recovery of immunologically reactive antibodies …...s. w. maidment, jr., et al. tbs with several...

[CANCER RESEARCH 41, 795-800, March 1981]0008-5472/81 /0041-OOOOS02.00

Recovery of Immunologically Reactive Antibodies and Antigens from

Breast Cancer Immune Complexes by Preparative IsoelectricFocusing1

Bert W. Maidment, Jr., Lawrence D. Papsidero, Takumo Nemoto, and T. Ming Chu2

Departments of Diagnostic Immunology Research and Biochemistry [B. W. M., L. D. P.. T. M. C.Â¡and Breast Surgery [T. N.Â¡.Roswell Park Memorial Institute,Buffalo, New York i 4263

ABSTRACT

A new technique for the recovery of immunologically reactiveantibodies and putative antigens from breast cancer-associ

ated immune complexes was described. Soluble immune complexes were isolated from extracellular fluids by 2.5% polyethylene glycol fractionation and affinity chromatography onProtein A:Sepharose CL-4B. The immune complexes bound toimmobilized Protein A were then subjected to preparative iso-

electric focusing. The dissociated immunoglobulins and putative antigens were recovered from the appropriate pH regionsof the preparative isoelectric focusing gel. G-type immunoglob

ulins recovered from immune complexes isolated from a breastcancer patient bound to the recovered putative antigen(s) andthree breast cancer cell culture lines, but these immunoglobulins did not bind to carcinoembryonic antigen or other cancercell culture lines originating in colon, pancreas, prostate, orlymphoblast. The recovered putative antigens had Â¡soelectricpoints between pH 3.0 and 5.0 and molecular weights of20,000 and 42,000.

INTRODUCTION

The study and characterization of immune complexes areimportant to the understanding of their role in the pathogenesisof immune complex-associated diseases. Immune complexes

have been detected in various malignant and nonmalignantdiseases (22, 28, 29). Immune complex levels have beencorrelated with prognosis of breast cancer patients (11, 24),and increased levels may be associated with disseminateddisease (10). Data also indicate that immune complex measurements may be useful in the differential diagnosis of benignand malignant breast diseases (20). Immune complexes havebeen implicated as one of the possible factors involved in"blocking" or "inhibition" of cell-mediated immunity in cancer

patients (3, 23, 26) and immunoregulation (9, 12, 18).Evidence has been provided to show that immune complexes

may contain tumor-associated antigens or antibodies (2, 19,29). The existence of tumor-associated immune complexes has

not been unequivocally established, and the identity of theantigen component(s) of such immune complexes remainsobscure in most instances (1, 25).

Therefore, the isolation and characterization of the components of immune complexes may lead to the elucidation of therole of immune complexes in human pathology, the develop

ment of new concepts in the pathogenesis of immune complex-associated diseases, and the identification of new tumor-re

lated products which may be candidates as markers for diagnosis and monitoring.

Recently, a technique using analytical IEF3 was developed

which dissociated antigen-antibody complexes and separatedthe antigen and antibody components, based upon differencesin isoelectric points (16). Furthermore, the dissociated immunecomplex components were recovered, identified, and found toretain immunological or biological reactivity. Subsequently, thisIEF*technique was used to examine immune complexes isolated

from extracellular fluids of cancer patients. The immune complexes were purified by a combination of PEG fractionation andProtein A:Sepharose CL-4B affinity chromatography. IgG and

putative antigens were detected in clinical specimens obtainedfrom cancer patients (17). As a continuation of this study,preparative IEF has been used for the isolation of antigen andantibody components from immune complexes purified fromthe extracellular fluid of a breast cancer patient.

MATERIALS AND METHODS

Purification of Immune Complexes from Clinical Specimens. Pleural fluids were obtained aseptically from a breastcancer patient by thoracentesis. Cytological examination confirmed the presence of malignant cells in the specimens.

Purification of immune complexes from pleural fluid has beensummarized previously (17) and is described as follows. Briefly,pleural fluid was brought to 0.02 M disodium EDTA (EastmanKodak Co., Rochester, N. Y.) with solid EDTA at room temperature for 2 hr and was clarified by centrifugation at 3000 x gfor 30 min at room temperature. Ten ml of 25% (w/v) PEG(M.W. 6000; Sigma Chemical Co., St. Louis, Mo.) in 0.05 MTBS (pH 8.4) were mixed with 90 ml of EDTA:pleural fluidsolution and incubated with continuous stirring for 90 min atroom temperature. After centrifugation at 2000 x g for 10 minat room temperature, the precipitate was washed with 2.5%PEG in TBS (pH 8.4) and then centrifuged at 2000 x g for 10min. The supernatant was discarded. The precipitate was re-

dissolved in 15 ml of 0.05 M TBS (pH 7.5) and dialyzed against

' This work was aided by Institutional Research Grant IN-54-S-13 of theAmerican Cancer Society and by Research Grant CA-25653 awarded by theNational Cancer Institute, Department of Health, Education and Welfare.

2 To whom requests for reprints should be addressed.

Received August 22, 1980; accepted November 10, 1980.

3 The abbreciations used are: IEF, isoelectric focusing; PEG, polyethylene

glycol; TBS, 0.05 M Tris;0.14 M NaCI:0.1 % NaN3, pH 7.5 unless otherwise noted;PEG-PPT, polyethylene glycol-precipitated materials; Protein A, purified Staph-ylococcus aureus Protein A; PrepAb, putative antibodies recovered by preparative isoelectric focusing at pH 6.0- to 8.0-region; Ag3-5, putative antigensrecovered by preparative isoelectric focusing at pH 3.0- to 5.0-region; TTB, 0.08M Tris:0.024 M W-tirs(hydroxymethyl)methylglycine:0.024 M barbital:0.003 Mcalcium lactate, pH 8.8; IEP, immunoelectrophoresis; PBS, 0.05 M NaH2PO4:Na2HPO4:0.14 M NaCI, pH 7.0; BSA, bovine serum albumin; CEA, carcinoembryonic antigen; RPMI Medium 1640, Roswell Park Memorial Institute Medium1640; SDS:PAGE, sodium dodecyl sulfaterpolyacrylamide gel electrophoresis;SOS, sodium dodecyl sulfate.

MARCH 1981 795

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S. W. Maidment, Jr., et al.

TBS with several changes of buffer. The dialyzed solution,PEG-PPT, was clarified by centrifugaron at 3000 x g for 30min. Ten ml of Protein A:Sepharose CL-4B (Pharmacia Fine

Chemicals, Inc., Piscataway, N. J.) in 50% TBS (v/v) stocksuspension were mixed with 15 ml of PEG-PPT at room temperature for 2 hr and then at 4Â°for 18 hr. Nonbinding materials

were removed by repeated washing of the immobilized ProteinA with TBS until no protein was detected in the 2000 x gsupernatant, as determined spectrophotometrically at 280 nm.This preparation of immune complexes bound to immobilizedProtein A was used for preparative IEF.

Preparative Flat-Bed IEF of Immune Complexes Bound toProtein A. Granular gel bed preparation and sample applicationwere performed by modifying the procedures outlined in LKBApplication Note 198 (30). The granular gel bed (11 x 24.5cm) was prepared with 5% IEF Sephadex (w/v) with 2%ampholytes (pH 3 to 10). A sample application trough wasprepared by placing the 9.5- x 2.5-cm sample applicator

template 1.5 cm away from the parallel to the anode. The gelincluded within the sample applicator template was then removed. Five ml of Protein A:Sepharose CL-4B with bound

immune complexes in 85% TBS (v/v) were placed in thesample applicator template. The template was removed, andthe gel bed was allowed to equilibrate hydrostatically for 5 min.IEF was performed using a Buchler Model 3-1500 power unitset at 2 watts, constant power, for 40 to 42 hr at 4Â°.Anode

buffer was 1 M H3PC>4,and cathode buffer was N NaOH. A 30-

zone fractionating grid was pressed into the gel bed at thecessation of the run. The pH gradient was determined usingBio-Lyte Gel Pro-pHiler (Bio-Rad Laboratories, Richmond,

Calif.) and a Fisher Accument Model 140 pH meter.Recovery of Isoelectrically Focused Material. Isofocused

materials were separated from the gel media by filtrationthrough Whatman no. 1 filter paper. The materials were washedfrom the gel by applying approximately 2 bed volumes of TBSto the gel. The materials recovered from the pH 6.0 to 8.0region of the gel bed (prepAb) and the materials from the pH3.0 to 5.0 region of the gel bed (Ag3-5) were each dialyzedagainst distilled water at 4Â°with several changes of distilled

water. The dialyzed materials were then lyophilized.Gel Filtration on Sephadex G-200. PEG-PPT or PrepAb

reconstituted with TBS were chromatographed over a Sephadex G-200 (Pharmacia) packed column (0.9 x 60 cm) equilibrated with TBS; 1-ml fractions were eluted with TBS, col

lected, and monitored for absorbance at 280 nm. Fractionswere also analyzed for IgG by immunodiffusion against rabbitanti-human IgG (y chain-specific) antiserum (Behring Diagnos

tics, Somerville, N. J.) in 1% agarose (Sigma type 1) in 0.08M TTB at room temperature for 24 hr. Agarose gels werewashed extensively in 0.14 M NaCI and then distilled H2O,press dried, stained with crocein scarlet (5), and destained in0.3% acetic acid.

IEP. IEP was performed using 1% agarose in TTB buffer.Samples (5 /ul) were placed in sample wells and electropho-resed at 4Â°with 150 V constant voltage. Electrophoresis was

monitored with bromophenol blue-dye marker. The anode andcathode buffers were TTB. Eighty jul of rabbit anti-human IgG(y chain-specific) antiserum or rabbit anti-human serum anti-

serum (DAKO, Copenhagen, Denmark) were transferred bypipet into a 6-mm-long trough cut 5 mm away from the sample

and parallel to the direction of electrophoretic migration. The

IEP plate was incubated at room temperature for 24 hr, washed,and destained as described for the immunodiffusion agarosegels.

Radioiodination of Ag3-5 and Protein A. Approximately 50jug Ag3-5 were reconstituted with 100 /xl 0.05 M PBS, mixedwith 0.3 mCi dried 125Ias Bolton and hunter reagent for protein

iodination (Amersham/Searle, Arlington Heights, III.) in a glasstest tube (1x10 cm), and incubated at 4Â°for 18 to 20 hr. One

hundred ^g purified Protein A (Pharmacia) were reconstitutedwith 100 n\ PBS, mixed with 1 mCi dried 125Ias Bolton and

Hunter reagent in a glass test tube (1x10 cm), and incubatedat room temperature for 15 min. Five hundred jul of 0.2 Mglycine in PBS were added to each tube to block unreactedreagents. Free 125Iwas removed by desalting the mixtures

separately over a PD-10 (Pharmacia) column (1.5 x 5 cm)

equilibrated with 0.05 M sodium phosphate:0.5 M NaCI, pH7.2, or PBS:3% BSA (Sigma). Fractions (1 ml) were collectedand monitored for 125Iby using a Packard Auto-Gamma scintil

lation spectrometer. The fractions corresponding to the voidvolume were pooled. I25l-labeled Protein A was diluted withPBS:3% BSA, aliquoted, and stored at -20Â° until use. The

specific activity was approximately 8 /iCi/ftg. RadiolabeledAg3-5 was mixed with Pansorbin (Calbiochem-Behring, La

Jolla, Calif.) to remove nonspecifically binding materials, incubated for 18 to 20 hr at 4Â°,and centrifuged at 3000 x g for 10

min. The supernatant was used for the immunological recombination assay.

Immunological Recombination Assay. The PrepAb was reconstituted in TBS: 1% BSA (Sigma) to a concentration of 2.5/Â¿gPrepAb per ml. A 2.5-/ug solution of human IgG per ml

(monomeric 7S preparation) in TBS:1% BSA was prepared.Thirty /Â¿I125l-labeled Ag3-5, 30 /il TBS:1% BSA, and 30 /il

PrepAb diluted with TBS:1% BSA (1:1; 1:10; or 1:100) weremixed in a polypropylene conical test tube and vortexed. After3 hr of incubation at room temperature, 30 jul of Pansorbinwere added to each tube, vortexed, incubated for 1 hr at roomtemperature, centrifuged at 3000 x g for 10 min, and washedtwice with 2 ml TBS:1% BSA:0.5% Triton X-100 (Sigma).

Nonspecific binding to normal human IgG was measured bysubstituting the same concentrations of human IgG in TBS:1 %BSA for PrepAb in TBS:1 % BSA. Also, 125l-labeled CEA (Roche

Diagnostics, Nutley, N. J.) was substituted for radiolabeledAg3-5 to measure nonspecific binding. After the last centrifu-

gation, the supernatant was aspirated and discarded, and thetubes were counted in a Packard Auto-Gamma spectrometer.Specific binding was calculated as follows: (PrepAb cpm) â€”

(human IgG cpm). Both PrepAb and the human IgG were thesame concentration. The specific binding was graphed versusPrepAb dilution.

Cell Culture. Human cell cultures were grown, unless otherwise indicated, in plastic culture flasks under standard conditions in PRMI Medium 1640 supplemented with 15% fetalbovine serum (Grand Island Biological Co., Grand Island,N. Y.) and 1-fold solution of insulin, minimum essential medium

with nonessential amino acids, and glutamine (Grand IslandBiological Co.; supplied as 100-fold solution). Cell lines ofhuman adenocarcinoma origin included BT-20 (14), MCF-7(27), SK-Br-3 (from Dr. J. Fogh, Sloan-Kettering MemorialInstitute for Cancer Research), CaPan-2 (from Dr. J. Fogh),PC-3 (13), HT-29 (6), and a human lymphoblastoid cell line,

796 CANCER RESEARCH VOL. 41



Breast Cancer Immune Complexes

125l-IabeledProtein A Assay for Cell Surface Antigens. Theassay was performed in triplicate as modified from the procedunedescribed by Brown et a!. (4). Adenocarcinoma cells weretested as monolayers in flat-bottomed Microtest 2 tissue cultureplates (Falcon Plastics, Oxnard, Calif.). Monolayers were prepared by adding the appropriate cells (5 x 10@)suspended inRPMI Medium 1640 with 15% bovineserumto each well andincubating at 37Â°in a humid incubator with 5% CO2in air. Theassay was performed the next day after cells had formed aconfluent monolayer.

At the start of the assay, the culture medium was removedby aspiration, and the cells were washed twice with fresh RPMIMedium 1640:1 % BSA. One hundred ,sl of the appropriateantibody test solution, PrepAb (250 jzg/ml), human lgG (250@tg/ml),or buffer (RPMI Medium 1640:1 % BSA) were added to

the wells. After incubation at room temperature for 2 hr, theantibody test solution was removed, and the cells were washedwith RPMI Medium 1640:1 % BSA. One hundred @zl1251-labeledProtein A (1O@cpm) were then added to each test well. After a1-hr incubation at room temperature, the reagent was removed,and the cells were washed twice with 200 @dRPMI Medium1640:1 % BSA. The cells were then dissolved in 100 @tlof 2 NNaOH and transferred to polypropylene tubes for@ 251determination in a Packard Auto-Gamma scintillation spectrometer.The assay was modified for the Raji cells. Raji cells wereincubated with the appropriate reagents in polypropylene conical test tubes in suspension, washed repeatedly by centrifugation at 1000 x g for 10 mm, and then aspirated. The finalpellet of cells was counted in a Packard Auto-Gamma scintillation spectrometer for 1251determination. A specific bindingindex was then calculated by the following formula:

(PrepAbcpmâ€” buffercpmâ€” (humanlgGcpmâ€” buffercpm)(PrepAbcpm â€”buffercpm)

SDS-PAGEof â€˜261-labeledAg3-5. Ninety @zlof â€˜251-labeledAg3-5 absorbed previously with Pansorbin, 90 @zlof TBS:i % BSA,and 90 @lof PrepAb (2.5 pg/mI) were mixed in a polypropyleneconical test tube and vortexed. After a 3-hr incubation at roomtemperature, 90 @sIof Pansorbin were added to the tube toprecipitate lgG and lgG-bound â€˜25l-labeledAg3-5, vortexed,incubated for 1 hr at room temperature, washed with TBS:1BSA:0.5% Triton X-i 00, and centrifuged until the supennatantcontained no detectable 1251radioactivity. The Pansorbin precipitate was mixed with 100 @iI1% SDS:0.05 M Tris:0.38 Mglycine, pH 8.4, and incubated for 18 hr at room temperature.

Polyacrylamide gels with 7.5% acrylamide (total concentration, 7.5%; cross-linking agent, 2.5%) and 0.05 M Tnis:0.i %SDS were prepared (16). The Pansorbin precipitate was mixed

with 100 @lglycerol with bromophenol blue dye marker andapplied to the top of 0.5 x 10-cm gels. The sample wasovenlayered with running buffer (0.1 % SDS:0.OS M Tnis:0.38M glycine, pH 8.4) and electrophoresed using LKB Model2i 03

power unit with 2 ma/tube constant current at 22Â°until thedye indicator migrated to within 5 mm of the bottom of the gel.The gel was removed from the glass tube and sliced into 2-mmsegments. Each 2-mm gel segment was placed in a separatetube and counted in a Packard Auto-Gamma scintillation spectrometen for 1251determination.

Rheumatoid Factor and Erythrocyte Agglutination Tests.PrepAb was tested for rheumatoid factor activity using a latexagglutination test (Rapi/tex-RF kit; Behning Diagnostics) andtested for hemagglutination activity with freshly obtained typeA and type B human erythrocytes in a tube hemagglutinationassay (7).

Isolationof Human igG. MonomerichumanIgG was isolatedfrom normal human serum by ammonium sulfate precipitationfollowed by a DEAE anion exchange chromatography as descnibed previously (15). Purity of isolated lgG preparation wasassessed by Sephadex G-200 gel filtration and by IEP withrabbit antisera to human lgG (â€˜ychain specific) and rabbitantisera to human serum protein (DAKO) and further confirmedby ultracentnifugation (8). No aggregated lgG was detected.

RESULTS

isolationÃ§ifimmunoglobulins.Fractionationof the pleuralfluid from a breast cancer patient by 2.5% PEG precipitationresulted in the isolation of high-molecular-weight (greater than232,000) proteins as shown by Sephadex G-200 gel filtrationchromatography. High-molecular-weight lgG, presumably inthe form of immune complexes, was detected in the fractionscorresponding to the void volume of the Sephadex G-200column, and no lgG (M.W. 150,000) was detected. The matenials containing high-molecular-weight lgG were allowed toreact with immobilized Protein A and then subjected to preparative IEF (pH 3 to 10). The proteins (PrepAb) recovered fromthe immunogiobulin region (pH 6.0 to 8.0) of the gel werecharacterized by IEP and Sephadex G-200 gel filtration chromatography. The recovered PrepAb was shown to be humanlgG as determined by IEP with antiserum against human IgGand antiserum against normal human serum (Fig. 1). By gelfiltration chromatography, the recovered PrepAb eluted as asingle symmetrical protein peak corresponding to a molecularweight of 158,000 (Chart 1). The amount of PrepAb recoveredrepresented approximately 0.08% of the total protein presentin the original pleural fluid (3,750 mg total protein).

Fig. 1. Immunoelectrophoretic analysis of isolated IgG(PrepAb). Proteins (PrepAb) recovered from the pH 6.0-to 8.0-region of the preparative IEF gel were analyzed byIEP as described in â€œMaterialsand Methods.' â€W̃ell 1,human serum albumin; Well 2, PrepAb. Trough A, rabbitanti-normal human serum protein antiserum; Trough B,rabbit anti-human lgG (â€˜i'chain-specific) antiserum.

L

+

B.@t4(

MARCH 1981 797



S. W. Maidment, Jr., Ã©tal.

No rheumatoid factor activity or agglutination of type A ortype B human erythrocytes was detected when the PrepAbwas analyzed by the rheumatoid factor latex agglutination testor hemagglutination tube assay.

Immunological Recombination of Recovered Immunoglob-

ulin and Putative Antigen. In order to show recombination ofthe recovered PrepAb and the recovered putative antigen, thePrepAb was mixed with radiolabeled Ag3-5. Pansorbin wasthen added to the mixture to precipitate IgG and IgG-bound125l-labeled Ag3-5. Nonspecific binding to human normal IgG

was measured by substituting human IgG for the PrepAb at thesame concentration. Binding of 126l-labeled CEA to PrepAb and

human IgG was also determined. The recovered PrepAb and125l-radiolabeled recovered antigen (125l-labeled Ag3-5) were

shown to recombine (Chart 2). It was shown that the specificbinding of 125l-labeled Ag3-5 to PrepAb decreased with increasing PrepAb dilution. No significant binding of 125l-labeled

CEA to PrepAb was observed.SDS-PAGE Analysis of Putative Antigens. SOS-PAGE was

used to obtain approximate molecular weights of immunologi-

SEPHADEX G-200

8-

eooi 70Â°' 600

1 5OO

2 400<* 300

20O

IOO

Vo

-IO if)

o

Â§CDUJ

IO 12 14 16 IB 20 22 24 26 28 30 32 34 36 38 40

FRACTION NUMBER

Chart 1. Gel filtration analysis of isolated IgG (PrepAb). PrepAb was chro-matographed over a packed Sephadex G-200 column (0.9 x 60 cm). One-mifractions were collected and analyzed for protein by absorbance at 280 nm.Markers were: catalase (M.W. 232,000); aldolase (M.W. 158,000); and BSA(M.W. 68,000). Vo, void volume; V,, total bed volume.

' no i loo

ANTIBODY DILUTION

Chart 2. Immunological recombination of PrepAb and radiolabeled Ag3-5.PrepAb was incubated with 125l-labeled Ag3-5, precipitated with Pansorbin, andwashed, and bound 125Iradioactivity was counted. The nonspecific binding to

human IgG was determined by the identical experiment substituting human IgGfor PrepAb. PrepAb was tested also for its ability to react with radiolabeled CEA.Specific binding (PrepAb cpm - human IgG cpm) was calculated for 125l-labeledAg3-5 and 125l-labeled CEA at various dilutions of PrepAb and human IgG.

cally reactive radiolabeled putative antigens. 125l-labeled Ag3-

5 was incubated with PrepAb, and the resultant complexeswere precipitated with Pansorbin. The Pansorbin precipitatewas analyzed by SDS:PAGE. The SDS:PAGE gel was slicedinto 2-mm segments, and each segment was counted for 125I-labeled Ag3-5 radioactivity. Two 126l-labeled Ag3-5 radioactiv

ity peaks were detected; a minor radioactive peak corresponding to a molecular weight of 42,000 and a major radioactivepeak corresponding to a molecular weight of 20,000 weredemonstrated (Chart 3).

12Sl-labeled Protein A Assay for Cell Surface Antigen De

tection. The recovered PrepAb was analyzed for binding specificity to various malignant cultured cells. Malignant cells weregrown to confluence on flat-bottomed Microtest 2 plates. Cells

were incubated with PrepAb, human IgG, or buffer (RPMIMedium 1640:1% BSA) and washed. The cells were thenincubated with 125l-labeled Protein A to detect cell surface-

bound IgG. A specific binding index was calculated as described in "Materials and Methods." A high specific binding

index of PrepAb was observed with 3 breast cancer cell lines(MCF-7, BT-20, and SK-BR-3) (Table 1). A low specific bindingindex of PrepAb was calculated when HT-29 colon cancer,CaPan-2 pancreas cancer, PC-3 prostate cancer, or Raji lym-

phoblastoid cell lines were used.

DISCUSSION

Immune complexes purified by 2.5% PEG fractionation andProtein A:Sepharose CL-4B affinity chromatography are dissociated and separated by IEF (17). The present data showthat immunologically reactive immune complex componentscan be recovered from preparative IEF of immune complexespurified from the pleural fluid of a breast cancer patient. Immune complex components (antibody and antigen) are recovered and are immunologically reactive. The supportive dataare as follows: (a) IgG (M.W. 150,000) is recovered from thepreparative IEF of high-molecular-weight IgG obtained by 2.5%

PEG fractionation and then bound to immobilized Protein A; (D)the recovered antibody (PrepAb) and the recovered antigens(125l-labeled Ag3-5) are able to recombine immunologically as

1234567MIGRATION IN cm

8 9 10

Chart 3. SDS:PAGE analysis of immunoreactive radiolabeled Ag3-5; 125l-la-beled Ag3-5 was incubated with PrepAb. PrepAb with bound 125l-labeled Ag3-5

was then precipitated with Pansorbin. The Pansorbin precipitate was analyzedby SDS:PAGE. After electrophoresis, the gel was sliced into 2-mm segments,and each segment was counted for 125Iradioactivity. 125Icpm were plotted versus

cm distance of migration into the SDS:PAGE gel. Arrows, markers: BSA (M.W.68.000): ovalbumin (M.W. 44.000); and RNase (M.W. 13,700).




Breast Cancer Immune Complexes

Table 1

Reactivity of isolated IgG (PrepAb) with cell surface antigens of human tumorcells

Human tumor cells grown to confluence were incubated with PrepAb, humanIgG, or buffer and then washed. In order to detect cell surface-bound IgG, cellswere incubated with radiolabeled Protein A, washed, and counted. The specificbinding index was calculated for each cell line tested. Increased binding ofPrepAb to breast cancer lines compared to other cancer cell lines was detectedby Student's t test ( p < 0.05).

Mean of replicate determinations(cpm)Cell

lineBT-20

MCF-7SK-Br-3CaPan-3HT-29PC-3RajiClassification

typeBreast

cancerBreast cancerBreast cancerPancreas cancerColon cancerProstate cancerLymphoblastoidPrepAb527

4896402790421199869Human

IgG179

867167759

436180

2415Buffer146

18369476

41084

712Specific

binding index"0.91

0.860.710.01

<0.010.16

<0.01

Specific binding index

_ (PrepAb cpm - buffer cpm) - (human IgG cpm - buffer cpm)

(PrepAb cpm â€”buffer cpm)

determined by the recombination assay; and (c) the recoveredantibody (PrepAb) binds cell surface antigens associated withmalignant breast tissue culture cells.

Antigen and antibody components of immune complexes areisolated by the physicochemical methods described. Data presented in this report provide evidence to substantiate thepotential importance of these moieties. The antibodies isolatedfrom the pleural effusion of a breast cancer patient showspecific binding to cell surface antigen(s) present on MCF-7,BT-20, and Sk-Br-3 breast cancer tissue cell lines, and they

do not bind to other cancer cell lines of pancreas, prostate,colon, or lymphoid origin. Whether this reactive breast cancercell surface antigen(s) is tumor associated or organ specificremains to be determined. Initial characterization of the putativeantigen(s) recovered by these methods provides informationon their physicochemical properties. The immunologically reactive putative antigen(s) recovered exhibits isoelectric focusing points between pH 3.0 and 5.0. The major putative antigencomponent demonstrated a molecular weight of 20,000, andthe minor putative antigen component, a molecular weight of42,000 by SDS:PAGE. The relationship of these putative antigen moieties with breast cancer is the subject of continuingstudy. Additional characterization of these moieties may leadto the identification of new breast tumor-related products.

Although data from pleural fluid of a breast cancer patientare presented, immune complex components have been detected by using an identical analytical IEF procedure in pleuralfluid or ascites fluid from 12 of 15 breast cancer samples; 3 of4 pancreas cancer (ascites fluid) samples; one of one coloncancer (ascites fluid) sample; one of one kidney cancer (ascitesfluid) sample; and none of two lymphoma, one leukemia, or 4nonmalignant samples (2 each of congestive heart failure andpneumonia) (17). These results indicate further the validity ofthe methods presented.

In conclusion, the preparative IEF of immune complexesisolated by 2.5% PEG precipitation and Protein A:Sepharoseaffinity chromatography results in the recovery of immunologically active antibody and putative antigen components. Theisolated antibody and putative antigen components can becharacterized physicochemically and immunologically. Further,

the isolated antibody can be used as a reagent for the detectionand isolation of antigens, and the isolated antigen can be usedfor the production of heteroantisera or hybridoma antibodiesthat may be useful in the development of specific immunoas-says for diagnosis and monitoring of immune complex-associ

ated antigens.

ACKNOWLEDGMENT

We would like to thank J. Ogledzinski for her secretarial assistance.

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1981;41:795-800. Cancer Res Bert W. Maidment, Jr., Lawrence D. Papsidero, Takumo Nemoto, et al. Isoelectric Focusingfrom Breast Cancer Immune Complexes by Preparative Recovery of Immunologically Reactive Antibodies and Antigens

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