Proc. Natl. Acad. Sci. USAVol. 80, pp. 124-128, January 1983Biochemistry

Detection of unique antigenic determinants on human plasma lowdensity lipoprotein and on delipidated apolipoprotein B

(enzyme-linked immunosorbent assay/hybridomas/detergent-solubilized apoprotein/biotinylated monoclonal antibodies)

TERRI S. WATT AND ROBERT M. WATTDepartment of Microbiology, State University of New York, Upstate Medical Center, Syracuse, New York 13210

Communicated by Charles Tanford, October 7, 1982

ABSTRACT To obtain detailed information on the role playedby apolipoprotein B (apo B) in determining the structural and func-tional properties of human plasma low density lipoprotein, wehave initiated immunochemical studies of the polypeptide. Wereport here the establishment of six hybridoma lines that secretemonoclonal antibodies to low density lipoprotein. In addition torecognizing antigenic determinants on low density lipoprotein, allsix monoclonal antibodies react with epitope(s) on very low density,but not high density, lipoproteins. The immunoreactivity of theseantibodies with low density lipoprotein and with detergent-deli-pidated apo B was compared in an enzyme-linked immunosorbentassay. Although all six of the antibodies reacted with the apopro-tein when it was prepared in a nonionic detergent known to main-tain the secondary structure of the protein, three of the six anti-bodies showed partial or total loss of activity with NaDodSO4-delipidated apo B. The specificity of these antibodies was testedby the ability of affinity-purified biotinylated antibodies to com-pete with unlabeled antibodies for antigenic sites on low densitylipoprotein in a competition enzyme-linked immunosorbent assaydeveloped with avidin-peroxidase. This competition assay allowedus to divide the antibodies into a minimum of two groups (I andH) based on the antigenic determinants on apo B that they rec-ognized. The epitope on apo B recognized by group II antibodieswas perturbed in NaDodSO4, whereas the determinant(s) on theprotein reactive with group I antibodies was unaffected.

Solubilization of cholesterol and other water-immiscible lipidsfor intravascular transport is accomplished by packaging themwith specific proteins into lipoprotein particles. Low densitylipoproteins (LDL) serve as the main carriers of plasma cho-lesterol. Two distinct subclasses of LDL, termed LDL1 andLDL2, can be obtained by centrifugal flotation at densities of1.006-1.019 g/ml and 1.02-1.063 g/ml, respectively. LDL2,the major subclass of plasma LDL, is a spherical particle com-posed of 78% lipid and 22% protein. A single species of poly-peptide, apolipoprotein B (apo B), is the sole protein constituentof LDL2 (1).

Information concerning biochemical and structural featuresofapo B is extremely limited (2). However, these data are vitallyimportant in achieving an understanding of the structure ofLDL and of its function(s) in vivo. For example, the catabolismof LDL is mediated largely by high-affinity receptors presenton hepatic and extrahepatic cells that recognize critical deter-minant(s) on apo B (3). Efforts to delineate the salient featuresof LDL catabolism have been impeded both by the paucity ofbiochemical information concerning apo B and by the com-plexity of the catabolic processes, including observations that:(i) the liver and intestine may produce distinct forms of apo Bheretofore presumed to be identical (4, 5); (ii) there is hetero-geneity in LDL (6) and there may be molecular heterogeneity

in apo B (7); and (iii) apolipoprotein E, normally a componentof very low density lipoprotein (VLDL), also is bound specifi-cally by the extrahepatic LDL receptor (8). To advance our un-derstanding ofLDL structure and to potentially correlate thesestructural features with its functional properties, we have ini-tiated immunochemical studies of LDL and apo B.We present here details ofthe production and immunological

specificity of six monoclonal antibodies to LDL2. Binding ofthese antibodies to LDL2 is inhibited by VLDL but not by highdensity lipoprotein (HDL), suggesting that the antibodies cross-react with antigenic determinant(s) on VLDL but not on HDL.Moreover, although all six antibodies recognize antigenic de-terminant(s) on apo B, three ofthem exhibit partial or total lossof activity with apo B prepared from LDL2 with NaDodSO4,a detergent known to alter the secondary structure of the pro-tein (9). Competition studies between biotinylated and unla-beled antibodies demonstrated that the six monoclonal anti-bodies may be divided into at least two distinct groups basedon the epitopes on apo B that they recognize. Antibodies in onegroup interact with the same NaDodSO4-sensitive epitope,whereas antibodies in the other group appear to interact withanother site(s) on apo B.

MATERIALS AND METHODSAnimals. Male, adult BALB/c mice were obtained from The

Jackson Laboratory.Preparation ofVLDL, LDL, HDL, and apo B. Lipoproteins

were isolated from the plasma of a single volunteer by centrif-ugal flotation. After adjusting the plasma to final concentrationsof 1.3 mM EDTA (pH 7.0) and 4.0 mM sodium azide, chylo-microns were removed by centrifugation in a Beckman SW 27rotor (82,000 x gav; 60 min at 150C). VLDL was obtained byflotation at p = 1.006 g/ml and HDL was collected at p =1.063-1.21 g/ml. LDL2 was prepared at p between 1.02 and1.05 g/ml, as described by Steele and Reynolds (9). Immedi-ately after isolation, free sulfhydryls were alkylated with io-doacetamide, followed by extensive dialysis against phosphate-buffered saline (Pi/NaCl) at pH 7.2. LDL2 samples then werepassed through 0.22-,um filters (Millipore) and were stored at4°C. Delipidated apo B was obtained from LDL2 by detergentextraction with either NaDodSO4 (BDH) or polyoxyethylene 9lauryl ether (C12E9) (Sigma), followed by gel filtration chro-matography (10). The purity of lipoprotein and apo B prepa-rations was assessed on NaDodSO4/polyacrylamide gels by us-ing the buffer system of Weber and Osborn (11). Proteinconcentrations were determined by using the method of Lowry(12), with NaDodSO4 added to a final concentration of 2% in

Abbreviations: LDL, low density lipoprotein; LDL2, low density li-poprotein isolated at densities of 1.02-1.05 g/ml; HDL, high densitylipoprotein; VLDL, very low density lipoprotein; apo B, apolipoproteinB; C12E9, polyoxyethylene 9 lauryl ether; ELISA, enzyme-linked im-munosorbent assay; Pi/NaCl, phosphate-buffered saline.

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both experimental samples and bovine serum. albumin stan-dards. All lipoprotein concentrations used here are given interms of protein content.Hybridoma Preparation. Hybridomas were generated by

using the method of Gefter et aL (13), with some modifications.Several BALB/c mutant myeloma parent cell lines (Sp2/0-Agl4, MOPC 21 NS1, and P3/X63-Ag8) were grown in Iscove'smodified Dulbecco's medium (GIBCO) supplemented with10% fetal calf serum, hypoxanthine (0.1 mM), thymidine (0.38mM), bovine insulin .(0.2 unit/ml), nonessential amino acids(0.1 mM; GIBCO), sodium pyruvate (1 mM), and 10% NCTC109 medium (M. A. Bioproducts, Walkersville, MD). Parentspleen cells to be used for fusion were obtained from BALB/c mice that had been immunized with an intravenous injectionof 100 Ag of human LDL2. Secondary intravenous injections of100 Ag ofLDL2 were administered approximately 30 days later.For each fusion, a mouse was sacrificed 3-10 days after the sec-ond immunization and a spleen cell suspension free ofred bloodcells was-prepared in Hanks' medium (GIBCO) supplementedwith glucose (0.45%). Spleen cells then were mixed with themutant myeloma cells at ratios of either 10:1 or 1:1 and werefused with 30%-polyethylene glycol 6,000 (13). Fused cells weresuspended in supplemented Iscove's medium containingamethopterin (0.4 AuM), plated at low densities in 96-well tissueculture plates (Costar), and incubated at 370C in an atmosphereof 5% C02/95% air. After 10-14 days wells that were positivefor cell growth were' screened for human LDL2-specific anti-body production. Cells from wells secreting anti-LDL2 anti-bodies were cloned at limiting dilution in liquid media in thepresence of 1.5 x 104 peritoneal macrophages per well. Allhybridoma cell lines were cloned at least two times, or until 98-100% of growth-positive cloning wells also secreted specific an-tibodies. Cloned cells were grown in supplemented Iscove'smedium.The class (or subclass) of antibody produced by cultured hy-

bridomas was determined by Ouchterlony double-immunodif-fusion by employing murine immunoglobulin class-specificantisera (Meloy Laboratories, Springfield, VA).

Enzyme-Linked Immunosorbent Assays (ELISAs). Thepresence of anti-LDL2 antibodies in hybridoma cell superna-tants and analyses of their immunoreactivity were assessed byusing an ELISA. A 100-,ul portion of a solution of LDL2 (10 ,ug/ml in Pi/NaCl) was introduced into individual wells of a 96-wellmicrotiter dish (Nunc immuno plate I) and was allowed to in-cubate overnight at 4°C. Sensitized plates then were washedfour times with Pi/NaCl and 100 ,ul of bovine serum albumin(10 mg/ml in Pi/NaCl) was added to each well. After a 3-hrincubation at room temperature, 100 /.d of hybridoma super-natants (or purified antibodies) was added to appropriate wellsand allowed to incubate overnight at 4°C. After washing all wellsfour times with Pi/NaCl containing 0.05% Triton X-100, theinteraction ofmouse antibodies with the immobilized LDL2 wasdetected by addition of 50 ,l of F(ab')2 fragments of sheep anti-mouse IgG to which 8-galactosidase was covalently coupled(Bethesda Research Laboratories). Two hours later the platesagain were washed four times and then p-nitrophenyl /-D-ga-lactoside (1 mg/ml) was added in aliquots of 50 ,ul per well.Immediately after a 1-hr incubation at room temperature, theOD414 of the contents of each well was measured.The interaction of monoclonal antibodies with detergent-de-

lipidated apo B was measured in an ELISA, performed essen-tially the same way as the LDL2-ELISA described above, butwith the following minor modifications. Microtiter wells weresensitized with either 100 ,ul of NaDodSO4-delipidated apo B(1.0 mg/ml in Pi/NaCl containing 2.5 mM NaDodSO4 at pH7.2) or C12E9-delipidated apo B (0.54 mg/ml in Tris-bufferedsaline at pH 9.2, containing 1.0 mM C12E9). Assays with

NaDodSO4-'delipidated apo B-sensitized plates then proceededexactly as those described for the LDL2-ELISA; NaDodSO4 wasnot present after the initial sensitization of the wells. The pro-tocol followed for tests employing C12E9-delipidated apo B-sen-sitized plates also was identical to that used in the LDL2-ELISA, except that 1 mM C12E9 was included in all washes andincubations-after the initial coating.The ability of either VLDL or HDL to inhibit the binding

of monoclonal antibodies to LDL2-sensitized plates was testedby using a protocol that varied slightly from that employed forthe LDL2-ELISA. Microtiter plates were sensitized withLDL2, washed four times with Pi/NaCl, incubated with bovineserum albumin (10 mg/ml) for 3 hr, and washed again with Pi/NaCi as described above. Then 100-Al portions of serial dilu-tions of either VLDL, HDL, LDL2 (a positive control), or Pi/NaCi (a negative control) were added to a row ofeight sensitizedwells, followed by a 50-pli addition ofan equal amount ofa givenmonoclonal antibody to each well. After an overnight incubationat 4'C, the assay was developed in the same manner as theLDL2-ELISA.

Purification of Monoclonal Antibodies. Monoclonal anti-bodies were purified from either cell culture supernatants ormouse ascitic fluid by affinity chromatography. Protein A-Agar-ose (Bethesda Research Laboratories) was used in the isolationof antibodies of the IgGl class from cell culture supernatants(14), whereas LDL-Sepharose 4B was employed in purifyingantibodies from murine ascitic fluid. The eluant used in bothprocedures was 0.1 M glycine HCI (pH 3.2). Purity of the an-tibodies obtained was assessed on 12.5% NaDodSO4/poly-acrylamide slabgels by using the buffer system of Laemmli (15).

Biotinylation of Purified Antibodies. After determining theprotein concentration of the purified monoclonal antibodies,aliquots of each preparation were biotinylated. After dialysisagainst 0. 1 M sodium carbonate atpH 8.3, a50-fold molar excessof N-hydroxysuccinimidobiotin (Pierce) dissolved in dimethylsulfoxide (1 mg/ml) was added to appropriate antibody samples.The reactions were terminated after a 4-hr incubation at roomtemperature by exhaustive dialysis of the labeled antibodiesagainst Pi/NaCl. Titrations ofunlabeled and biotinylated mono-clonal antibodies in the LDL2-sensitized ELISA demonstratedthat the labeling protocol had very little effect on the immu-nological reactivity of these antibodies.

Competition ELISA. Before beginning competition studies,biotinylated antibodies were titered on LDL2-sensitized platesin an ELISA, essentially as described above, except that avidin-peroxidase (Vector Laboratories, Burlingame, CA) was substi-tuted for f3-galactosidase-conjugated sheep anti-mouse IgG.Peroxidase activity was measured by addition of the "Trinderreagent" (H202, 4-aminoantipyrine, and phenol) and the for-mation of the resulting quinonimine was monitored at 492 nm(16). Competition studies then were performed by first makingserial dilutions of a purified, unlabeled "competitor" antibodyon-LDL2-coated wells. A prescribed amount ofa given biotinyl-ated antibody (as determined by the titration) then was intro-duced into each well. After an overnight incubation at 40C,the assay was developed with avidin-peroxidase. Data are ex-pressed as OD492 vs. log2 dilution of unlabeled competitor.Generally, a 50- to 100-fold excess of unlabeled antibody wasemployed in the most concentrated competition well.

Solid-Phase Radiobinding Assay. The apparent affinity ofmonoclonal antibodies for immobilized LDL2 was estimated ina solid-phase radiobinding assay. Antibodies, affinity-purifiedon LDL-Sepharose, were labeled with Na'"I (Amersham) tospecific activities of 1-9 x 106 cpm/pug by using lactoperoxidaseaccording to the protocol of Olender and Stach (17) with minormodifications. After termination of the 1-hr labeling reaction,iodinated antibody was separated from free iodine by passage

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of the reaction mixture over a 12-ml gel filtration column (30x 0.75 cm) of BioGel P-60 (Bio-Rad) equilibrated with Pi/NaCl containing bovine serum albumin at 1 mg/ml. The ra-

dioactivity in 10-A.l portions ofeach 350-,ul column fraction wasmeasured and generally the two or three most highly labeledantibody fractions were pooled. "2I-Labeled antibodies were90-100% precipitable with trichloroacetic acid.

Solid-phase radiobinding assays were performed by firstcoating flat-bottom wells on 8-well vinyl assay strips (Costar)with 200 pil of LDL2 (10 Ag/ml in Pi/NaCl) overnight at 40C.After three washes with Pi/NaCl, 200 ,x1 of bovine serum al-bumin (10 mg/ml in Pi/NaCl) was added to each well and was

allowed to incubate for 3 hr at room temperature. After washingthree times with Pi/NaCl, 100 Al ofvarying amounts ofan '25i-labeled monoclonal antibody in saline/albumin was introducedinto individual wells. Fifty microliters of bovine serum albumin(10 mg/ml in Pi/NaCl) then was added to all wells to bring thetotal volume of the reaction to 150 til and the assay was placedat 40C for 12 hr. Assay strips then were washed four times withPi/NaCl containing 0.1% Triton X-100 and were cut into in-dividual wells, and the radioactive content of each well was

measured. A parallel experiment performed on uncoated wellswas always used to access the extent of nonspecific binding ofthe "'I-labeled antibodies. This nonspecific binding never ex-

ceeded 5% of that observed in the experimental samples.

RESULTS AND DISCUSSION,To date, we have performed five fusions between LDL2-sen-sitized murine splenocytes and myeloma cells that have resultedin the production ofa large number ofcell lines secreting LDL2-specific antibodies. We have partially characterized the mono-clonal antibodies produced by six of these cloned lines. Five ofthese six hybridomas (TRW-3, -5, -6, -8, and -13) synthesizeantibodies of the IgGi subclass, whereas one (TRW-1) secretesIgM antibodies.An ELISA was used both in the detection ofantibodies in the

media of hybridomas cultured in vitro and in the assessmentof their immunoreactivity. Results of a typical assay are shownin the first column of data in Table 1. The OD414 of wells in-cubated with hybridoma supernatants was significantly greaterthan that measured in those wells that received either ofthe twonegative controls, normal mouse serum (diluted 1:400) or 11-A5, an immunoglobulin-producing hybridoma of undefinedspecificity. Differences in color development seen from sampleto sample could be accounted for by variations in concentrationor affinity (or both) of the monoclonal antibodies secreted byeach clone. For purposes ofcomparison, polyclonal mouse anti-LDL antiserum (diluted 1:400) was included as a positive con-

trol and yielded an OD414 of 0.958. Normal or immune serum,as well as hybridoma supernatants, placed in uncoated wellsresulted in little color development, indicating that nonspecificbinding was minimal in these assays (Table 1, column 3).

The effect of inclusion of either VLDL or HDL on purifiedmonoclonal antibodies incubated with LDL2-sensitized platesin the ELISA was used as a measure of the reactivity of the an-tibodies with these lipoprotein classes. Representative resultsof these inhibition studies are presented in Fig. 1. As shown inthis titration with TRW-8 antibodies, although VLDL andLDL2 (included as a positive control) clearly inhibited antibodybinding to immobilized LDL2, HDL had no effect on the ob-served interaction. This general pattern of strong inhibition byVLDL and complete lack of inhibition by HDL also was seen

with the other five antibodies. These data suggest that all sixanti-LDL2 monoclonal antibodies crossreact with antigenic de-terminants present on VLDL but absent from HDL.

Interaction of Monoclonal Antibodies with apo B. apo B isinsoluble in aqueous solution in the absence of bound amphi-

Table 1. Reactivity of monoclonal antibodies with LDL2 andNaDodSO4-delipidated apo B

WellsNaDodSO4-delipidated

LDL2- apo B-Sample sensitized* sensitizedt Controlt

SupernatantTRW-1 0.890-± 0.016 0.333 ± 0.02 NDTRW-3 0.293 ± 0.033 0.242 ± 0.01 0.021 ± 0.009TRW-5 0.178 ± 0.019 0.500 ± 0.019 0TRW-6 0.165 ± 0.045. 0.714 + 0.036 0.013 ± 0.006TRW-8 0.496 ± 0.025 0 0.011 ± 0.002TRW-13 0.397 ± 0.019 0 0.008 ± 0.002

Mouse polyclonalanti-LDLantiserum(1:400) 0.958 ± 0.069 0.408 ± 0.016 0.031 ± 0.004

Hybridomasupernatant11-A5§ 0.008 ± 0.004 ND 0.014 ± 0.003

Normal mouseserum (1:400) 0.015 ± 0.001 0 0

All data are expressed as the OD414 measured in an ELISA 1 hr afteraddition of substrate to the assay. Assays were performed in triplicate.The mean value ± SD is presented. ND, not determined.* LDL2-sensitized wells were pretreated with 100 u.l of a solution ofLDL2 at 10 ,ug/ml before addition of cell supernatants.

t NaDodSO4-delipidated apo B-sensitized wells were pretreated with100 Aul of a solution of purified apo B at 1.0 mg/ml in a buffer con-taining 2.5 mM NaDodSO4 before addition of cell supernatants. Notethat NaDodSO4 was present only during the incubation period usedfor coating, not during the assay itself

t Control wells were untreated before the addition of cell supernatants.§ Supernatant 11-A5 was a control supernatant from an actively pro-liferating culture that contained no anti-LDL activity, but which se-creted immunoglobulin, and is included as a negative control.

philes or strong denaturant. However, the protein can be ob-tained from LDL2 by detergent solubilization of the holo par-ticle with either ionic (9, 18) or nonionic (10, 19, 20) detergents,followed by a chromatographic separation of apo B from deter-gent/lipid mixed micelles. Although extraction of LDL2 witheither type of detergent yields a delipidated apo B/detergentparticle, in general, the secondary structure and immunologicalproperties ofthe protein are maintained in nonionic detergents(10, 19) and perturbed in micellar solutions of ionic detergents(9, 10). We have demonstrated previously by circular dichroismthat the denatured conformation adopted by apo B in Na-DodSO4 is readily reversible to the native form when Na-DodSO4 is exchanged with a suitable nonionic detergent (10).To access the state of epitopes on detergent-delipidated apo B,the ability of monoclonal antibodies to bind the solubilizedapoprotein was investigated.The interaction of the six monoclonal antibodies with epi-

tope(s) on NaDodSO4-delipidated apo B was measured in anELISA assay performed in exactly the same manner as theLDL2-sensitized ELISA, except that microtiter dishes werecoated with apo B in 2.5 mM NaDodSO4. As shown in the sec-ond column ofTable 1, the immunoreactivity of three of the sixantibodies was substantially decreased (TRW-1) or totally abol-ished (TRW-8 and TRW-13) compared with their reactivityagainst holo-LDL2. However, under identical experimentalconditions, TRW-5 and TRW-6 showed significantly greaterreactivity with apo B than with LDL2, whereas the apparentactivity of TRW-3 was only affected slightly. The control, het-erologous mouse anti-LDL2 antiserum, showed about 50% ofthe reactivity with the apoprotein as compared to LDL2.

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0.3

0.2

0.1

0 2 4 6log2 (dilution of inhibitor)

FIG. 1. Inhibition of the binding of TRW-8 monoclonal antibodiesto immobilized LDL2 by soluble HDL (e), VLDL (o), or LDL2 (o). Mi-crotiter wells were precoated with 100 ul of LDL2 (10 ug/ml). Onehundred-microliter portions of either VLDL (610 ,ug/ml), HDL (100jug/ml), or LDL2 (100 pg/ml) then were added to individual wells andwere serially diluted over seven wells, followed by the addition of 50,ul of purified TRW-8 antibodies (5 pg/ml) to each well. The ELISAwas developed by using ,-galactosidase-copjugated sheep anti-mouseIgG andp-nitrophenyl P-D-galactoside. A series of eight wells that re-

ceived 100 ,ul of buffer and 50 Al of purified TRW-8 antibody was in-cluded as a control; the OD414 (mean ± SD) of the contents of thesewells after 1 hr was 0.442 ± 0.019.

The observed decrease in binding of TRW-1, TRW-8, andTRW-13 to NaDodSO4-solubilized apo B could be attributableto several factors. Likely possibilities are: (i) the reversible al-teration in secondary structure ofapo B in solutions of micellarNaDodSO4 (10) may be rendered irreversible when the proteinis immobilized on microtiter dishes; (ii) NaDodSO4-delipidatedapo B may lack components (i.e., glycolipids) that partially or

totally constitute antigenic determinants on LDL; (iii) the threeantibodies may possess relatively low affinities for LDL2;hence, even slight structural changes in apo B could result insubstantially decreased binding; or (iv) residual NaDodSO4bound to apo B may sterically block epitope(s) on the protein.An assay of "I-labeled monoclonal antibody binding to LDL2-sensitized plates demonstrated that TRW-8 possessed an ap-

parent affinity for LDL2 comparable to or greater than TRW-5 (for example), thus diminishing the likelihood that affinitydifferences account for the observed results. Furthermore, be-cause all six antibodies react with C12E9-delipidated apo B (seebelow), the second alternative also seems improbable.The increases in observed apo B reactivity measured with

TRW-5 and TRW-6 are more difficult to reconcile. This in-creased binding may reflect a greater accessibility ofantibodiesto site(s) on apo B that are sterically blocked when the poly-peptide is in LDL. Enhanced binding also may represent fur-ther exposure ofpartially masked sequential determinants in thedelipidated protein that are not exposed on native LDL2. Thisargument is consistent with some recent biochemical (21, 22)and immunological (23) studies that were interpreted as sug-gesting that apo B may be composed of repetitive subunits.Alternatively, the capacity of individual microtiter wells for apoB may exceed the saturating levels of LDL2 used in these stud-ies. Nonetheless, under identical experimental conditions, thebehavior of TRW-5 and TRW-6 antibodies was markedly dif-ferent than that of TRW-I, TRW-8, and TRW-13.The interaction of monoclonal antibodies with apo B pre-

pared in C12E9, a nonionic detergent known not to perturb thesecondary structure of the apoprotein (10), was tested in an

ELISA. This assay was performed exactly as outlined for LDL,except that 1 mM C12E9 was present continuously during the

Table 2. Reactivity of monoclonal antibodies with LDL2 andC12E9-delipidated apo B

Wells

C12E9-delipidated

LDL2- apo B-Sample sensitized* sensitizedt Controlt

SupernatantTRW-1§ 0.627 0.420 0.122TRW-3 0.146 ± 0.010 0.197 ± 0.007 0.012 ± 0.001TRW-5 0.115 ± 0.014 0.193 ± 0.007 0.010 ± 0.001TRW-6 0.145 ± 0.008 0.174 ± 0.011 0.014TRW-8 0.170 ± 0.030 0.156 ± 0.035 0.014 ± 0.004TRW-13 0.186 ± 0.006 0.174 ± 0.010 0.011 ± 0.001

Mouse polyclonalanti-LDLantiserum(1:400) 0.217 ± 0.001 0.201 ± 0.040 0.028 ± 0.005

Normal mouseserum (1:400) 0.006 ± 0.004 0.008 ± 0.004 0.007 ± 0.002

All data are expressed as the OD414 measured in an ELISA 3 hr afteraddition of substrate to the assay. Assays were performed in triplicate.The. mean value ± SD is presented.* LDL2-sensitized wells were pretreated with 100 Al of a solution ofLDL2 at 10 pug/ml before addition of cell supernatants.

t C12E9-delipidated apo B-sensitized wells were coated with 100 /.l ofa solution of purified apo B at 540 Ag/ml in a buffer containing 1.0mM C12E9. Note that 1.0 mM C12E9 was present throughout the as-say.

t Control wells were untreated before the addition of cell supernatants.§ TRW-1 was assayed separately.

test. Data presented in Table 2 demonstrate that all six anti-bodies reacted with C12E9-delipidated apo B and that five ofthesix possess reactivities comparable to that observed with LDL2.It is noteworthy that although mouse anti-LDL2 antiserumshowed somewhat less binding to NaDodSO4-delipidated apoB than to LDL2, C12E9-delipidated apo B and LDL2 wereequally reactive in this assay. These data suggest that the sixmonoclonal antibodies to holo-LDL2 are directed to antigenicdeterminants on apo B and not to other nonprotein componentsofthe particle. Further, these epitopes on apo B are maintainedwhen the protein is in C12E9, in agreement with our previouswork (10). The generally decreased activities seen in this ex-periment compared to those reported in Table 1 were due toa lower-titer ,Bgalactosidase anti-mouse antibody conjugate.

Competition ELISA. To study all apo B epitopes expressedon LDL it will be necessary to produce a family of monoclonalantibodies, each with a unique specificity for one of the anti-genic determinants. To identify individual epitopes of apo Brecognized by the six antibodies, a competition ELISA was usedthat measured the ability of a purified unlabeled antibody toinhibit the binding to LDL2 ofa second, biotinylated antibody.Representative results of these experiments are shown in Fig.2 A and B. The binding of labeled TRW-8 to LDL2-sensitizedwells in the presence ofunlabeled TRW-8, TRW-5, or TRW-13is shown in Fig. 2A. Binding ofTRW-8 was inhibited by TRW-13 or by itself (a positive control), but not by TRW-5. Similarly,although unlabeled TRW-8 and TRW-13 blocked the bindingofbiotinylated TRW-13, no inhibition was observed with TRW-5 (Fig. 2B). The simplest interpretation of these results is thatTRW-13 and TRW-8 recognize either the same epitope on apoB (or epitopes in close proximity) and that TRW-5 interacts withanother antigenic determinant on apo B. These competitionassays were performed on -all possible combinations of the sixantibodies, over a wide range of concentrations, and have pro-vided a basis for a tentative division ofthese antibodies into twogroups according to the epitopes on apo B with which they in-

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0.5

0.4

9 0.30

0.2

0.1

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8log2 (dilution of unlabeled competitor)

FIG. 2. Competition ELISA using a fixed amount of biotinylated monoclonal antibody and varying amounts of unlabeled competitor anti-bodies. Microtiter wells first were coated with 100 1.l of LDL2 at 10 Ag/ml. Fifty microliters of a preparation of unlabeled purified competitormonoclonal antibody then was serially diluted in the LDL2-sensitized wells. Identical 50-,ul portions of a biotinylated antibody were added to eachwell, followed by development of the assay with avidin-peroxidase. The figure shows the OD492 measured 1 hr after the addition of substrate. (A)Binding of biotinylated TRW-8 in the presence of unlabeled TRW-5 (e), TRW-13 (o), and TRW-8 (i). Biotinylated TRW-8 was tested at 0.25 ,.g/ml, whereas the first dilutions of unlabeled antibodies contained TRW-5 at 26 pg/ml, TRW-13 at 48 /Ag/ml, and TRW-8 at 45 ,ug/ml. (B) Bindingof biotinylated TRW-13 in the presence of unlabeled TRW-5 (e), TRW-8 (i), and TRW-13 (o). Biotinylated TRW-13 was tested at 0.34 ,4g/ml.Unlabeled antibodies were used at the same concentration as in A.

teract. TRW-1, TRW-3, TRW-5, and TRW-6 comprise groupI, whereas group II consists ofTRW-8 and TRW-13. As shownin Fig. 2, antibodies in group II possess specificity for the sameepitope on, apo B, whereas attempts to subclassify group I an-tibodies have thus far yielded equivocal results. Further, groupII antibodies fail to react with NaDodSO4-delipidated apo B.

Solid-Phase Radiobinding Assay. The interaction of three"2I-labeled monoclonal antibodies with immobilized-LDL2 was

studied in a solid-phase radiobinding assay. Binding data for thetwo group I antibodies, TRW-5 and TRW-6, and one group IIantibody, TRW-8, yielded apparent half-saturation values of172, 11, and 3 nM, respectively. However, we note that it wasclear from the binding data that interaction of "2I-labeled an-tibodies with solid-phase LDL2 was extremely complex andheterogeneous. Many factors undoubtedly contributed to thiscomplexity; obvious considerations for solid-phase -assays in-volving LDL are the often-reported instability of proteins ad-sorbed to surfaces (24) and the potential that some epitopes on

LDL were either partially or totally inaccessible to antibodybinding. Nonetheless, these studies provided us with additionaluseful information. For example, the failure of group II anti-bodies to react with NaDodSO4-delipidated apo B appears tobe unrelated to their apparent affinity for LDL2.

In summary, the reactivities of six monoclonal antibodiesproduced to LDL2 have been analyzed by utilizing an ELISAand a solid-phase radioimmunoassay. The interaction of all sixof these antibodies with solid-phase LDL2 was inhibited byVLDL but not by HDL. All ofthe antibodies recognize epitopeson apo B when the apoprotein is prepared in the benign, non-ionic detergent C12E9. Further, although. it is not possible toassess the conformation ofthe immobilized polypeptide in thesesolid-phase assays, three ofthe six antibodies exhibit substantialloss of binding to apo B prepared in the denaturing ionic de-tergent NaDodS04. Competition binding studies have allowedus to divide the antibodies into two groups (I and II) based ontheir reactivity with LDL2. Antibodies in group II are directedto the same (or crossreacting) antigenic determinant(s) on LDL2and it is this epitope that is perturbed in NaDodSO4. Studiesof the specificity of these and other monoclonal antibodies toapo B will allow analysis of individual epitopes on LDL andother apo B-containing lipoproteins. Additionally, monoclonalantibodies should prove to be valuable tools in the isolation ofproteolytic and chemical cleavage products of apo B.

The authors express their gratitude to Drs. Jacqueline A. Reynoldsand D. Bernard Amos, in whose laboratories some of the work pre-sented here was conducted. This study was supported by National In-stitutes of Health Grant HL-29340.

1. Tanford, C. & Reynolds, J. A. (1979) in The Chemistry and Phys-iology of the Human Plasma Proteins, ed. Bing, D. H. (Perga-mon, New York), pp. 111-126.

2. Bradley, W. A., Rohde, M. F. & Gotto, A. M., Jr. (1980) Ann.N.Y. Acad. Sci. 348, 87-103.

3. Brown, M. S., Kovanen, P. T. & Goldstein, J. L. (1981) Science212, 628-635.

4. Krishnaiah, K. V., Walker, L. F., Borensztajn, J., Schonfeld, G.& Getz, G. S. (1980) Proc. Nati Acad. Sci. USA 77, 3806-3810.

5. Wu, A.-L. & Windmueller, H. G. (1981) J. Biol Chem. 265,3615-3618.

6. Shen, M. S., Krauss, R. M., Lindgren, F. T. & Forte, T. M.(1981) J. Lipid Res. 22, 236-244.

7. Sparks, C. E. & Marsh, J. B. (1981)J. Lipid Res. 22, 519-527.8. Innerarity, T. L., Mahley, R. W., Weisgraber, K. G. & Bersot,

T. P. (1978) J. Biol Chem. 253, 6289-6295.9. Steele, J. C. H., Jr., & Reynolds, J. A. (1979)J. Biol Chem. 254,

1633-1638.10. Watt, R. M. & Reynolds, J. A. (1980) Biochemistry 19, 1593-

1598.11. Weber, K. &.Osborn, M. (1969)1. Biol Chem. 244, 4405-4412.12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.

(1951) J. Biol Chem. 193, 265-275.13. Gefter, M. L., Margulies, D. H. & Scharff, M. D. (1977) Somatic

Cell Genet. 3, 231-236.14. Ey, P. L., Prowse, S. J. & Jenkin, C. R. (1978) Immunochemistry

15, 429-436.15. Laemmli, U. K. (1980) Nature (London) 227, 680-685.16. Gallati, V. H. (1977) J. Clin. Chem. Clin. Biochem. 15, 699-703.17. Olender, E. J. & Stach, R. W. (1980) J. Biol Chem. 255, 9338-

9343.18. Helenius, A. & Simons, K. (1971) Biochemistry 10, 2542-2547.19. Ikai, A. & Hasegawa, M. (1978)J. Biochem. 83, 755-759.20. Socorro, L. & Camejo, G. (1979) J. Lipid Res. 20, 631-638.21. Bradley, W. A., Rohde, M. F., Gotto, A. M., Jr., & Jackson, R.

L. (1978) Biochem. Biophys. Res. Commun. 81, 928-935.22. Deutsch, D. G., Heinrikson, R. L., Foreman, J. & Scanu, A, M.

(1978) Biochim. Biophys. Acta 529, 342-350.23. Goldstein, S. & Chapman, M. J. (1979) Biochem. Biophys. Res.

Commun. 87, 121-127.24. Pesce, A. J., Ford, D. J. & Gaizutis, M. A. (1978) Scand. J. Im-

munol Suppt 7, 8, 1-6.

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