LIN. CHEM. 36/7, 1333-1338 (1990)

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nterference by Human Anti-Mouse Antibodies in CA 125 Assay after Immunoscintigraphy:nti-Idiotypic Antibodies Not Neutralized by Mouse lgG but Removed by ChromatographyJrsula Turpelnen, Penttl Lehtovlrta, Henrlk Alfthan, and UIf-H#{225}kanStenman

:alsely increased concentrations of the ovarian carcinoma-

ssociated antigen, CA 125, were measured by a monoclo-ial antibody (MAb)-based double determinant immunoradio-netric assay (IRMA) in patients who developed antibodies tonouse immunoglobulins (IgGs) after receiving injections ofhe same MAb as is used in the CA 125 IRMA. Addition ofndiluted mouse serum or purified mouse lgG to the assaynixture failed to eliminate the falsely increased CA 125oncentrations in most of the samples, owing to the presence)f anti-idiotype antibody. Because of their anti-idiotypic na-ure, the human anti-mouse antibodies (HAMA5)had only littleaffect on other immunometnc assays, and this effect could beompletely eliminated by addition of mouse lgG. To eliminatehe effect of HAMAon the CA 125 assay, we studied the ability)f various chromatographic methods to separate the interfer-ng HAMA from CA 125. For measuring HAMA in serum andhromatographic fractions we developed a time-resolvedluoroimmunoassay. Adequate separation of CA 125 andIAMA was achieved by affinity chromatography of patients’;era with solid-phase Protein A, Protein G, cation-exchangehromatography on Mono S. and gel filtration on Superose 6.Ihese results demonstrate that the interference can effec-ively be removed by rather simple chromatographic proce-lures.

ddItIonaI Keyphrasee: immunoradiometric assay time-esoIved fluoroimmunoassay . chromatography, cation-exchange

gel filtration

“Sandwich”-type immunoradiometric assays (ifiMAs))ased on mouse monoclonal antibodies (MAbs) give falselyncreased results when human anti-mouse immunoglob-un antibodies (RAMAS) are present in the patients’ seraJ_,3)l These antibodies simulate the behavior of the anti-[en by linking the radiolabeled IgG to the solid-phase.Inch artificial sandwiches are observed in immunoassays)ased on both monoclonal and polyclonal antibodies andre indistinguishable from the specific sandwiches (4-6).)evelopment of HAMA is a common complication afterntravenous injections of murine MAbs in connection withadioimaging of ovarian tumors. Induction of HAMA re-ponse has been found in 62% of the patients (7). Low

ncentrations of HAMA have also been found in 9% of bloodonors (8).

The m of CA 125 has been developed with use of aturine MAb, 0C125, (9) and optimized for quantification

Helsinki University Central Hospital, Department of Obstetricsnd Gynecology, Laboratory, Haartmaninkatu 2, 00290 Helsinki,inland.‘Nonstandard abbreviations: HAMA, human anti-mouse anti-dy; MAb, monoclonal antibody; IRMA, immunoradiometric as-ty; TK-IFMA, time-resolved fluoroimmunoassay; AFP, a-fetopro-in; CEA, carcinoembryonic antigen; and hGC, choriogonadotro-

in.Received February 14, 1990; accepted May 16, 1990.

of the MAb-defined determinant, CA 125 (10). Klug et al.(10) have discussed the possibility that at least someincreases in CA 125 concentrations can arise from serumfactors other than the defined antigenic determinants andrecently identified (6) an interfering human 1gM-type an-tibody in a patient for which the adverse effects wereeliminated by using a different iodination technique of theantibody. Addition of murine serum has also been used todiminish false-positive CA 125 results caused by HAMA (10,11). In other immunoassays, affinity chromatography onProtein A-Sepharose and murine MAb adsorbed onto vi-nylidene fluoride floccules have proved effective for remov-ing HAMA (8, 12, 13).

We analyzed serum samples from patients who hadundergone immunoscintigraphy with the MAb 0C125.Falsely increased concentrations of CA 125 in the “sand-wich”-type IRMA were found regularly, and the interferencecorrelated directly with the concentration of HAMA in thesera. In most of the samples, addition of mouse serumblocked only part of the interference. We therefore charac-terized the reactivity of HAMA and evaluated the ability ofvarious chromatographic methods to separate the interfer-ing factor from CA 125 antigen. On the basis of theseresults we propose methods for separating HAMA from CA125 antigen.

MaterIals and Methods

CA 125 analysis, immunoscintigraphy, and blocking ar-periments. The CA 125 IRMA kits (Abbott Laboratories,North Chicago, IL) were used according to the manufactur-er’s instructions. In the CA 125 IRMA, the same murineMAb to CA 125 is used both as capture and tracer antibodyin a simultaneous sandwich format.

Immunoscintigraphy was performed with ‘311-labeledF(ab’)2-fragments of 0C125 monoclonal antibody Imacis-2(ORIS, Gif-sur-Yvette, France).

To attempt to block or reduce the false increase in CA125 in the patient’s serum, we pre-incubated samples,directly or after dilution, for 1 h at room temperature with0.02-1.0 mL of mouse serum (Dakopatts AJS, Glostrup,Denmark) per milliliter of serum. Alternatively, mouseIgG, purified by Protein G chromatography (MAbTrap G;Pharmacia LKB, Uppsala, Sweden), was added at finalconcentrations of 1-50 mg of IgG per milliliter of serum.After this, we performed the CA 125 assay as usual.

Apparatus. All chromatographic separations, except Pro-tein G chromatography, were performed with a liquidchromatography system consisting of two 2150 HPLCpumps combined with a high-pressure mixer and a 2152HPLC controller (all from LKB-Produkter AB, Bromma,Sweden) and a Wisp 710B injector (Waters Associates,Milford, MA). All chromatographic runs were performed atroom temperature.

Affinity chromatography. Serum samples, 25 or 100 .tL,were applied to a Protein A-Superose HR 10/2 (10 X 20mm) column (Pharmacia LKB) in 0.1 mol/L sodium phos-phate buffer, pH 8.5, at a flow rate of 2 mL/min. Fractions

of 2 mL were collected. The bound protein was eluted with0.1 mol/L citric acid-NaOH buffer, pH 3.0, starting 6 mmafter sample application.

“Protein G fast flow” (Pharmacia LKB) was packed in an8 x 35mm polypropylene column (Bio-Rad, Richmond, CA)to total volume of 1.8 mL and equilibrated with 20 mmol/L

sodium phosphate buffer, pH 7.0. Then, 100 p.L of serumwas applied to the column. The flow rate was 0.5 mL/minwith use of hydrostatic pressure. After sample applicationthe column was washed with 10 mL of equilibration buffer,and elution was performed with 0.1 molJL glycine HC1solution, pH 2.7. Fractions of 1 mL were collected.

Ion-exchange chromatography. Anion-exchange chroma-tography was performed with aS x 50 mm Mono Q HR 5/5column (Pharmacia LKB). The column was equilibratedwith 20 mmol!L Tris HC1 buffer (pH 8.0), and 100 L ofserum was applied. Elution was achieved with a saltgradient from the starting buffer to buffer B: Ti-is . HC1 20minollL, NaC1 0.5 molJL, pH 8.0. Three minutes aftersample application, the concentration of buffer B waslinearly increased to 100% in 17 mm. Fractions of 1 mLwere collected.

A 5 x 50 mm Mono S HR 5/5 column (Pharmacia LKB)was used for cation-exchange chromatography. The columnwas equilibrated with 20 mmolIL sodium acetate buffer, pH5.0, pH 5.75, pH 6.5, or pH 6.75 (buffer A), and then elutedwith a gradient to 20 mmoIJL sodium acetate of the samepH containing NaC1, 0.5 molIL (buffer B). Elution with alinear gradient reaching 100% B in 18 mm was started 2mm after sample application. Serum samples of 50 L werediluted fourfold with buffer A, and 200 L of the dilutedsample was applied to the column. The flow rate was 1mL/min and fractions of 1 mL were collected. Centricon-lOmicroconcentrators (Amicon, Danvers, MA) were used ac-cording to the manufacturer’s instructions for concentra-tion of chromatographic fractions.

Gel filtration. This was performed with a 10 x 300 mmSuperose 6, HR 10/30 column (Pharmacia LKB) at a flowrate of 0.4 mL/min. The buffer was 50 mmol/L phosphate,0.5 mol/L NaCl, pH 7.2. Serum samples of 30-50 L werediluted with an equal volume of buffer before application.The column was calibrated using low- and high-molecular-mass standard proteins and Blue Dextran 2000 (Pharma-cia).

Hydrophobic interaction chromatography. This was per-formed with a 7.5 x 75 mm Ultropac TSK Phenyl-5PWcolumn (LKB-Produkter AB) at a flow rate of 1 mL/min.Buffer A was ammonium sulfate 1.7 molIL, phosphate 10mmolIL, and buffer B, phosphate 10 mmol/L, pH 7.0. Afterapplication of a 25-FL serum sample, the concentration ofbuffer B was linearly increased from 30% (starting buffer)to 100% in 20 mm. Fractions of 1 mL were collected.

Assay for anti-murine IgG antibodies. Crude mouse IgGswere isolated from mouse serum by precipitation at 45%ammonium sulfate saturation. The precipitate was dis-solved in Od moIiL sodium carbonate buffer, pH 9.0.

Mouse IgG was adsorbed onto polystyrene microtiterwells (Eflab Oy, Helsinki, Finland) by incubation overnightat room temperature. To each well we added 250 &Lof a 10mg/L solution of IgG in 0.1 mol/L sodium carbonate buffer,pH 9.0. After washing the strips with the wash solution,containing 9 g of NaCl, 0.2 mL of Tween 20, and 1 g ofGermall II as preservative per liter, we stored the strips ina moist atmosphere at 4#{176}C.

We added 25 L of fourfold-diluted serum samples, or

1334 CLINICAL CHEMISTRY, Vol. 36, No. 7, 1990

undiluted chromatographic fractions, and 200 tL of as&buffer (50 mmolIL Tris buffer containing 9 g of NaCI, 1 gbovine IgG, 0.1 mL of Tween 20, and 0.5 g NaN3 per liteto the coated microtiter wells and incubated for 2 h at roo:temperature. The wells were washed four times with wasolution, and to each well 20 ng of Eu-labeled polyclonalmonoclonal anti-human IgG (Pharmacia Wallac, Turk’Finland) in 200 L of assay buffer was added and incubatat room temperature for 1.5 h. Again, six washings weiperformed and 200 jL of enhancement solution (Pharmcia Wallac) was added (14). After gentle shaking for 5 miwe measured the fluorescence with a 1230 Arcus fluoroneter (Pharmacia Wallac), using a measuring time of 1 s.positive and negative control serum was included in eveidetermination. The antibody concentration was expres&in arbitrary units, one arb. unit corresponding to 10 OCcounts/s. The mean fluorescence (±SD) in sera from 4

apparently healthy subjects was 8040 ± 2460 counts/corresponding to 32 ± 10 kilo-arb. unitsfL with the po1:clonal Eu-label.

Quantitative assays. Human a-fetoprotein (AFP), carcnoembryonic antigen (CEA), and choriogonadotropi(hCG) were measured by time-resolved fiuoroimmunoasss(TR-IFMA) with commercially available kits (PharmaciWallac).

Binding studies. Binding of HAMA to different mouse asrat antibodies was determined by TR-IFMA. Polystyrerbeads coated with mouse MAb to CA 125, CEA, or with nMAb to estrogen receptor (Abbott Laboratories) were useWe added 25 pL of fourfold-diluted sera followed by 200 zof assay buffer and performed the assay as for the antmurine IgG antibodies. Before counting we added 500 /LLenhancement solution and transferred 200 L to microtitwells for counting. Results were expressed in counts/seond. To study the specificity of the antibodies, we preincibated the polystyrene balls coated with OC125 with 500arb. units of CA 125 (per ball) that had been obtained frorovarian cyst fluid after removing cells and debris by centrilugation. To these, 25 iL of fourfold-diluted serum samplewas added.

ResultsApparent concentrations of CA 125 and HAMA in patien

injected with MAb. We observed dramatic increases iapparent CA 125 concentrations in six of nine patienafter immunoscintigraphy. Figure 1 shows serum CA 1and HAMA concentrations in two patients after injectionthe MAb 0C125. About one month after the injectiondetected an increase in the apparent CA 125 concentrtions, which rapidly increased by 20-450-fold over the netwo months. This increase in apparent antigen concentrtions paralleled a similar rise in HAMA content. Generallboth the CA 125 and antibody concentrations decreaseda similar fashion three to six months after the immunocintigraphy. The similar pattern of these two suggestthat bridging of the two MAbs by HAMA in the CA 1immunoassay was responsible for the increase in CA 1concentrations.

We first attempted to suppress the false increase of C125 in patients’ sera by adding increasing amountsnormal mouse serum. Concentrations as high as 1.0 mLmilliliter of sample serum only slightly suppressed tapparent CA 125 concentrations (Figure 2). To evaluate tproportion of the antibodies that could be inhibitedaddition of mouse IgG, we diluted the patient’s serum to

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CLINICAL CHEMISTRY, Vol.36, No. 7, 1990 1335

Weeks

Fig. 1. l-lAMftand CA 125 before and after (CA 125, net) removal ofHAMAby cation-exchange chromatography in two patients who hadundergone immunoscintigraphy with MAb OC1 25Results expressed in kU/L = kilo-arb. units/L in Fig. 1, 2, and 4-6

Fig. 2. Effect on apparent #{231}A125 concentrations of addition ofvarious volumes of mouse serum per milliliter of four differentpatients’ sera containing HAMA

CA 125 concentration in the measuring range of the assayand added increasing amounts of purified IgG. The maxi-mum reduction was only 28%, attained with 10 mg ofmouse IgG per milliliter of serum. Larger amounts ofmouse IgG did not further reduce the apparent CA 125concentrations.

Interference in other assays. To study the specificity of the0C125-induced HAMA, we measured the concentrations ofCEA, AFP, and hCG by MAb-based immunometric assays.HAMA did not interfere in the Th-WIvIA of CEA. Minorinterferences were observed in the AFP and hCG assays. Inthe AFP assay HAMA decreased the true AFP values, and inthe hCG assay it increased them. The interference in thehCG assay was totally eliminated by adding 0.3 mL ofmouse serum to 1 mL of patient serum.

Binding of HAMA to various antibodies. To further studythe specificity of the 0C125-induced HAMA, we measured itsbinding to solid-phase mouse MAbs raised against CA 125

and CEA, to a rat MAb raised against the estrogen recep-tor, and to polyclonal mouse IgG isolated by affinity chro-matography. HAMA bound strongly to purified polyclonalmouse IgG and to OC125 (Figure 3). In two samples HAMA

bound significantly to the CEA MAb. HAMA did not reactwith the rat MAb to the human estrogen receptor. Thebinding to mouse IgG is not directly comparable with thatof the other assays because different solid phases wereused, microtitration strips for mouse IgG and polystyreneballs for the other assays. The background in the assayswith coated beads was much higher than in our standardassay with microtiter wells.

The idiotypic nature of HAMA was further studied byadding 5000 arb. units of partially purified CA 125 to 25 Lof fourfold-diluted sample in the HAMA assay with 0C125as solid-phase. CA 125 reduced the binding of HAMA to0C125 by 40%.

Separation of CA 125 and HAMA

Cation-exchange chromatography. On the cation-exchanger Mono S at pH 6.5, CA 125 did not bind to thecolumn, whereas all the interfering immunoreactivity(HAMA) was retained (Figure 4a). At pH 6.75 part of HAMA

(40%) eluted together with CA 125 as a broad peak withbuffer B at 7-11 mL. When the pH was lowered to 5.75 and5.0, part of CA 125 (30% and 75%, respectively) wasretained together with HAMA. At pH 6.5, CA 125 wasrecovered in the first 0.5-2 mL (Figure 4a) and this caused

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1336 CLINICAL CHEMISTRY, Vol. 36, No. 7, 1990

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Fig. 4. (a) Cation-exchange chromatography on Mono S at pH 6.5and (b) anion-exchange chromatography on Mono Q at pH 8.0 of apatient’s serum with HAMA(CA 125, total)For comparison the elution pattern of CA 125 of a control serum is shown (CA125, true)

a 30-fold dilution of the original CA 125 concentration.These results indicate a lower isoelectric point of CA 125compared with HAMA, thus complete separation at pH 6.5 ispossible.

To study the reproducibility of cation-exchange chroma-tography for removal of lWs, we studied serum samplesfrom patients who had undergone immunoscintigraphy4-30 weeks earlier. In all samples the CA 125 concentra-tion in the nonretained fraction was decreased and ap-proached prescintigraphic values (Figure 1). The MAMA

concentration in this fraction was not measurable. Afterchromatography at pH 6.5 of serum from the controlpatient, 90% of CA 125 was recovered in the nonretainedfraction, demonstrating that the effects on CA 125 concen-trations noted for the immunized patients were related tospecific removal of MAMA.

Anion-exchange chromatography. The results obtainedby anion-exchange chromatography on Mono Qat pH 8.0 ofHAMA and CA 125 are demonstrated in Figure 4b. The mainnative CA 125 peak eluted after IgG as a broad peak atabout 0.5 mol/L NaCl. MAMA was mainly recovered in thenonretained fraction but there was some overlapping withnative CA 125.

Affinity chromatography on Protein A and Protein G. Byaffinity chromatography on Protein A (Figure 5a) or Pro-tein G (Figure Sb) MAMA was bound and could be eluted atlow pH, whereas CA 125 was not retained. This treatmentcaused a marked decrease in apparent CA 125 concentra-tion of patients’ sera, CA 125 concentrations approachingpre-treatment values. In the patient’s samples (Figure 5)most of the apparent CA 125 reactivity and the antibodyconcentrations were in the retained fraction, whereas noMAMA was detected in the nonretained fraction. After

5 10 15 20Eiution volume (mL)

Fig. 5. Affinity chromatography on Protein A (a) or on Protein G (bof a patient’s serum with HAMA (CA 125, total)The elution patternof CA 125 in a control serum is shown for comparison (C�125, true). Bound protein is etuted (at arrow) with 0.1 mol/L citric acid-NaOl-buffer, pH 3.0 (a), or with 0.1 mol/L glycine . HCI, pH 2.7 (b)

affinity chromatography 80-90% of the CA 125 was recovered. Protein A chromatography caused a 20-fold dilutiorof original serum CA 125 when a 100-.tL sample was used:that with Protein G chromatography was 30-fold. Afteichromatography of serum from control patients, the recovcry of CA 125 was complete, demonstrating that the effect!noted for the patients were related to specific depletion olinterfering immunoglobulins.

Gel filtration. In gel filtration on Superose 6, native CA125 antigen eluted at a volume of 7-9 mL corresponding tcan Mr of more than 106 and HAMA at 12-15 mL correspond.ing to an Mr of about 150 000 (Figure 6a). The recovery oCA 125 was 78%. This shows that MAMA can be separatetifrom native CA 125 by gel filtration on the basis of thdifference in molecular mass.

Hydrophobic interaction chromatography. The hydrophbic properties of CA 125 antigen and HAMA were quitsimilar as the main immunoreactive peak of both components eluted at 17 mL, corresponding to 0.17 molt(NH4)2S04. The broad elution proffle of CA 125 antige:indicated a fairly high degree of heterogeneity in hydrphobicity, overlapping that of HAMA (Figure 6b).

DiscussionThis study demonstrates that the antibodies that develo

after injection of MAb 0C125 can be only partially neutralized by absorption with mouse serum and mouse IgCbecause a major part of HAMA consists of anti-idiotypantibodies. Several studies claim that addition of mousserum eliminates the false increase of CA 125 caused bMAMA (10, 11, 15). In sera with only moderately increaseconcentrations of HAMA, this treatment may reduce thfalsely increased CA 125 concentrations, but it is importan

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CLINICAL CHEMISTRY, Vol. 36, No. 7, 1990 1337

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Fig. 6. (a) Gel filtration on Superose 6 and (b) hydrophobic interac-tion chromatography on Ultropac TSK Phenyl-5PW of a patient’sserum with HAMA(CA 125, total), with the elution pattern of “true” CA125 in a control serum shown for comparisonSame symbols are used in (a) and (b)

o recognize that, in most cases, the effect is only partial forpatients who have received injections of 0C125. Anti-idiotype HAMA was recently described in a patient who hadreceived 0C125 (16).

The CA 125 assay is especially prone to MAMA interfer-ance because the same MAb is used both on the solid-phaseand as tracer. Studies of the effect of HAMA on various otherimmunometric assays have shown that the adverse effectswere minimal, except for CA 125, and could be eliminatedby addition of mouse serum or mouse IgG. The smallinterference is partially explained by the fact that mouseEgG is included in the buffers used in the sandwich assaysstudied. In addition, all the sandwich assays, except for A125, used different antibodies on the solid-phase and as abracer. Therefore, the likelihood that HAMA binds to bothsolid-phase and labeled antibody is reduced if the sameantibody is used for both purposes. Thus our results suggestthat immunoscintigraphy with 0C125 causes major prob-lems only for the assay in which this antibody is used.

Four weeks after injection of 0C125, an antibody re-sponse was observed as a simultaneous increase in CA 125and MAMA concentrations. This finding agrees with theresults of Moseley et al. (7). CA 125 concentrations re-rnained falsely increased for several months and thus madereliable measurement of actual CA 125 concentrationsimpossible. To solve this problem, we applied variousthromatographic methods to separate the CA 125 antigenom the interfering substances.

Of the chromatographic methods used, good separationwas achieved by affinity chromatography on Protein A andProtein G, by cation-exchange chromatography, and by gelftltration. Chromatography on Protein A and Protein G is

rapid and simple to perform. The complete binding of thenonspecific immunoreactivity to these columns (Figure 5)and especially to Protein G suggests that the interferingfactor in the patient serum was IgG. Gel filtration could beused because the molecular mass of CA 125 is greater thanthat of IgG; however, this procedure is slow and requires alarger dilution, about 40-fold, than the other methods.

Cation-exchange chromatography at pH 6.5 was a suit-able method for routine removal of HAMA. Because CA 125was not retained by Mono S at this pH, the chromatographywas simple to perform and CA 125 recovery was good. Thedilution of serum CA 125 in cation-exchange, Protein A,and Protein G chromatography was similar, about 30-fold.Before quantifying CA 125 after chromatography, we con-centrated the nonretained fractions about fivefold, but thetotal dilution of the original serum reduced the sensitivityto 30-50 kilo-arb. umtsfL. The recovery of the procedure,determined with sera containing native CA 125 antigen,was about 90%. However, a larger sample volume and alarger column can be used. An economical approach wouldbe to use cation-exchange chromatography with open-column technique. Preliminary studies suggest that 200 Lof serum can be processed on an 8 x 55 mm column ofS-Sepharose with the same recovery, but with a smallerdilution, as a Mono S column. This allows concentration ofthe sample to its initial volume.

The background of the MAMA assay was relatively highand varied from patient to patient, probably due to the highsensitivity of our assay technique in combination with thepresence of natural antibodies reacting with mouse IgG (8).Because of this, each patient should serve as his owncontrol when the assay is used to monitor MAMA afterimmunoscintigraphy.

The best specificity and sensitivity of the MAMA assaycould apparently be achieved by using the same MAb onthe solid-phase as that given to the patient. We tested thisby using the solid-phase bead from assays for CA 125, CEA,and estrogen receptor, and europium-labeled anti-humanIgG. This comparison showed that the HAMA of our patientsreacted much more strongly with 0C125 than with theother MAbs. However, H.AMA of different patients showedlarge differences in reactivity with various MAbs. A ratMAb showed the weakest reaction.

As the use of diagnostic immunoscintigraphy with MAbsincreases, the presence of MAMA in sera should be deter-mined, as should methods devised to eliminate the falselyincreased results. Although cation-exchange chromatogra-phy is effective for CA 125, removal of MAMA with Protein Aor Protein G, as reported here, may be more universallyapplicable. However, problems may be expected with seracontaining antibodies of IgA or 1gM type, which apparentlywas the case with some of our samples (not shown). ProteinA and Protein G affinity matrices are also relatively expen-sive, which will be important in large-scale use. Cation-exchange chromatography is more economical, especiallywhen applied as a simple open-column method, but re-quires that the antigen being measured have a clearlylower isoelectric point than MAMA. Consequently, the use ofthis method will be limited to relatively acidic antigens.Although most proteins are acidic, the applicability of themethod must be tested for each antigen.

The dilution of the sample caused by the chromato-graphic methods used in the present study poses a problem;e.g., normal concentrations of CA 125 could not be mea-sured without concentration of the fractions obtained by

1338 CLINICAL CHEMISTRY, Vol. 36, No. 7, 1990

chromatography. This problem may be solved by concen-trating the samples.

Although the adverse effects of HAMA can be reduced, theadditional work required is considerable. Therefore, thebenefits of using immunoscintigraphy have to be carefullyweighed against its potential adverse effects.

References1. Boscato LM, Stuart MC. Incidence and specificity of interfer-ence in two-site immunoassays. Clin Chem 1986;32:1491-5.2. Boscato LM, Stuart MC. Heterophilic antibodies: a problem forall immunoassays. Clin Chem 1988;34:27-33.3. Hansen HJ, LaFontaine G, Newman ES, et a!. Solving theproblem of antibody interference in commercial “sandwich”-typeimmunoassays of carcinoembryonic antigen. Cliii Chem1989;35:146-51.4. Bock JL, Furgiuele J, Wenz B. False positive immunometricassays caused by anti-immunoglobulin antibodies: a case report,Clin Chim Acts 1985;147:241-6.5. Clark PM, Raggatt PR, Price CP. Antibodies interfering inimmunometric assays [Letter]. Clin Chem 1985;31:1762.6. Klug TL, Green PJ, Zurawski VR, Davis HM. Confirmation ofa false-positive result in CA 125 immunoradiometric assay causedby human anti-idiotypic immunoglobulin. Clin Chem 1988;34:1071-6.7. Moseley KR, Knapp RC, Haisma HJ. An assay for the detectionof human anti-murine immunoglobulins in the presence of CA125antigen. J Immunol Methods 1988;106:1-6.8. Thompson RJ, Jackson AP, Langlois N. Circulating antibodiesto mouse monoclonal immunoglobulins in normal subjects-mci-

dence, species specificity, and effects on a two-site assay foicreatine kinase-MB isoenzyme. Clin Chem 1986;32:476-81.9. Bast RC, K1ugTL, St. John E, et al. A radioimmunoassay usina monoclonal antibody to monitor the course of epithelial ovariarcancer. N Engi J Med 1983;309:883-7.10. Klug TL, Bast RC, Niloff JM, Knapp RC, Zurawski YRMonoclonal antibody immunoradiometric assay for an antigenitdeterminant (CA 125) associated with human epithelial ovariarcarcinomas. Cancer Res 1984;44:1048-53.11. Mogensen 0, Moller J. False positive results in an enzymtimmunometric assay for the ovarian cancer associated antigen CA125. Eur J Cancer Clin Oncol 1989;25:129-31.12. Primus FJ, Kelley EA, Hansen HJ, Goldenberg DM. “Sandwich”-type immunoassay of carcinoembryonic antigen in patient1receiving murine monoclonal antibodies for diagnosis and therapyClin Chem 1988;34:261-4.13. Newman ES, Moskie LA, Duggal RN, Goldenberg DM, Hansen HJ. Murine monoclonal antibody adsorbed onto vinylidenCfluoride floccules used to eliminate antibody interference in “sandwich”-type immunoassays. Clin Chem 1989;35:1743-6.14. Hemmil#{225}I, Dakubu S, Mukkala V-M, Siitari H, LOvgren TEuropium as a label in time-resolved immunofluorometric assaysAnal Biochem 1984;137:335-43.15. Bouvier J-F, Pernod J, Rivoire M, Maiassi N. Serum levels oltumor markers and presence of human antimouse antibodiesCancer Detect Prevent 1988;13:251-62.16. Reinsberg J, Heydweiller A, Wagner U, Pfeil K, Oehr P, KrebsD. Evidence for interaction of human anti-idiotypic antibodieswith CA 125 determination in a patient after radioimmunodetec’tion. Clin Chem 1990;36:164-7.