APPLIED MICROBIOLOGY, Dec. 1974, p. 935-942Copyright i 1975 American Society for Microbiology

Vol. 28, No. 6Printed in U.S.A.

Criteria for Preparing, Evaluating, and Standardizinglodinated Globulins for Radioimmunoassay Procedures

HARRIET D. HUTCHINSON AND DONALD W. ZIEGLERCenter for Disease Control, Atlanta, Georgia 30333

Received for publication 19 August 1974

Procedures were examined for labeling immune globulins with radioactiveiodine using chloramine-T as the oxidizing agent. The chloramine-T method wascritically evaluated to establish the optimal conditions for preparing iodinatedglobulins with high specific radioactivities without impairing their immuno-specificities for use in in vitro radioimmunoassays. The results showed that theuse of 100 ,g of chloramine-T per ml, 500 to 1,000 ACi of Na 12"I per mg of protein,and a 10-min oxidation reaction time produced globulins of both high specificradioactivities and immunospecificities. Criteria were established for evaluatingand determining optimal concentrations of iodine-labeled globulin for use inradioimmunoassays. The results of this investigation indicated that the amountof labeled indicator globulin used in radioimmunoassays should be based uponprotein concentration rather than radioactivity.

Radioimmunoassay (RIA) methods are gain-ing acceptance as diagnostic tools for viralagents, and RIA will probably become thestandard procedure for many serological deter-minations as definitive methods are estab-lished. Assays have been designed which areoperationally simple yet provide sensitive,quantitative antibody determinations (8, 13; H.D. Hutchinson, D. W. Ziegler, G. A. Baer, P.A. Yager, J. S. Smith, and T. K. Lee, manu-script in preparation; D. W. Ziegler, H. D.Hutchinson, J. P. Koplan, and J. H. Nakano,submitted for publication).The success of RIA methodology depends,

however, upon the integrity of the immuneglobulin reagents. The radioactively labeledimmune globulins must have high specific anti-body activity and high specific radioactivity.After immune globulins of high antibody con-centration are obtained, radioactive atomsmust be introduced into the protein moleculeswithout altering the immunological specificity.

Several methods of labeling protein withradioactive iodine have been described. Amongthese procedures are the electrolytic method ofRosa et al. (12), the microdiffusion method ofGruber and Wright (6), and the peroxidasemethods of Thorell and Johansson (14) andDavid (3).Because of their simplicity, chemical

methods of converting anionic iodine to anoxidized state are used in many laboratories.Among the chemical methods are the iodinemonochloride method described by McFarlane

93E

(11) and the chloramine-T procedure describedby Greenwood et al. (5). Both of these methodshave gained considerable acceptance and areextensively used. The chloramine-T iodinationprocedure is easily controlled by precise termi-nation of the oxidation period by addition of areducing agent, sodium metabisulfite. This pre-cludes the possibility of protein denaturationdue to prolonged exposure of the protein tooxidizing conditions. Because of this distinctadvantage of the chloramine-T method, it wasadopted as the standard procedure in our labo-ratory.The Greenwood chloramine-T oxidation pro-

cedure was designed for labeling microgramquantities of protein hormones with high spe-cific radioactivities. McConahey and Dixon (10)later modified the procedure so that largerquantities of partially purified serum proteins,such as albumin and gamma globulin of variousanimal species, could be trace-iodinated withminimal denaturation for in vivo applications.The optimal conditions for iodinating theseproteins may not be the same as those requiredfor iodinating partially purified immune globu-lins for use in in vitro RIA procedures. Hence, inthis study we determined the oxidation condi-tions and the quantity of radioactive iodineyielding high levels of iodination while main-taining satisfa.ctory immunological specificityfor use in in vitro serological determinations.When radioactively labeled antiglobulin is

used as an indicator in RIA, the sensitivity ofthe assay is greatly influenced by the quantity

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of globulin added to the assay. Definitive infor-mation regarding the optimal quantity of io-dine-labeled globulin for use in RIA is generallyunavailable. To meet this need we investigatedthe effect of concentration of the labeled indica-tors on the RIA results. From this investigation,criteria for preparing, evaluating, and usingRIA reagents are proposed.

MATERIALS AND METHODSPreparation of antiglobulins. Antisera to human

and rabbit globulins were prepared in guinea pigs.The guinea pigs were initially inoculated intrader-mally with 2 mg of sodium sulfate-precipitatedhuman or rabbit globulin emulsified with Freundcomplete adjuvant. The animals received intraperito-neal booster inoculations of 2 mg of globulin everysecond week until high antiglobulin precipitin titerswere obtained. The titers were tested with the Ouch-terlony procedure of double diffusion in agar. Speci-ficities of the globulins were tested by immunoelectro-phoresis.The globulin from the guinea pig antiglobulin

serum was precipitated by the sodium sulfate methodof Kekwick (9). After three sodium sulfate precipita-tions, the globulin was dissolved in borate salinebuffer (0.15 M NaCl, 0.002 M H,B03, pH 7.4) anddialyzed against 0.0175 M phosphate buffer, pH 7.4.Estimations of the protein concentrations of thepartially purified globulins were made spectro-photometrically at 280 nm (extinction coefficient =15.0).

Sulfate-precipitated guinea pig antirabbit or an-tihuman globulins were usually iodinated in 5.0-mglots. If different amounts of protein were used, thevolumes of the reactants were adjusted accordingly.

Chloramine-T iodination. Globulin was routinelyiodinated by the McConahey and Dixon (10) modifi-cation of the Greenwood, Hunter, and Glover method(5) with chloramine-T as the oxidizing agent. Chlora-mine-T and sodium metabisulfite solutions were pre-pared in 0.05 M phosphate buffer, pH 7.5, at aconcentration of 500 gg/ml. Fresh solutions wereprepared on the day of each iodination.

Samples of 5.0 mg of antiglobulin were diluted to4.0 ml with 0.05 M phosphate buffer. The dilution andiodination were performed in a 12-ml Wheaton vialfitted with a rubber-sleeve stopper and containing asmall magnetic stirring bar. The diluted proteinsample was chilled in an ice bath on a magnetic stirrerbefore the iodination reaction was started, and it wasmaintained in the ice bath during the reaction. All ofthe reagents were injected through the sleeve stopperwith syringes.

Carrier-free Na125I (New England Nuclear) wasadded at a concentration of 1,000 MCi/mg of proteinwith a Hamilton syringe. Immediately after the addi-tion of Na 2"I, 1.0 ml of chloramine-T (500 Ag/ml) wasinjected slowly (about 30 s) into the reaction mixture.The mixture was stirred vigorously for 10 min. Fi-nally, 1.0 ml of sodium metabisulfite (500 ug/ml) wasadded, and the reaction mixture was stirred for 1 min.

The iodinated protein was then transferred to dialysistubing (8 mm diameter) and dialyzed against 0.1 Mphosphate buffer (pH 7.0) to remove the unreactedNa'21I. This procedure was adopted as the standardmethod after variations of the conditions were investi-gated to ascertain optimal conditions. To determinethe amount of protein-bound 1251, we added a portionof iodinated protein to 0.2 ml (approximately 2 mg) ofcarrier protein and precipitated it with 0.2 ml of 10%trichloroacetic acid. The precipitate was then washedwith three 1.0-ml portions of 10% trichloroacetic acid.The radioactivity in the precipitate, the supernatant,and the three rinses was measured. If 90% or more ofthe radioactivity was present in the precipitate (pro-tein-bound), the protein was considered satisfactoryfor use in the RIA procedure.RIA procedure. The RIA procedures used in this

study have been described by Hutchinson and Ziegler(8), Hutchinson et al. (manuscript in preparation),and Ziegler et al. (submitted for publication). EitherEscherichia coli, vaccinia virus, or rabies virus wasused as the primary antigen in the reaction and wasprepared as described by these authors. The RIAprocedures were performed either on cover slips (8) orin microtiter wells (Hutchinson et al., manuscript inpreparation; D. W. Ziegler, H. D. Hutchinson, J. P.Koplan, and J. H. Nakano, submitted for publica-tion). In each case the RIA was an indirect test inwhich specific rabbit or human antiserum was allowedto react with the antigen which was affixed to thecover slips or microtiter wells.

After the primary antigen-antibody reaction, adilution of guinea pig antirabbit or antihuman 125I-labeled globulin was allowed to react with the primaryantigen-antibody complex. The guinea pig anti-globulin used was specific for the antiserum speciesreacting in the primary immunological reaction. Afterthe second reaction, the excess antiglobulin wasremoved, and the cover slips or microtiter plates werewashed with phosphate-buffered saline (pH 7.2),dehydrated with 95% ethanol, and air dried. Thesamples were then transferred to counting vials, andthe incorporated radioactivity was measured in anautogamma scintillation spectrometer. The results ofantiserum titrations by the RIA were recorded as thecounts per minute per cover slip or counts per minuteper microtiter well (counts min per sample).

RESULTS AND DISCUSSIONRIA methodology depends upon the availabil-

ity of indicator globulin of both high specificradioactivity and high immunospecificity.Therefore, it was desirable to evaluate availablemethods for iodination. Several methods havebeen reported but were not evaluated in thisinvestigation because they required uniqueequipment and materials.

In this study the optimal conditions for iodi-nation of protein with the McConahey andDixon (10) modification of the chloramine-Tprocedure were critically evaluated. The choices

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of the optimal concentrations of the reagentsand conditions for the reactions were based ontwo considerations: the amount of iodine intro-duced into the protein and the in vitro immuno-specificity. Factors which may alter the effi-ciency of iodination of the protein or its immu-nospecificity include: (i) the chloramine-T con-centrations, (ii) the Nal25I concentration, and(iii) the oxidation reaction time. Each of thesefactors was considered in this investigation.Optimal concentration of chloramine-T.

Although chloramine-T is a mild oxidizingagent, it may alter the protein and result in adiminution of the immunospecificity of theglobulin (7). In these trials conditions wereexplored which would consistently produce ""I-labeled indicator globulin of high specific radio-activity and freedom from denaturation. Theoptimal concentration of chloramine-T for iodi-nation of immunoglobulin was estimated byiodinating six separate 2-mg portions of a singlelot of guinea pig antirabbit globulin and usingvariable amounts of chloramine-T (0.5 to 250gg/ml). The volume of the reaction mixture was2.0 ml in each trial. For each trial reaction,carrier-free Na126I was added at a concentrationof 100 uCi/mg of protein. Each reaction wasterminated by adding a reducing agent, sodiummetabisulfite, in the amount equal to theamount (weight/weight) of chloramine-T. Inas-much as chloramine-T has a higher molecularweight than sodium metabisulfite (281.7 and190.1, respectively), an excess of the reducingagent was assured when equal concentrations ofthe two reactants were used.The results (Table 1) show that the efficiency

of iodination increased from 0.19 to 20.7%, anincrease of approximately 100-fold, as the con-centration of chloramine-T was increased from0.5 to 250 ug/ml. The maximal efficiency ofiodination was only 20.7% with the highestconcentration of chloramine-T; this suggeststhat even greater concentrations of chlora-mine-T could increase the efficiency of thereaction.However, in addition to the efficiency of

iodination, the immunological integrity of theiodinated protein must be considered. There-fore, the effect of chloramine-T on the immuno-specificity of the labeled antiglobulin was testedby RIA with each of the six antiglobulin sam-ples (Table 1) which were iodinated with differ-ent concentrations of chloramine-T. Eachiodinated antiglobulin was tested by RIA withE. coli as the antigen and a 1:10 dilution ofspecific E. coli antiserum. In the controls,phosphate-buffered saline diluent was substi-

tuted for the antiserum in the primary reaction.The immunospecificity was revealed by com-

paring the 25I-labeled antiglobulin adsorbed bythe specific reaction with that adsorbed by thecontrol reaction (Fig. 1). The specific and con-trol reactions adsorbed about the same amountsof radioactivity when the antiglobulins

TABLE 1. Results of iodinating protein with varyingconcentrations of chloramine-T

Chloramine-T 1 Protein-bound 12SIb Efficiency ofconcn (e-g/Ml)a (counts/mn per mg iodination (cof protein)

0.5 2.10 x 105 0.192.5 1.69 x 10. 1.50

10.0 7.62 x 10" 6.7340.0 1.46 x 107 13.0100 1.78 x 107 15.8250 2.34 x 107 20.7

aThe concentration represents that which waspresent in the reaction mixture during the oxidationreaction. The volume and protein concentrations wereconstant in each reaction.

b Over 90% of the 125I was protein bound as deter-mined by precipitation with 10% trichloroacetic acid.

c The efficiency of iodination was calculated as theratio of the amount (counts/min) of radioactivitywhich was protein bound versus the amount (counts/min) of radioactivity which was added to each sam-ple. Na125I was added to each sample to obtain aconcentration of 100 ACi/mg of protein. After adjust-ing for the counting efficiency of the scintillationspectrometer, Na'25I = 1.12 x 108 counts/min per mgof protein.

' pe c lf c Reoc t,on,

8~

t/ Control Ret1~~~~~~IoIo ec

Teoonr rrg P,,ote,,

FIG. 1. Estimation of the optimal chloramine-Tconcentration. Guinea pig antirabbit globulin wasiodinated with the same amount of Na 125I (100MCi/mg) but with different concentrations of chlora-mine-T. Each lot of 125I-labeled indicator globulin wasreacted with an E. coli antigen-antibody immunecomplex. Symbols: 0, control reaction (diluent); 0,specific antigen reaction. CPM, counts/min.

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iodinated with 0.5 and 2.5 pg of chloramine-Tper ml were used as indicators. When anti-globulins iodinated with 10, 40, and 100 ,g ofchloramine-T per ml were used, the iodinebound in both the specific and control reactionsincreased as the concentration of chloramine-Twas increased. However, the specific reactionsadsorbed proportionately more of the `I-labeled antiglobulin than the controls at theseconcentrations of chloramine-T. Antiglobuliniodinated with 250 Ag of chloramine-T per mlexhibited a decreased specific adsorption of"2II-labeled antiglobulin and an increased ad-sorption in the control reaction. Thus, theresults suggest that immunoglobulins iodinatedwith 250 pg of chloramine-T per ml are partiallydenatured, with a concomitant loss of immuno-specificity.

Despite the higher efficiency of labeling atthis chloramine-T concentration, it was neces-

sary to sacrifice iodination efficiency so that theimmunological specificity of the protein couldbe retained. Therefore, chloramine-T was usedat a concentration of 100 ,g/ml of protein in thestandard procedure.Optimal concentration ofNa 1251. In a simi-

lar trial we examined the relative effects ofvarying concentrations of Na125I on the iodina-tion of globulins. The optimal concentration ofNa125I was estimated by varying the concentra-tion of Na125I from 25 to 500 piCi/mg of proteinin the iodination reaction. The antiglobulin (2mg), chloramine-T (100 pg/ml), and sodiummetabisulfite (100 ug/ml) concentrations were

constant in each iodination reaction with thedifferent Na125I concentrations.The results show that as Na125I was increased

from 25 to 500 pCi, the specific activity of theiodinated protein increased about 20-fold(Table 2). Likewise, the efficiency of iodinationincreased from 20 to 31% as the concentrationwas increased from 25 to 50 pCi/mg of protein.Although 50, 100, and 250 pCi showed about thesame efficiencies of iodination, the efficiencydeclined about 10% with 500 pCi of Na125I per

mg of protein. These results suggest that interms of maximal iodination efficiency 50 to 250,uCi of Nal25I per mg of protein represent thenear optimal concentration of iodine. In thisexperiment we observed that when Na 125I con-centrations were varied (Table 2) the efficien-cies of iodination were higher than the efficien-cies attained when chloramine-T concentra-tions were varied (Table 1). This apparentdiscrepancy may be caused by the use of differ-ent lots of Na125I. Anomalous iodination reac-

tions have been attributed to unknown contam-

TABLE 2. Results of iodinating protein with varyingconcentrations of Na125I

Na I"" concn Protein-bound 1251a Efficiency of(pCi/mg) (pCi/mg) iodination ()b)

25 5.05 20.150 15.7 31.2100 32.0 32.0250 69.2 27.5500 100.0 20.0

a Over 90% of the 125I was protein bound as deter-mined by precipitation with 10% trichloroacetic acid.

b The efficiency of iodination was calculated as theratio of the amount (CCi/mg) of radioactivity whichwas protein bound versus the amount (;Ci/mg) ofradioactivity which was added to each sample.

inants in different lots of Na125I when lac-toperoxidase is used for iodination of protein(4). It is conceivable that chloramine-T iodina-tions might be similarly affected.To further assess the optimal concentration of

Na125I for labeling protein we evaluated thespecificity of 125I-labeled indicator globulin bymeasuring the competition between radioac-tively labeled and unlabeled antiglobulin forthe specific antibody bound in the primaryreaction. In these studies the primary reactionconsisted of a 1:10 dilution of antigen-specificantiserum and a diluent control which wasadsorbed to the antigen. The RIA procedure wasthe same as the routine RIA method, exceptthat unlabeled indicator globulin in varying con-centrations was combined with the 125I-labeledindicator globulin. The 1251-labeled indicatorglobulin was maintained at a constant concen-tration (0.02 mg/0.1 ml), whereas the concen-tration of unlabeled globulin was varied (0.01mg/0.1 ml to 0.167 mg/0.1 ml). The results ofthe competitive titrations were recorded graph-ically by a semilogarithmic plot. The radioac-tivity (counts/min per sample) was plotted onthe ordinate, and the logarithm of the concen-tration of the unlabeled globulin (milligrams persample) was plotted on the abscissa.The adsorption of 1251-labeled indicator glob-

ulin was inhibited by the simultaneous additionof unlabeled antiglobulin. With each separatelot of antiglobulin labeled with either 100, 250,or 500 pCi of Na125I per mg of protein, thequantity of adsorbed 1251-labeled antiglobulindecreased as the amount of unlabeled anti-globulin was increased (Fig. 2). Although thecontrols are not plotted in Fig. 2, the minimaladsorption of the 1251-labeled indicator globulinby the specific reaction was always three- tofourfold greater than the diluent control reac-tion. Moreover, none of the controls showed

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10

8

6'0

x2

u 4

2

500pC I/mg Globulin

__.!250pC "1/mg Gl n \

IOOpC I/mg Globulin

X --X I X X - X -

0 0010 0.021 0.042 0.084 0.167Unlabeled Antirobbit Globulin (mg/sa mple)

FIG. 2. Reactivity of 125I-labeled guinea pig anti-rabbit globulin labeled with different concentrationsof Na125I. Antirabbit globulin labeled with differentconcentrations of 125I and unlabeled antirabbit globu-lin were allowed to competitively react with immunecomplexes of E. coli and its specific antiserum. Aconstant amount of 1251-labeled indicator globulin(0.02 mg/reaction) and varied amounts of unlabeledantirabbit globulin'(amount indicated on the ab-scissa) were added to the immune complexes on coverslips. Symbols: x, 100 ACi of 12511/mg of globulin; 0,

250 MCi of 1251/mg of globulin; 0, 500 ACi of 125I/mgofglobulin.

marked inhibition when the 1251-labeled indica-tor globulin was combined with increased con-

centrations of unlabeled globulin.The inhibition of the specific reaction was

linear over the entire range (16-fold) tested foreach of the three 125I-labeled antiglobulin prep-arations. However, the competition betweenlabeled and unlabeled globulin appeared to berelated to the amount of iodine introduced intothe globulin molecule. This is indicated by thechanges in the slopes of the curves in Fig. 2. Theglobulin preparation labeled with 500 MCi ofNa125I per mg of protein had a negative inhibi-tion slope of 1.62 x 104, whereas those labeledwith 250 and 100 tCi of Na125I per mg of proteinhad negative slopes of 8.23 x 101 and 1.10 x 101,respectively. The smaller negative slope withdecreasing specific activities is directly relatedto a decrease in the sensitivity of the globulinpreparation. As the specific activity of theiodinated preparation decreases, fewer mole-cules of protein are labeled; hence, there arefewer labeled molecules to compete with theunlabeled globulin for the antibody-bindingsites. The linear regression lines suggest thatthe antibody specificity was not altered. There-fore, greater quantities of Na125I could be addedto the protein without impairing its immunolog-ical integrity.

In subsequent iodination trials with higher

concentrations of Na12I (250 to 1,000 1Ci/mg ofprotein), the mean efficiencies of iodinationhave remained about the same. However, thevariation in the iodination efficiencies amongthe individual trials with higher Na125I concen-trations was greater than the variation amongindividual iodination trials with lower concen-trations of Na 121I. Nevertheless, when anti-globulins iodinated with higher concentrationsof Na125I (750 or 1,000 ,Ci/mg of protein) weretested in competition RIA procedures, it wasshown that the immunological integrity of thelabeled protein was maintained.High specific radioactivities are required be-

cause the labeled antiglobulin must be dilutedto obtain low nonspecific adsorption and highspecific adsorption in RIA methods. Subse-quent results will show (Fig. 4) that eitherexcess or insufficient concentrations of 1215Ilabeled antiglobulin markedly depressed thesensitivity of the RIA. Because high specificradioactivities are required for the RIA proce-dure, we adopted 500 to 1,000 liCi/mg as thestandard Na125I concentration.

Effect of varying the oxidation reactiontime on the efficiency of iodination. Anotherfactor considered in the investigation of theiodination of proteins was the oxidation reac-tion time. A single sample of protein wasiodinated with chloramine-T as the oxidizingagent. After 2, 5, 10, 20, and 30 min, samples ofthe protein-iodine reaction mixture were trans-ferred to vials containing sodium metabisulfite.The samples were dialyzed, and trichloroaceticacid precipitations were used to determine theprotein-bound iodine of each.Samples removed at 2, 5, 10, 20, and 30 min

after the start of the oxidation reaction con-tained 81, 84, 97, 92, and 95 MCi of 125I per mg ofprotein, respectively. Because reaction timesbeyond 10 min produced no additional increasesin the quantity of 1251-labeled protein, thisreaction time was used routinely.The immunological integrity of these radioi-

odinated proteins was assessed in two RIAexperiments. First, the specificity was tested bycompetitively inhibiting the 121I-labeled globu-lin with unlabeled globulin as described above(see Fig. 2). Pronounced differences were notexhibited by the five labeled globulins in thecompetition assay. Next, each of the five 125I-labeled globulins were used in RIA serum titra-tions. A human antiserum of known anti-vac-cinia potency was titrated with each of the125I-labeled globulin preparations. The range oftiters obtained with five preparations was 80 to102, with an arithmetic mean titer of 100. The

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titers observed for the five samples did notexhibit any apparent pattern as a function ofiodination reaction time. This suggests theglobulin was not altered by increases in theiodination reaction time.

Criteria for determining the optimal con-centration of 12-labeled globulin for use inRIA. After the optimal conditions for iodina-tion of globulin protein had been investigated, itappeared advisable to study and subsequentlystandardize the procedures used for determin-ing the optimal concentration of 12-labeledantiglobulin which should be used in indirectRIA. Investigators have shown that dilution ofthe radioactively labeled globulin reduced thenonspecific adherence and yielded increasedtiters (13). Some laboratories have reportedthat the amount of radioactively labeled globu-lin they added to their RIA procedures wasbased on the amount of radioactivity in thelabeled indicator (1, 2). However, the basis forthe addition was not established.To develop a criterion for determining the

optimal concentration of an 125-labeled indica-tor globulin for use in RIA, we prepared threeseparate lots of 125I-labeled guinea pig antihu-man globulin for trial reactions with antigen-human antibody complexes. Two of the lots (Aand B) were prepared from the same globulinpreparation, but with different shipments ofNa125I. The third lot (C) was prepared from adifferent antihuman globulin preparation. Theantihuman titers as determined by immunodif-fusion were the same for each of the purifiedunlabeled globulin preparations. After the pro-teins were iodinated, lots A and B were evalu-ated in reactions with vaccinia-human antibodycomplexes. Lot C was evaluated in a similarreaction, but with rabies-human antibody com-plexes.The optimal concentrations of each of the

three lots of 125I-labeled globulin were deter-mined by reacting serial dilutions of lots A, B,and C with antigen-human antibody com-plexes. As controls, diluent was substituted forthe human antiserum in the primary reactionfor each dilution of 125I-labeled antihuman glob-ulin. After appropriate, dilutions each lot of125I-labeled antiglobulin were reacted, the ra-dioactivity adsorbed by both the specific reac-tions and the control reactions was measured. Aratio of the specific and the control radioactivity(counts/min) observed at each dilution wascalculated and evaluated by two procedures.

First, the ratios were evaluated on the basis ofthe radioactivity of 125I-labeled globulin(counts/min) added to each sample. The calcu-

20 A

S 16

2..

20 40 80 16 32 64 128 256 512 1024

CPM 10

FIG. 3. Radioactivity as a criterion for selectingoptimal amounts of 1251-labeled indicator globulin foruse in RIA. The optimal amounts for each of three lotsof 12I_-labeled guinea pig antihuman globulin were theconcentrations which produced the highest specific(counts/min) to nonspecific (counts/min) ratios ofradioactivity adsorption with radioactivity (counts/min) used as a basis for comparison. A constantamount of each immune complex or control reactionwas reacted with the amounts (counts/min) of 125J_labeled indicator globulin indicated on the abscissa.Symbols: (A) x, vaccinia-human antivaccinia reac-tion measured by using 125-labeled antihuman globu-lin with a specific activity of 70 jACi of 125I per mgof globulin; (B) 0, vaccinia-human antivaccinia reac-tion measured by using 121-labeled antihuman globu-lin with a specific activity of 280 MCi of 125I per mgof globulin; (C) 0, rabies-human antirabies reactionmeasured by using 125I-labeled antihuman globulinwith a specific activity of 85 ACi of 125I per mg ofglobulin.

lated ratios were plotted on the ordinate, andthe added radioactivity was plotted on theabscissa (Fig. 3). The amount of radioactivityyielding the maximal ratio was different foreach lot of 125I-labeled indicator globulin. Lot Bhad the highest specific radioactivity (280 ,Ci/mg), yet it required a larger amount of radioac-tivity to attain the maximal ratios. Lots A andC had lower specific activities (A = 70,MCi/mg,C = 85 MCi/mg), and they required approxi-mately equal but smaller amounts of radioac-tivity to attain maximal ratios.

Next, the same data were examined by plot-ting the ratios on the ordinate and the proteinconcentration of the 1251-labeled antiglobulin onthe abscissa (Fig. 4). These plots show that themaximal ratios for each lot of I21-labeled anti-globulin were attained at approximately thesame protein concentration.

It is important to note that the optimalconcentration of the indicator globulin in thereaction with the primary ai gen-antibodycomplex is independent of the antigen involvedin the complex. This was shown by the fact that

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// =\1A. ,

c/;l~~~~c

z 16~

6E *,.4_' o 12,

2

2 0 40 l '6; 32 5 650 130 260 520

. g * f0' Lbed 9hoW-1-9srn0l

FIG. 4. Protein concentration as a criterion forselecting optimal amounts of 125I-labeled indicatorglobulin for use in RIA. The same data (Fig. 3) were

evaluated for each of three lots of 11I-labeled guineapig antihuman globulin with protein concentration(jgg) used as a basis for comparison. A constantamount of each immune complex or control reactionwas reacted with the amounts (jug) of 125I-labeledindicator globulin indicated on the abscissa. Thespecific activities of the 1251-labeled indicator globu-lins were: (A) 70 gCi of 125I per mg of globulin; (B)280 ,Ci of 125I per mg of globulin; and (C) 85 AtCi of125I per mg of globulin. Symbols: (A) x, vaccinia-hu-man antivaccinia reaction; (B) 0, vaccinia-human an-

tivaccinia reaction; and (C) 0, rabies-human antira-bies reaction.

lots A and B were reacted with complexes ofvaccinia and its specific antibody, whereas lot Cwas reacted with complexes of rabies antigenand its antibody.The results of these studies indicate that the

quantity of 125I-labeled indicator globulin whichshould be used in RIA is very important. Eitherinsufficient or excess amounts of 15I-labeledantiglobulin will impair the sensitivity of thetest. Thus, an arbitrary choice of an amountequivalent to a specific radioactivity contentmay cause failure of the RIA. The optimalamount, based upon radioactivity as in Fig. 3,could be determined; however, each lot ofiodinated antiglobulin would have to be evalu-ated. Furthermore, periodic adjustments fordecay of the radioactivity would be necessary.An alternate and better procedure would be

to determine the optimal amount of 125I-labeledantiglobulin on the basis of its protein content(Fig. 4). By this procedure an initial determina-tion of the optimal protein concentration of an

iodinated antiglobulin lot will be valid forsubsequent iodinations of the same globulin.Relationship of the concentration of 125I_

labeled indicator globulin to RIA titers. Afterthe importance of adding 121I-labeled indicatorglobulin in optimal amounts was observed, the

* effect of using less than or greater than optimalamounts in an actual RIA procedure was con-sidered. The antivaccinia titer of a humanserum was determined by using six differentconcentrations of 125I-labeled guinea pig antihu-man globulin as indicator. After the primaryantigen-antibody reaction, the 125I-labeled indi-cator globulin was added to the complexes intwofold increments, from 0.16 to 5.2,ug/sample.Thus, an antiserum titration curve for eachconcentration of 125I-labeled indicator globulinwas established. The results obtained with eachconcentration of 125I-labeled indicator globulinwere evaluated by plotting the logarithm of theradioactivity (counts/min) observed for eachantiserum dilution on the ordinate versus thelogarithm of the antiserum dilution factor onthe abscissa. From these antiserum titrationcurves the slope and titer were calculated ateach 125I-labeled indicator globulin concentra-tion (Table 3).The results showed that with decreasing con-

centrations of the radioactively labeled proteinthe slope of the antiserum titration curvesincreased. Concomitant with the increasedslopes of the titration curves, the radioactivityadsorbed by the diluent control (nonspecificadsorption) decreased. The increased slopesand decreased nonspecific adsorption resultedin increased antiserum titers. In fact, at thethree highest 125I-labeled indicator globulin con-centrations (5.2, 2.6, and 1.3 jug/sample) thenonspecific adsorption was so great that errone-ous negative or nonsense titers were obtained.As the slope of the titration curves increasedfrom 1.04 to 2.76, the apparent titers increasedfrom <10 to 70 (Table 3). Thus, to accurately

TABLE 3. Relationship of '25I-labeled indicatorglobulin concentration to titers and slopes of

antiserum dilution curves

Concn of "I-labeled lope0 Titer0indicator globulina

5.2 1.04 <102.6 1.56 <101.3 1.49 <100.65 2.06 380.32 2.44 480.16 2.76 70

aQuantity (Mg of "II-labeled indicator globulin/sample) added to each primary antigen-antibodycomplex and to each diluent control.'The method of least squares was used to deter-

mine the slope of each tftration curve.cThe titer is the antiserum dilution factor which

adsorbed twice the radioactivity (counts/min) ad-sorbed by the control.

941VOL. 28, 1974

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942 HUTCHINSON

titrate sera with RIA methods, it is necessary toselect an optimal concentration of the 1251-la-beled antiglobulin as well as to use iodinatedglobulins of high specific activity and immuno-logical integrity.

LITERATURE CITED

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2. Catt, K., and G. W. Tregear. 1967. Solid-phase radioim-munoassay in antibody-coated tubes. Science158:1570-1572.

3. David, G. S. 1972. Solid state lactoperoxidase: a highlystable enzyme for simple, gentle iodination of proteins.Biochem. Biophys. Res. Commun. 48:464-471.

4. David, G. S. 1974. Quality of radioiodine. Science184:1381.

5. Greenwood, F. C., W. M. Hunter, and J. S. Glover. 1963.The preparation of 1'3I-labelled human growth hor-mone of high specific activity. Biochem. J. 89:114-123.

6. Gruber, J., and G. G. Wright. 1967. Iodine-131 labeling ofpurified microbial antigens by microdiffusion. Proc.Soc. Exp. Biol. Med. 126:282-284.

AND ZIEGLER APPL. MICROBIOL.

7. Hughes, W. L. 1966. The chemical requirements of asatisfactory label for proteins, p. 3-16. In L. Donato,G. Milhaud, and J. Sirchis (ed.), Labelled proteins intracer studies. Proc. Conference on Problems withPreparation and Use of Labelled Protein in TracerStudies. EURATOM, Pisa.

8. Hutchinson, H. D., and D. W. Ziegler. 1972. Simplifiedradioimmunoassay for diagnostic serology. Appl. Mi-crobiol. 24:742-749.

9. Kekwick, R. A. 1940. The serum proteins in multiplemyelomatosis. Biochem. J. 34:1248-1257.

10. McConahey, P. J., and F. J. Dixon. 1966. A method oftrace iodination of proteins for immunologic studies.Int. Arch. Allergy 29:185-189.

11. McFarlane, A. S. 1958. Efficient trace-labelling of pro-teins with iodine. Nature (London) 182:53.

12. Rosa, U., G. A. Scassellati, F. Pennisi, N. Riccioni, P.Giagnoni, and R. Giordani. 1964. Labelling of humanfibrinogen with 1311 by electrolytic iodination. Biochim.Biophys. Acta 86:519-526.

13. Rosenthal, J. D., K. Hayashi, and A. L. Notkins. 1972.Rapid microradioimmunoassay for the measurement ofantiviral antibody. J. Immunol. 109:171-173.

14. Thorell, J. I., and B. G. Johansson. 1971. Enzymaticiodination of polypeptides with "'I to high specificactivity. Biochim. Biophys. Acta 251:363-369.

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