with the antigen-coated sol idphase. Specif ic antibody, i f present in thesample, wil l compete with the conjugated anti-body for binding sites on the sol id phase andlead to a reduction in signal. The signal gener-ated wil l be inversely proport ional to theamount of sample ant ibody.

Antibodies against the antigens of interestcan be prepared in animals or purif ied fromhuman serum. Alternatively, specif ic mono-clonal antibodies can be developed. The anti-bodies must be purif ied, conjugated with t l ' ieappropriate label ( f luorochrome, enzyme,lanthanide, or radioisotope), and r igorouslyevaluated before use. An important aspect ofconjugate evaluation involves the serial t i tra-t ion of the conjugate against serial di lut ions ofantigen to measure activi ty and define theti trat ion curve of the conjugate across severalantigen concentrat ions. Each conjugate t i tra-t ion curve wil l exhibit a point at which the nextdi lut ion of conjugate wil l exhibit a substantialdrop in signal (Fig. 7.1). Several conjugate-antigen di lut ion pairs that meet the basic cr i-ter ia should be selected then further charac-terized with respect to sensit ivi ty, usingpreparat ions containing known amounts ofantigen-specif ic antibody. The goal is toselect the conjugate antigen pair that con-tains the highest di lut ion of conjugate andantigen which wil l :(i) perm it the gen eration of high signal(ii) exhibit a substantial reduction in signal in

the presence of sample ant ibody(i i i ) al low the measurement of small amounts

090.80.7

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0,30.20.1

050 t00 200 400 800 1600Conjugate Dilution

Figure 7 .1 . Titration of an ti IgG conjugate. BKvirus antigen (top line) or control antige n (bottomline) we re coated onto w ells of a 96-well microtiterplate. Mouse an ti BK virus antiserum diluted 1:1000was the detection antibody. Goat anti-mouse IgGantibody conjugate was serially diluted anddetected with OPD substrate.of sample ant ibody against al l importantantigenic determinants.

Competit ive assays are based on a l imit ing-reagent concept where sol id-phase ant igenand conjugate concentrat ions are minimizedso that even small quantit ies of sample anti-body can ef fect ively block the binding of con-jugate. An advantage to using the compet i t iveformat for ant ibody measurement is the rela-t ive ease with which highly specif ic antibodies,especial ly monoclonal antibodies, can be puri-f ied and conjugated. In general, antibodies areeasier to purify than antigens, and since thespeci f ic i ty of the test is conveyed by the con-jugate relat ively impure antigen can be used onthe sol id phase. Another advantage is a reduc-t ion in the number of procedural steps sincesample and conjugate can be incubated wi ththe antigen on the sol id phase. Competit iveassays are general ly more sensit ive than indi-rect assays. Competit ive RIAs and EIAs havebeen used to measure ant ibody to HB core Ag(anti-HBc). The anti-HBc assay has been com-mercial ized by Abbo tt Diagnost ics and has

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V i r o l o g y m e t h o d s m a n u a lbeen used extensively in clinical laboratoriesfor over twenty years (Mushahwar et al 1978;Purcell et al 1984).The fol lowing procedure describes thedetection of anti-HBc by competit ive RIAusing plastic beads.

1. Viral antigen is used to coat 6 mmpolystyrene beads by passiveadsorption using carbonate bufferpH 9.6 (with or without the use ofinit ial capture antibody coated to thesolid phase).2. An aliquot of serum undiluted (0.1 ml)is mixed with 0.1 ml of 1251-anti-HBcsolution, the mixture is added to anantigen coated bead and incubatedovernight at room temperature.

3. The bead is washed with water andcounted in a gamma counter. Areduction of 50% in the countcompared to a negative controlsample is considered posit ive for anti~HBc.

Co m m e n tThe format of this assay has beenchanged over the years from RIA to EIAand more recently to microparticle EIAbut the principle of the assay hasremained unchanged. The competit iveassay format can be used to measureantibody to any virus and provides asimple (one-step) sensitive assay.

Non-competitive IgG ortotal antibodymeasurementEither direct or indirect approaches may beused to construct a SPIA to measure anti-bodies in a patient's specimen. The solidphase is usually coated with either antigen(s),or anti- immunoglobulins (anti-total or anti-

class, i.e . anti-lgG , -IgM , -IgA etc.). If the solidphase is coated with antigen all classes ofantibodies in the specimen wil l be trapped. Aconjugated DA which is anti-species can thenbe used to detect al l antibody classes trappedor the DA can be specif ic for a class such asanti-human IgM or IgG. This approach usuallyrequires the pre-adsorption of the sample withanti-rheum atoid factor (RF) or aggregated IgGto eliminate RF interference (described in moredetail below). When anti-species antibodiesare used on the solid phase they are usuallyanti- lgM. This approach wil l capture only IgM,which wil l be available for the DA which can belabeled antigen available to act on an addedsubstrate. Alternatively at this stage an unla-beled antigen is added then detected by avirus-specif ic DA (which may be labeled for adirect assay) or followed by a labeled anti-species antibody.A further modif ication of the antibody classcapture assay, and one that will permitthe measurement of ant igen-speci f ic IgG,involves the use of labeled antigen and asolid phase coated with RF-IgM. A samplecontaining antigen-specif ic igG is mixed withlabeled conjugate and incubated with thesolid phase coated with RF-IgM. Only anti-gen-specif ic IgG complexed with labeled anti-gen is bound by the RF-IgM; therefore, only asingle incubation step is required. Antigenquality, as well as the quality of RF-IgM coat-ing the solid phase, must be carefullyassessed.The noncompet i t ive technique has beenwidely used to measure specif ic anti-viral anti-body because it requires only a few reagents,is easy to perform and provides an objectivequantifiable result. The assay is most oftenperformed in microtiter plates or on plasticbeads although it can be adapted to plastictubes, microparticles, paramagnetic beads orother solid surfaces. This format has allowedcommercial companies to develop automatedinstrumentation with increased throughput forprocessing several hundred specimens perday.The fol lowing procedure describes the detec-tion of BK papovavirus antibody (Mahony et al1989) but it can be used to detect either IgG or

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A n t i b o d y d e t e c t i o nigM antibody to any virus for which antigen canbe obtained:

Procedure1. BK virus purified by CsCIultracentrifugation from infected cellculture is diluted in carbonate buffer,pH 9.6 and passively adsorbed towells of 96 well polystyrene microtiterplates for 2 h at 37~2. The plates are wash ed with phosp hatebuffered solution (PBS) and blockedwith PBS containing 10% FBS for 2 hat 37~ The blocking reagent is

removed and the plate dried bytapping on paper towels. Controlantigen-coated wells are prepared inthe same way using mock-infectedcultures.3. Serial two fold dilutions of sera to betes ted (50 ~1) dilu ted in Buffer G(0.01 M pho sph ate buffer pH 7.2, 0.3 MNaCI, 0.001 M MgCI2, 0.5% gelatin ,0.1% FBS) are incubated in duplicatein BKV antigen and control antigencoated wells for 3 h at 37~4. BKV IgG or IgM antibody is detectedby a dding 50 ILtl of HR P-co njug atedgoat anti human IgG or IgMimmunoglobulin diluted optimally inbuffer G (usually 1:400-1:1200depending on the supplier) for 1 h at37~5. OPD substrate (50 ~1)is added andthe plates are incubated for 30 minfollowed by 3 N HCI stop solution(50 !~1). The pla tes are read in amicrotiter plate ELISA reader at492 nm. The cutoff for positivity isestablished as twice the mean (or X +3 SE) of 5-10 representative negativespecimens (obtained from a similarpatient population).

Co m m e n tThere are many variations of this EIAprotocol. Some use blocking solutions

and diluents containing Tween-20 orNP40 detergents, some incubate at roomtemperature, 37~ or 4~ some useHRP-conjugate, while others use alkalinephosphatase conjugates (see below). Wehave successfully used the aboveprotocol with minor modification tomeasure specific antibody to BKV,Chlamydia trachomatis, Parvovirus B19and CMV.

ImmunoblottingImmunoblotting or Western blotting has beenused to detect specific antibodies to a numberof viruses. Viral proteins are first separated bypolyacrylamide gel electrophoresis and trans-blotted to nitrocellulose or nylon membranesthen reacted with clinical specimens usuallysera, but saliva or urine can be tested. Recom-binant imm unob lot assa ys (RIBA) use recom-binant proteins expressed in prokaryotic oreukaryotic expression systems instead of viralpolypeptides in the form of purified viral anti-gen prepared from infected cell culture. Wes-tern blot and RIBA have been successfullycommercialized and supplementary or confir-matory kits are available for detecting antibod yto a number of viruses including HIV-1, HTLV-Iand H CV. These assays can also be used fortyping herpes simplex virus and HIV. The follow -ing protocol describes a Western blot assay fordetecting HSV-1 and HSV-2 specific an tibody asdescribed by Ashley and Militoni (1987).

1. HSVol (strain KOS) and HSV-2 (strain333) viruses are used to infectseparate cultures of human diploidfibroblast ceils (MOI of 1). Cells areharvested at 3+ CPE by gentlyscraping with a rubber policeman.2. Cells are washe d in PBS, collected b ylow speed centrifugation (5 min at500 g) and suspended in a smallvolum e (0.25 m l for each 75 cm 2 flask)of lysis buffer (0.125 M tris, pH 6.8,20% glycerol, 8% SDS, 10%

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V i r o lo g y m e t h o d s m a n u a l

.

.

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2-mercaptoethanol, 0.025%Bromphenol Blue) and frozen inal iquots at -70~Aliquots of HSV-1 and HSV-2 arethawed, boiled for 10 min, andelectrophoresed on a 9%polyac rylamide gel with a 4~ stackinggel at 100 volts for 2 h. Proteins areelectrophoretically transferred tonitrocellulose at 100 V for 1 h (Bio Radmini Protein gel transblotter).Reactive sites are blocked byincubating the NC paper in 5% (w/v)Blotto in PBS for 1 h at roomtemperature on an orbital shaker.Rinse with 0.1% Tween-20 in PBS(PBST) twice for 10 min each and cutNC into str ips 3 mm wide.Dilute serum to be tested 1:50 in PBSand incubate for 1 h at roomtemperature with one HSV-1 and oneHSV-2 str ip. Rinse the str ips twice inPBS then incubate in HRP-conjugatedgoat anti human IgG antibody diluted1:500 in PBS for 1 h with gentleshaking. Rinse the str ips again andincubate in freshly prepared 4-chloro-1-naphthol (0.5 mg m1-1) substratecontaining 0.025% hydrogen peroxidein PBS and allow color to develop for5-10 min then stop with disti l ledwater. Dry and store strips in thedark.

Co m m e n tFor the detection of HSV-1 and HSV-2specif ic antibody each serum must betested on an HSV-1 and HSV-2 str ip (Fig.7.2). Interpretation is based on thefollowing criteria (Ashley et al 1993). Apredominance of ant ibody to ei therHSVol or HSV-2 blots is interpreted asinfection with HSVol or HSV-2respectively. HSV-1 posit ive sera wil lcontain antibodies to 11-18 HSV-1proteins (gB, gG, gC-GE, VP16, gD, p45)and lack antibody to the 92 kDa HSV-2glycoprotein G (gG-92) on the HSV-2blot. HSV-2 posit ive sera generally show

1 3 0

HSV-I HSV-2

; =

ill" a

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Figure 7.2 . Immu noblot detection of HSV-1 andHSV-2 type-specific antibody. Serum from two indi-viduals with culture-documented HSV-1 and HSV-2infections w ere reacted with immuno blot strips ofHSV-1 (1) or HSV-2 (2)in fected ce ll lysates. Anti-bodies were visualized wi th HRP-anti human IgGantibody conjugate and 4-chloro-l-nap hthol.

fainter bands than HSV-1 posit ive seratherefore, some sera showing strongreactivity on the HSV-2 blot will requirepreadsorption to clarify the HSV-1 type.HSV-2 posit ive sera react primari ly withproteins in the HSV-2 str ip, have a gG-92band and wi l l show 16-24 HSV-2 bands(major bands of 150 kDa, gG-92, gG-70,52 kDa, 49 kDa, 47 kDa, 45 kDa) andfewer than 6 HSV-1 bands. Sera withboth HSV-1 and HSV-2 antibodies havefull antibody profi les on both str ips and aclear gG-92 band. Some sera wil l requireadsorption with type 1 or 2 virus fortyping.

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Antibody detectionRadioimmuno-precipitation (RI PA)in R IP A specific anti-viral an tibodies aredetected by immunoprecipitation of radio-labeled viral proteins followed by either scintil-lation counting of washed precipitates orimmune complex dissociation, polyacryla-mide gel electrophoresis and autoradiographyto identify specific proteins. This method hasnot had widespread application in virology butis still used by some laboratories in place ofWestern blotting as a confirmatory test for HIV.The following protocol for HIV antibod y hasbeen adapted from Tersmette et al (1988).

1. HIV-1 is propagated in H9 cells in thepresence of 35S-methonine or 3SS-cyste ine (10 ILtCi m1-1) for 8 h and acell lysate is prepared by scraping andhom oge nizing cells in 0.125 M TFis-HCI, pH 6.8, 8% SDS, 0.1 M2-mercaptoethanol.An alternative approach is to purifyHIV on a sucrose gradient, treat with0.2% Triton X-100 and label with 1251using chloramine-T (the preparationcan be enriched for HIV glycoproteinsby adsorption to lenti l- lectinsepharose beads and elution with0.3 M 0~-methyI-D-mannoside).2. An aliquot of viral lysate containing5 x 104 cpm of labeled protein isincu bate d w ith 5 ~1 of serum for 16 hat 4~3. An aliquot of protein A-Sepharosebeads (5 mg)is added and the mixtureincubated 90 min, pelleted andwashed four times with PBS followedby a final wash in 10 mM Tris-HCI, pH6.8.4. Bo und immun oprecipitates are elutedwith 8% SDS and 0.1 M2-mercaptoethanol and analyzed bypolyacrylamide gel electrophoresisand autoradiography.

Co m m e n tRIPA is a labour intensive test employ ingmetabolic or surface radiolabeling andimmunoprecipitation techniques that areusually restricted to researchlaboratories. It has the advantage ofbeing more sensitive and specific thanWestern blot especially for the detectionof high molecular weight glycoproteins ofHIV.

IgM measurementThe most common assays for IgM use astandard indirect solid-phase ELISA with theantigen immobilized and an IgM-specific sec-ondary DA. As mentioned above false positiv-ity is common due to the presence of IgMrheumatoid factor (RF) in the pa tient sample.False negativity can also occur from competi-t ive inhibit ion of IgM binding in the presence o fhigh levels of specific IgG. One way toapproach these problems is to use an isolatedIgM preparation from the patient sample,removing IgG which acts as a substrate forIgM RF and competes with the specific IgMfor binding to the antigen or remove all of theIgG with the addition of a precipitating anti-igG. An alternative approach is to remove theRF itself by using an aggregated IgG prepara-tion. RF false positives can be eliminated bytreatment of serum with commercial adsor-bents such as RF sorbent (Behringwerke)(Zapata et al 1984).

A second approach is to modify the type ofimmunoassay by using an igM capture assay.In this procedure, a polyclonal anti-lgM anti-body is bound to the solid phase. Upon incu-bation of the patient sample, all IgM will becaptured on the plate. The test antigen isthen added, binding any specific IgM presenton the plate. An enzyme-labeled secondaryantibody is then added, and the reaction iscompleted. This assay obviates the problemswith false-negative results due to competit iveinhibition with IgM, as all of the IgG in the

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V i r o lo g y m e t h o d s m a n u a lpatient sample is washed away in the f irststep. False positive results, however, may stilloccu r as a result of bound IgM RF either react-ing with the IgG conjugate or binding anyantigen-specif ic IgG in the sample. One wayto avoid the problem with conjugate binding isto use F(ab')2-conjugated antibodies. Alterna-tively, the assay can be modified to a directtechnique by employing enzyme-labeled ant i -gen in the second step, thus eliminating anyimmunoglobulin that could bind RE Even withthese modif ications, problems can sti l l occurwith borderl ine and low posit ive IgM results.

Antibody capture assays can be used tomeasure antiviral IgG, IgM or IgA antibody.The fol lowing RIA procedure has been usedto detect IgM antibody to parvovirus B19(Cohen et al 1983), but the method can bemodif ied to detect either IgG or IgM antibodyto other viruses.

Procedure1. Wells of microtiter plates are coatedwith sheep ant i -human IgM ant ibody

diluted 1:200 in carbonate buffer, pH9.6 for 2 h at 37~ All reagentvolu me s are 50 ILtl per well an d w ashe sare with PBS containing 0.5% Tween-20 (PBST).

2. Serial serum dilutions beginning at1:200 are prepared in buffer G (asabove but containing 2% normalsheep serum) and inoculated in platesovernight at 4~3. Parvovirus B19 antigen is prepared byultracentrifugation of viremic serum isdiluted optimally in buffer G (usually1:200-1:800) and added to the wells.The plates are incubated for 3 h at3~4. A mouse monoclonal ant ibody toparvovirus B19 viral capsid proteindiluted in buffer G is added at optimaldilution and the plate is incubated for1 h at 37~

5. HRP-conjugated aff inity purif ied goatanti mouse IgG antibody optimallydiluted in buffer G is added and the

plate is incubated for 1 h at 37~followed by OPD substrate.6. The plates are read in an ELISA plate

reader and the cut-off determined astwice the mean absorpance of 5-10IgM negative sera. IgM antibody t i tersare determined as the highest serumdilution with an absorpance greaterthan the cut-off.

Co m m e n tCapture assays can be formatted as anEIA or RIA. For RIA, a 1251-labeled antispecies antibody (2-10 #Ci ug-1; usually10,000 cpm per assay) is subst i tuted forenzyme-labeled conjugate and the assayis run on other polyvinyl chloride platesand individual wells are cut apart withscissors or on plastic beads and thebeads counted in a gamma counter.Specif ic reactivity is expressed as aBinding Ratio (cpm for viral antigendivided by cpm for control antigen).

Solid phasesA number of surfaces can be used for SPIAs:plastic, polyvinyl chloride, nitrocellulose, agar-ose, glass, cellulose, po lyacrylamide, and dex-tran. Plastic is by far the most popular,especially with the frequent use of 96-wellmicrotiter plates. These plates can be pur-chased in intact form or as 8- or 12-well indi-vidual str ips for running a small number ofsamples. Plastic beads are also popular, espe-cially wi th some com mercial companies. Mostantigens and antibodies are bound throughhydrophobic interactions. Proteins generallyare used at a concentration of 1-50 ~tg mi-1.A high-pH (9.6) carbona te coating buffer isoften used as the diluent (1.59 g of Na2CO3;2.93 g of NaHCO3; 0.2 g Na N3 mad e up to 1 Idisti lled water and stored at 4~ for 2 weeks);however, the optimal buffer should be deter-mined expe rimentally for each system. In addi-t ion to passive absorption, immunoreactants

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Antibody detectioncan be coupled covalently by a variety ofmethods.

Absorpt ion depends on the concentrat ionand type of antigen, surface characterist icsof the plastic, t ime, and temperature. Severalstudies have shown that use of too muchantigen may actual ly result in the stacking ofseveral layers of antigen, which results in aprotein-plast ic layer that is subsequent ly cov-ered by a second layer formed by protein-protein interactions. To avoid this problem, atleast three washes, and preferably more,should always be per formed between assaysteps. Both the isoelectr ic point and chemicalcomposit ion of the antigen may affect i ts abi l-i ty to adhere to the sol id phase. Lipid antigensmay pose dif f icult ies in obtaining reproduciblecoating to the wells of the sol id phase. I t iscr i t ical with these types of antigens that sev-eral plates be screened for optimal coating. Inaddit ion, the physical characterist ics of 96-well microti ter plates have important variablessuch as an 'edge effect ' in which the perimeterwells of plates may absorb more protein thanthe inner wells, thereby causing an assay bias.Different plates from several manufacturersshould be screened for this effect when anassay is set up. When the lot of a plate ischanged, they should be reevaluated toensure that none of the assay parametersneed to be modif ied. Coated plates general lyare stable for a few weeks to several months,depending on condi t ions. Most ant igens havethe greatest stabi l i ty when dessicated in a foi lpack. Commercial kits usually have platespackaged in this way, providing a longer shelfl i fe. For in-house assays in which stablepackaging may be more dif f icult to achieve,the integrity of the sol id phase should beclosely moni tored. A procedure which shouldalways be condu cted is to wash stored platesprior to use so as to remove any free antigenthat may have desorbed dur ing storage.

ConjugateDepending on the assay, if it is an EIA it mustcontain either an antibody or an antigen con-jugated with an enzyme. The desirable proper-

t ies of such an enzyme include a high turnoverrate, stabi l i ty, low cost, ease of conjugation,lack of endogenous enzyme in the pat ientsample, easy detection, and compatibi l i tywi th the standard condi t ions used. The mostpopular enzymes are alkal ine phosphatase(AP) and horseradish peroxidase (HRP). A vari-ety of other enzymes are also available, butthey are not as frequently used. These includeB-galactosidase, glucose oxidase, urease, andcarbonic anhydrase, which are primari ly usedin immunoh istochem istry and for assays wi th af luorometric or other end point.

There are a number of choices available foran ant ibody conjugate: whole polyclonal ant i -bodies, immunoglobulin G (IgG) fractions orF(ab')2 fragments of polyclonal antibodies,aff inity-purif ied polyclonal antibodies. I t is bestto screen several antibody preparations andchoose the conjugate that provides opt imalresults for the least cost. The least purifiedant ibodies, such as whole polyclonal conju-gates, are inexpensive, but they frequentlygive a high background. This problem canoften be al leviated with the use of an IgGfraction of the sam e antisera. I f one is perform-ing an IgM assay or using an antigen sourcecontaining Fc receptors, use of F(ab')2 anti-bodies may signif icantly improve results. Over-al l , aff inity-purif ied antibodies often give thebest results, with high detectabi l i ty and lowbackground levels. In addit ion, they are avail-able f rom several commercial companies andare relat ively inexpensive. AP conjugated aff i-nity-purif ied antibodies are general ly moreexpensive than HRP conjugates. Monoclonalant ibodies are the most expensive but someassays require the specif ici ty that they offer.

In choosing the proper di lut ion of an anti-body conjugate, one can run a checkerboardti trat ion using a high and low posit ive standardalong with buffer alone. The optimal conjugatedilut ion is one in which the absorpance of thehigh posit ive control is 1.0 or greater, coupledwith background levels of less than 0.1 absor-pance unit. A checkerboard t i trat ion is per-formed as fol lows:

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1. The plate is coated horizontally. Fourdifferent antigen or antibody coatingconcentrations can be tested, witheach concentration added toduplicate horizontal rows.2. Following this coating step, unreactivesites are blocked by using anon spe cific protein, i.e. BSA, gelatin,or nonfat dry milk. For test samples,we use a high posit ive patient control,negative control, and PBS alone. Forcontrol specimens, a 1:100 dilution isoften used since m ost analytes can bemeasured at this level and nonspecificbinding is decreased.

3. Following an incubation of 1-2 h atRT, the plate is washe d 3-6 t imes w ithPBS.4. The conjugate is added in a verticalfashion to the plate. Four di lutions intr ipl icate wells can be used per96-w ell p late, in rows 1 to 12. Theconjuga te dilution wil l vary greatly withthe type of antibody used (dilutionsmay range from 1:50 to 1:100,000).5. After incubation for 1-2 h, the enzymereaction is developed with theappropriate substrate. The optimalchoice for the assay is a combinationof antigen concentration andconjugate dilution that gives a PBSvalue of < 0.05, a negative control of1.0 absorpance unit.6. This checkerboard t i tration may haveto be modif ied if a posit ive controlserum is not available. In this case, theantibody conjugate t i tration can beperformed by coating the solid phasewith the immunoglobulin protein towhich the ant ibody conjugate isdirected. Following determination ofan appropriate conjugate dilution, thesolid phase can be coated in a rangefrom 1 to 50 l~g m1-1, as this is themost commonly used range ofconcentrations in al l EIAs.7. Once these optimal concentrationsare determined, a large number of

patient samples can be screened anda pool of posit ive controls can beobtained.

Excellent enzyme-labeled antibodies of var-ious specificities are available from severalcommercial sources. However, in some cir-cumstances it may be necessary to label anti-bodies in the laboratory. The procedure beginswith enrichment of the antibodies from serumor ascites fluid. This initial step is required sothat proteins other than antibodies wil l not belabeled and give false-positive results. Anti-body enrichment is conveniently performedby using ammonium sulfate fractionation, ion-exchange chromatography, or protein A col-umns. The second stage represents couplingof the enzyme to antibody. Several methods,including periodate oxidation and gluteralde-hyde procedures are widely used. The condi-tions used ordinarily result in optimum labelingof the antibody. Blocking solutions and washbuffers containing detergent are required todecrease nonspecif ic color intensity in EIAprocedures. When the concentration of pro-tein in the coating solution is suboptimal forsaturation of microwell binding sites, an immu-nologically irrelevant protein is used to oc cup y(block) the remaining plate surface. Bufferscontaining bovine serum-albumin or gelatinare commonly used for this purpose. To pre-vent nonimmunological interactions in themicrowell plate wells, al l subsequent reagentsare added in buffers containing detergent(usually 0.1% Tween-20). In addition, all wash-ing steps between reagent addit ions are alsodone in buffers with Tween-20.An example of an enzyme ant ibody conjuga-tion is the periodate method.

To 4 mg of peroxidase in 1 ml of distilledwater, add 0.2 ml of freshly prepared0.1 M Nal0 4 and stir gen tly for 20 min.

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Antibody detection(On addit ion of the periodate, the solut ionshould change color f rom gold to green;if i t does not do so, addit ional NalO4 isrequired.) Dialyze the mixture against1 mM sodium acetate buffer, pH 4.4,overnig ht at 4~ Raise the pH byaddit ion of 20 #1 of 0.2 M sodiu mcarbonate-bicarbonate buf fer (pH 9.5) .Immediately add 8 mg of antibody in 1 mlof 0.01 M sod ium carbo nate buffer, pH9.5. Stir for 2 h at room temperature. Add0.1 ml of freshly prepared sodiumborohydride (4 mg ml-1 in dist i l ledwater). Leave for 2 h at 4~ Prec ipitatewi th saturated ammonium sul fate.

SubstratesWhen deciding on a substrate for an enzymeconjugate, there are several factors to con-sider. First, the substrate must produce ameasurable soluble reaction product. Formost EIAs this is a colored reaction product,although f luorescent substrates can be used.Other variables to consider include sensit ivi ty,background absorpance, stabi l i ty of the com-pound, toxicity, and cost. Toxicity is a part icu-lar concern, since a number of substrates havebeen suggested to be potent ial ly carcinogenic.i f the color wi l l be moni tored spectrophoto-metr ical ly, the maximum wavelength of the

absorpance is also a factor, since readersgeneral ly come with a l imited number of f i l ters.

A variety of substrates are available for HRP(Table 7.1). The enzyme reduces hydrogenperoxide (H202) and oxidizes a second sub-strate, which produces a colored reaction pro-duct; thus, H202 s always required along withanother substrate. The most popular sub-strates include o-phenytenediamine (OPD),5-aminosalicyl ic acid, 2,2-asinodi-(3)-ethyl-benz-o- thiazol ine-6-sul fonate, and 3,3' ,5,5 'otetramethylbenzidine (TMB). Although variousstudies have shown some substrates to bepreferable to others, general ly they al l performadequately in most assays. In sett ing up a newprocedure, several substrates may be evalu-ated to determine the opt imal compound.Regardless of the secondary substrate used,the most cr i t ical variable in catalyzing HRP isthe concentrat ion of H202. This is usually keptas a 30% solut ion; however, i t has l imitedstabil i ty and may account for signif icant inter-assay variability.

For AP, the most popular substrate is p-ni t rophenyl phosphate (pNPP), which absorbsstrongly at 405 nm. pN PP is general ly dis-solved in a high-pH diethanolamine bufferwi th magnesium added. Use of phosphate-buffered sal ine (PBS) as a wash buffer in AP-based EIAs may result in low optical density(OD) values. Ph osph ate is a potent inhibitor ofAP, so any phosphate left in the plate wellsafter washing may cause an inhibit ion of theenzyme react ion. Al though many laborator iesreport few problems with use of PBS as the

Table 7.1. Comm only use d enzymes and substrates for enzyme immunoassays.Enzyme (Source) Substrate Colou r Change~ReadingPeroxidase(Horseradish)Alkaline phosphatase(Calf intestine)Beta lactamase(B. cereus or E. coil)Urease(Jack bean)

o-phenylenediamine (OPD)5-5'-tetramethylbenzidine ('I'MB)5-aminosalicylic acidp-nitrophenylphosphatestarch-potassium iodideurea and a pH indicatorbromocresol purple

Clear to yellow (490 nm)

Clear to yellow (405 nm)Purple to clea r (590 nm)Yellow to purple (590 nm)

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V i r o l o g y m e t h o d s m a n u a lwash buffer in AP-c atalyz ed EIA, i t is a variableto consider i f the color reaction does notdevelop suff iciently. One can substi tute a Trisbuffer for PBS, as Tris may actual ly increaseAP activity.

Enhancement of SPIAsignalsBacterial proteinsCell wall proteins in Cowan strains of Staphy-lococcus aureus bind to imm unoglobul ins f romseveral species in a nonimmunological fash-ion. Each protein A molecule has four regionsthat bind to the Fc port ion of immunoglobulins.The immunoglobul in-binding propert ies of pro-tein A make this molecule useful as a develop-ing reagent in enzyme immunoassays. ProteinA covalently bound to enzymes is availablefrom several commercial sources and can sub-st i tute for enzyme- labeled second ant ibodiesin EIA. Protein A binds poorly to hum an IgG3and some immunoglobul ins f rom other mam-mals. Protein G, a cel l wall consti tue nt of someStreptococcus strains, exhibits high-aff inityinteract ions wi th these immunoglobul ins. Pro-tein G is used in EIAs much like protein A andis available in a genetical ly engineered formdesigned to reduce nonspecif ic interactions.It is also used to detect IgA and IgMresponses to microbial antigens. In thismethod, igG is removed from sera by usingprotein G coupled to agarose. The residualantibodies are then incubated with the targetant igen. The use of enzyme- labeled secondantibodies that recognize the heavy chain ofIgM or IgA al lows a resolut ion of immuneresponses of these isotypes.

Avidin-biot inThe strong interaction between avidin fromegg white protein; and biot in, a low-molecu-lar-weight vitamin, has been used to amplifythe sensit ivi ty of EIA. Each of the four subunitsof avidin contains a site that interacts with the

ureido ring of biotin. The remaining valeric acidside chain of the biot in molecule can be che-mical ly modif ied to generate reactive groupswithout alter ing avidin interactions. The lengthof the spacer between the ureido group andthe chemically reactive group is suff icient inseveral commercial ly available forms of biot into al low at tachment of the biot in to ant ibodiesor enzyme and st i l l permit interactions withavidin. Avidin i tself may be covalently l inkedto enzyme reporter molecules by using sev-eral avai lable methods. Streptavidin fromStreptomyces avidinii as a result of its lowerisoelectr ic point, is used more widely than eggwhite avidin because of lower backgroundintensity. EIA using avidin-biot in interactionsis performed in two major variat ions. The f irstmethod makes use of a biot inylated secondant ibody. Avidin-enzyme conjugates are thenused to detect bound biot inylated ant ibodies.The increase in sensit ivi ty over standard EIAmethods resul ts f rom a pyramid- l ike enhance-ment of reporter group molecules. The othermajor format of avidin-biot in EIA takes advan-tage of the mult ivalency of avidin. In the pro-cedure, unconjugated avidin serves as abr idge between biot inylated ant ibody andbiot in-enzyme conjugates. Al ternat ively, com-plexes of avidin and biot inylated enzyme canbe used to detect the biot inylated antibody.

Use of chemi luminescentsubstratesThe cycl ic hydrazide luminol (5-amino-2,3-dihydro-1, 4-phthalazinedione) can beemployed directly as a label ( for example toavidin) in SPIA or as a substrate to interactwith peroxidase enzyme. Oxidation of luminolto a chemi luminescent product is catalyzed byfree iron ions or chelated iron in peroxidase.The electronical ly exited aminophthalate aniondecay provides the more sensit ive signal. Per-oxidase conjugated to ant ibody may act moreef fect ively on luminol than on chromogenicsubstrates because conjugat ion of the per-oxidase may destroy substant ial amounts ofenzymatic activi ty but st i l l al low the large

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A n t i b o d y d e t e c t i o nheme(iron) donation for al lowing luminol oxida-t ion to take place in a chemiluminescent test.

Use of radioactive isotopesAlthough the use of isotopes for diagnost ictests has fal len from popLtlar i ty the principlesare worth discussing. The use of a radioactivecon jugate in a SPIA ma y faci l i tate the provisionof a more sensit ive assay based on thekinetics of reaction of the components in thetest. The most commonly used isotope is 1251,a weak gamma-emit ter wi th a hal f - l i fe of 60days . 1251 can be in co rpo rate d effectiv ely intoprotein molecules of high specif ic activi ty byseveral methods wi thout markedly af fect ingthe immunological act iv i ty and speci f ic i ty ofthe proteins.

The most commonly used conjugat ion pro-cedure is the chloramine-T method of Hunterand Greenwood (1962).

in this method, the protein to be labeledis add ed to a m ixture of 1251 andchloramine-T (a mild oxidizing reagent).After an incubation of 2 min the reactionis stopped by the addi t ion of NaHSO3 orby the di rect appl icat ion of the react ionmixture to a Sephadex G 25 column,which is used to separate the labeledprotein. Other methods are also used. Toa lesser extent, beta-emitters 3H and ~4Calso have been used in sol id-phase RIAs.

Highly specif ic activi t ies of labeled immuno-globulins have been obtained by using themethod of reduct ive methylat ion wi th t i t ratedsodium borohydride. Due to the long half- l i feof these isotopes, the labeled reagents can bestored and used for years. However, becausethe l iquid scint i l lat ion counting is considerablymore compl icated than the gamma-count ing,these istopes have not found wide use indiagnost ic vi rus ant ibody assays.

The labeled immunoglobul ins are usual lydi luted in a buffer contain ing protein and deter-

gent to decrease their nonspeci f ic binding.Eagle minimum essent ial medium (E-MEM)supplemented wi th 10% heat- inact ivated cal fserum 0.5% lactalbumin hydrolysate, ant i -biot ics, and 1% Tween-20) is usual ly used.Because the act ivi ty of the labeled immuno-globul in preparat ions decrease cont inuouslydue to radioact ive decay, f requent standard-ization is necessary. This is done by di lut ingthe preparat ions to give a standard cpm value(e.g. 5000 cpm) bou nd whe n 200 #1 of the labelis incubated with the sol id phase coated withthe homologous ant igen.

RIA can be used to determine ant ibodiesfrom al l body f luids. Nonspecif ic inhibitors ofhemagglut inat ion, ant icomplementary factors,etc. which ser iously disturb some serologicantibody tests, do not interfere in the RIA.Like the labeled immunoglobul ins, the speci-mens should be di luted in a buffer containingprotein and detergent; PBS containing 0.5%bovine serum albumin and 0.5% Tween-20(PBS-BSA-Tween) is often used. The expres-sion of serum ant ibody concentrat ion wi th anendpoint t i ter value is the most commonlyused method of expressing RIA results. Thespecimens are tested in several serialdi lut ions, and the t i ter value is interpolatedfrom the cpm versus serum di lut ion curve asthe highest di lut ion where the cpm value of thetest specimen is 2.1 t imes higher than the cpmvalue of the negative control at the samedilut ion. As the negative control, either thetest specimen incubated wi th an uninfectedcontrol antigen or a standard negativespecimen incubated wi th the vi rus ant igen isused. Similar creation of standard curvesment ioned above for EIA apply to providing ameasurement of ant ibodies in a single testedserum.

Measuring responses inSPIAEach SPIA wil l have characterist ic methods formeasuring and interpreting the reaction. Wehave selected a typical EIA to i l lustrate thecomplexi ty involved. The color react ion of an

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V i r o l o g y m e t h o d s m a n u a lEIA can be monitored visual ly for a qual i tativeresult. For quanti tative readings or a moresensi t ive qual i tat ive assessment, a spectro-photometer is used. Photometers can be inter-faced wi th a microcomputer equipped wi th adata reduct ion package for d i rect calculat ionof results.

It is adequate for most assays to blank theOD m easureme nt wi th a i r . However, someinvestigators prefer to blank the plate byusing a wel l containing either substrate orsubstr ate-an t ibody conjugate alone. I f th isprocedure is used, i t is important to documentthe absolute absorpance of these wel ls usedfor blanking. An OD value much greater than0.1 may indicate a deterioration in some of theassay components. For report ing the analytein an absolute amount per ml, a referencepreparation is used as the source of cal ibra-tion. A number of these preparations are avai l-able from commercia l companies, professionalorganizat ions, and government agencies.Instead of uti l izing a valuable reference pre-paration, the test can be cal ibrated against astandard and used for regular assays. A dose-response curve is made, consist ing of theconcentrat ion of the reference preparat ion ver-sus i ts absorpance. When making this curve,one must careful ly evaluate the characteristicsof the data. Al though a standard curve canoften be constructed by s imply p lott ing theabsorpance against a log of concentrat ionand performing l inear regression, this maynot be the best treatment for a l l systems. Avariety of computer software packages offera range of curve-f it t ing techniques for evaluat-ing EIA data.

One common method of expressing data isto determine the mean absorpance of samplesfor normal individuals and report patientsamples as posi t ive when they are two orthree standard deviations above this value.Although this method requires running onlyone di lut ion of the pat ient sample, a numberof problems can arise. From a statistical pointof view, i t may not be correct to apply a meanand standard deviat ion to th is type of data, asthey are often not normally distributed andthere may be an insufficient sample size toapply nonparametr ic stat ist ics. The greatest

problem wi th use of absolute absorpancevalues is the poor reproducibi l i ty of results.To deal with this problem, one can use anadjusted absorpance in which the pat ientvalue is presented as a ratio or a percentageof a posi t ive contro l . These methods aresimple and incorporate an internal control, butmust be careful ly standardized. It is importantto moni tor the level of absolute absorbanciesto ensure that they are relatively constant fromday to day and within the l inear range of theassay. One can use a reference pool of serawith assigned arbitrary units to establ ish adose-response curve and then determinepatient values from the curve. In addition toextrapolating the values directly from thecurve, some invest igators have suggesteddetermining the ratio of the area under thedose-response curve of the test serum andthe reference serum. These techniques offermuch improved reproducibi l i ty and a quan-t i tat ive answer but the requi red calculat ionsare more t ime-consuming and may neces-sitate several di lutions of patient sera toobtain a value fal l ing on the l inear portion ofthe curve. Use of a microcomputer interfacedwith the reader greatly simpl i f ies the calcula-tions.

Controls and assaystandardizationThe basic controls in al l assays should includePBS, a negative control, and low and highpositive controls. However, additional con-tro ls should be incorporated when the assayis set up. Initially, one should assess the back-ground absorpance of al l reagents alone. Oneimportant control is to assess the binding ofpat ient sera to the uncoated sol id-phase ma-terial . In a minori ty of samples, one can findsignificant binding to the sol id phase evenwhen i t is coated with a nonspecific protein.This may be a problem, especial ly in IgMassays, and may require running al l samplesin both coated and uncoated wel ls, wi th sub-traction of the OD reading in the uncoatedwells. Whenever any new reagents are used,

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Antibody detectioncomparat ive studies should be per formed.There can be signif icant variabi l i ty withindifferent lots of the plates and the conjugate,and one can never assume that the assayparameters wil l remain unchanged. Factorssuch as alterat ions in RT and the q uali ty ofthe water can also signi f icant ly af fect day- to-day variabi l i ty in assays. For this reason,rel iance on standard curves wil l yield morereproducible results.

The absorpance in PBS control wel ls shouldbe less than 0.1 (optimally less than 0.05) ODunit. I f the ab sorp anc e is higher, the m ost l ikelyreasons are ei ther inadequate washes or aproblem with the enzyme conjugate. I t maybe important to increase the number and eff i-ciency of the wash steps. I f this is not effect ive,the di lut ion and pur i ty of the enzyme conjug ateshould be evaluated. I f the conjugate cannotbe di luted further without affect ing the sensi-t ivi ty of the assay, one should look for either ahigher-avidity or an aff inity-purif ied conjugate.Anoth er way to deal wi th the problem is to addnormal serum (1-5%) of the same species asthe conjugate to the conjugate di lut ion buf fer .In addit ion, use of 1-5% bovine serum albumin(BSA) or another nonspecif ic protein to bindunreacted sites on the sol id phase may cor-rect the problem. Another problem may occurwi th a h igh absorpance (>0.20D uni t ) o f anegat ive pat ient sample and can be deal twith in the same manner as with the PBScontrol.

An unexpe cted fal l in the am ount of an ana-lyte at the high end of the dose-responsecurve, (hook effect) result ing in a gross under-est imat ion of the analyte may be a problem insandwich immunoassays wi th pat ient samplesthat contain an extremely high level of an

analyte. The patient samples wil l give a lowto moderately high resul t when the standardassay di lut ion is used. Upon further di lut ion ofthe sample, the result wi l l either be out-of-range high or, if it is diluted far enough, willgive an extremely elevated value. Therefore, i fthe laboratory ran the sample only at the rou-t ine di lut ion, signif icant underestimation of thevalue would be reported. This problem mayarise from low-aff inity antibody, inadequatewashing, and subopt imal concentrat ions oflabeled ant ibody. Always ensure that ade-quate washing is per formed between al lsteps, especial ly between the steps fol lowingthe addi t ion of each ant ibody. When one isperforming new kit evaluations, test ing speci-mens with high levels of the analyte is impor-tant , as the f requency of the hook ef fect wi thdifferent kits may be variable.

,Heterophile antibodies may interfere withsandwich EIAs. These ant ibodies can befound directed to several dif ferent species(i.e. sheep, goat, mouse , an d rabbit) . Theirpresence can have a variable effect onSPIAs. I f the analyte is not present, a false-posit ive result may arise from the heterophilecross- l inking the two ant ibodies of the sand-wich. As monoclonal ant ibody-based assaysare of ten used for diagnost ical ly importantanalytes, erroneous results can cause signif i-cant prob lems in pat ient care. Addi t ion of non-immune immunoglobul in f rom the appropr iatespecies wil l el iminate this interference; how-ever, the amount and source of the normalserum may be crucial . By employing a sys-temat ic approach using some or al l of thesetact ics, the major i ty of background problemscan be solved.

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Quality controlQual i ty control is important because of thecomplexi ty and sensi t iv i ty of the procedures.These factors tend to magnify errors in perfor-mance or materials, and exact ing control stan-dards are needed to ensure accurate andprecise test results. Crit ical elements of a con-t rol program are standardizat ion of the sero-chemistry, cont inuous moni tor ing of the testprocesses, and pre- test inspect ion of testspecimens.

Opt imum ant igen-ant ibody concent rat ionsare establ ished by convent ional block t i t ra-t ions wi th reference specimens. From theseexper iments, the test reagent concentrat ionsfor maximum speci f ic react ivi ty and minimumnonspecif ic signal are establ ished. Test proto-cols are fur ther val idated by test ing of knownposi t ive and presume d normal specimens f romthe target-diagnost ic populat ion. From thesedevelopmental studies, the test f indings thatcorrectly dif ferentiate reactive-posit ive speci-mens f rom nonreact ive-negat ive specimens isidenti f ied. This cutoff value is expressed as: (i)a f ixed absorpance value determined on thebasis of the exper ience of the test developer;( ii ) an ab sorp anc e value that is a mult ipl ier of anumber of standard deviat ions above thenega tive pop ulat ion abso rpan ce; ( i i i) a fract ionor percentage of a known posi t ive referencevalue; or ( iv) a comb inatio n of these. In everyinstance, specimens wi th absorpance valuesgreater than the cutoff are reactive or posi-t ive, and those with values less than the cutoffare nonreact ive or negat ive. Some manufac-turers establ ish a 'grey or equivocal ' zonewhich is neither posit ive or negative. Thisapproach compl icates test interpretat ion andpat ient management.

The equipment manuals provided by manu-facturers must be consul ted for per formancecharacterist ics, cal ibrat ion specif ications, andoperat ing procedures of al l instruments usedwith EIA tests. These techniques are very sen-sit ive and therefore require accurate f luid mea-surements. Pipett ing devices, plate washers,and well readers must be cal ibrated, and sche-dules for cal ibrat ion checks and rout ine main-

tenance must be establ ished and recorded.Special attention must be paid to pipett ingdevices. Accuracy for these devices is maxi-mized by, ensuring correct f i t of the t ips to thedevice barrel, prewett ing t ips, and keepingvolume delivery below a reagent level so asnot to contaminate the pipette. Washers, l ikereagent del ivery devices, must perform exactlyas specif ied. Vacuum and f luid del ivery mustbe set for and matche d to the plate test wel ls.A simple measure of wash eff iciency is a plate-to-plate inspection for residual wash f luid.Either excess residual f luid or excessiveaspirat ion in wash cycles, and thus excessivedrying, may adversely affect test reactivity.Since al l EIA reactions are temperature andt ime sensi t ive, technical protocols mustinclude clocks and thermometers as standardmoni tor ing devices to ensure compl iance wi ththe parameters establ ished for each test .

Each EIA run should include controls fromthe reagent manufacturer, a set prepared in-house or obtained f rom other sources (exter-nal or supplemental controls), as well as,whenever possible, a set of pr imary standardsfrom a recognized reference laboratory. Thisset is used initially, and at appropriate inter-vals, to ensure the optimum level of reactivityin consensus wi th other laborator ies. Thecontrol set provided by the manufacturerdescr ibes the opt imum performance charac-terist ics of the test. The results of this controlset , however, apply only to the potency of thereagents in the set in use. Variations inreagents, kit to kit or lot to lot, are monitoredby the external control set, which shouldconsist of mult iple (duplicate or tr ipl icate)high- and mid-range react ive specimens orspecimen pools and a nonreact ive specimenor specimen pool . This external control thusserves not only to detect immediate fai lurebut also to moni tor both excessive run- to-runvariat ion and long-term trends. Valuesobtained at each test run are eff iciently moni-tored by charts that show, for example, non-reactive or background as well as levels ofspecif ic reactivity over t ime.

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References

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7 - Immunoassays Principles and Assay Design