Lp(a) Phenotyping by Immunoblotting withPolyclonal and Monoclonal Antibodies

Hans-Georg Kraft, Hans Dieplinger, Elaine Hoye, and Gerd Utermann

A new method that allows rapid phenotyping of genetic Lp(a) glycoproteln types Inlarge numbers of samples Is described. The method is based on sodium dodecylsulfate gel electrophoresls of reduced serum or plasma In horizontal slab gels fol-lowed by Immunoblotting with polyclonal antl-Lp(a) llpoproteln or monoclonal anti-Lp(a) glycoproteln antibodies. Phenotyping of 194 unrelated, healthy subjects result-ed In Lp(a) allele frequencies of Lp(a)B = 0.013, Lp(a)81 = 0.032, Lp(a)S2 = 0.106,Lp(a)33 = 0.096, Lp(a)34 = 0.156, and Lp{a)° = 0.600, and confirmed the recently recog-nized association of Lp(a) glycoproteln phenotype with Lp(a) llpoproteln concentra-tion. The new procedure is suitable for large-scale population, genetic, and epidemi-ologlc studies and may be Important for atherosclerotic risk assessment.(Arteriosclerosis 8:212-216, May/June 1988)

T he Lp(a) lipoprotein was first described by Berg1 as agenetic variant of low density lipoprotein (LDL), and it

was believed that this lipoprotein is inherited as an autoso-mal, dominant trait. After the introduction of quantitativeLp(a) measurement, it was shown that the Lp(a) lipopro-tein represents a quantitative trait and that Lp(a) concen-tration is under polygenic control, probably with a majorgene effect for high Lp(a) concentration.2"5 The interest inthis unusual lipoprotein was greatly stimulated by reportsof an association of high levels of Lp(a) lipoprotein withatherosclerosis.6"11

The Lp(a) lipoprotein is an LDL-like particle that floats ina density range of about 1.05 to 1.10 g/ml. The lipid com-position is similar to that of the LDL particle, but its proteinmoiety contains, in addition to apo B-100, the Lp(a)-spe-cific glycoprotein, which is linked to apo B by disulfidebonds.12-13> u The Lp(a) lipoprotein exhibits a density het-erogeneity within and between individuals, as well as asize heterogeneity of the Lp(a)-specific glycoprotein.1315

Recently, six different types of this Lp(a)-specific glycopro-tein were resolved in our laboratory by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE)followed by immunoblotting.16 Preliminary evidence sug-gests that Lp(a) phenotypes are genetically controlled and,moreover, are associated with Lp(a) lipoprotein concentra-tion in plasma.16 Hence, the interesting possibility arosethat the observed association of Lp(a) lipoprotein levelswith atherosclerosis might be due to differences in Lp(a)glycoprotein species, rather than to differences in the con-centration of the lipoprotein. To answer this and relatedquestions, a method that allows convenient phenotyping ofa large number of samples in epidemiologic studies isneeded. In this paper, we describe an improved method for

From the Institute for Medical Biology and Genetics, Universityof Innsbruck, Innsbruck, Austria.

Address for reprints: Dr. G. Utermann, Institute for Medical Biol-ogy and Genetics, University of Innsbruck, SchOpfstr. 41, 6020Innsbruck, Austria.

Received June 9,1987; revision accepted November 4, 1987.This work was supported by grants from the Deutsche Fors-

chungsgemeinschaft and the Thyssen Stiftung to Gerd Utermann.

Lp(a) glycoprotein phenotyping directly from serum orplasma by immunoblotting.

MethodsElectrophoretlc Procedures

Acrylamide, /V,/V'-methylene-i)/s-acrylamide (Bis) andsodium dodecyl sulfate (SDS) were purchased from BDHChemicals (UK) and were used without further purification.The buffer substances were from Merck (FRG) and wereall of analytical grade. The discontinuous buffer and gelsystem of Neville17 was used with minor modifications asmentioned below. The gels (25 x 12 cm) were cast into acassette consisting of a glass plate, onto which a PAG-Gelbond film (LKB, Bromma, Sweden) with its hydropho-bic side up had been rolled, a U frame (1.5 mm thick), anda second glass plate. This second glass plate was pro-vided with 40 slot formers by applying three layers of adhe-sive tape and cutting it to the dimension required ( 4 x 6mm, 2 mm distance between the slots). The plate wastreated with Repel-Silane (LKB, Bromma, Sweden).

The density of the stacking gel solution (pH = 6.14,T = 3.6%) was enlarged with glycerol (13.4%, vol/vol); thestacking gel solution was first poured into the precooled(4CC) cassette. The resolving gel solution (pH = 9.14,T = 7%) was added immediately afterward by using thefront chamber of a gradient mixer to provide a slow andeven flow of the solution. This procedure produces a veryeven, straight border line between the two parts.

The samples were prepared by mixing 7 fi\ of serum orplasma with 50 /il of a sample buffer prepared fresh everyday by mixing 2 ml of 5% SDS with 200 /il of B-mercap-toethanol and 300 fii of 1.5% Bromphenol Blue in glycerol.The well-mixed solution was boiled for 10 minutes in awater bath, and 8 /xl of this mixture was applied in thesample slots. The electrophoretic run was performed in aMultiphor II (LKB, Bromma, Sweden) cooled to 10°C. Aconstant power of 40 W was applied throughout the run.When the Bromphenol Blue marker had reached the end ofthe gel (at about 3 hours), the run was continued for anadditional hour. Afterward, the gel was transferred onto a

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LP(a) PHENOTYPING Kraft et al. 213

sheet of nitrocellulose membrane (BA 85,0.45 /j.m, Schlei-cher & Schull, FRG) and the proteins were electroblottedaccording to the method of Towbin et al.18 by using theTransblot cell (Biorad, USA). The blotting was performedovernight with a constant voltage of 80 V.

The Lp(a) bands were visualized on the membrane by adouble-antibody procedure. The first antibody, which wasdirected against Lp(a), was either a polyclonal or a mono-clonal one (their production, purification, and characteriza-tion are described below). The second antibodies, goatantirabbit or goat antimouse IgG from Bioyeda, Israel,were labeled with colloidal gold (20 nm diameter) accord-ing to the method of Lin and Langenberg.19 To increase thesensitivity of the immunoblot procedure, a physical devel-oper consisting of silver lactate (Fluka, Switzerland) andhydroquinone (Sigma, FRG) was used when necessary.Accurate phenotyping was achieved by placing a serumwith S1/S2 phenotype as an internal marker in every sev-enth position.

The sensitivity of the described method enables the de-termination of the Lp(a) glycoprotein phenotype in serawith Lp(a) lipoprotein concentrations as low as 5 mg/100ml. This is the same sensitivity that we achieved in theelectroimmunodiffusion assay that we used to determinethe serum Lp(a) concentration. The Lp(a) standard serum(83.7 mg/100 ml) from Immuno (Austria) and its serial dilu-tions were used as standards in this assay. This standardwas prepared in the manner described by Krempler et al.20

Production, Purification, and Characterization ofAntibodies against Lp(a)

The polyclonal antibodies were raised in rabbits by usingLp(a) lipoprotein as the antigen and the immunization pro-cedure described by Berg.1 The antibodies were tested forspecificity by reaction with human sera, LDL, and Lp(a)lipoprotein in double diffusion experiments by the Clark-Freeman technique.21 The antisera against Lp(a) showedtwo precipitation lines; one was identical to LDL and theother was the same as Lp(a), but not LDL. The antiserawere made specific by absorption with purified LDL and byaffinity chromatography on a CN-Br-Sepharose column(Pharmacia, Sweden) to which the Lp(a)-specific glycopro-tein had been coupled according to the manufacturer'sdescription.

Monoclonal Antibodies against Lp(a)

Immunization, fusion, cell culture handling, and screen-ing of hybridomas producing specific antibodies were per-formed according to standard protocols.22 Briefly, 20 /xg ofpurified Lp(a) antigen mixed with complete Freund's adju-vant were injected intradermally into 8- to 10-week-oldfemale BALB/c mice. The Lp(a)-specific glycoprotein wasisolated and purified as described by Utermann et al.16

After 3 weeks, an intradermal boost with another 10 ̂ ig ofantigen in incomplete adjuvant and 7 days later, a last(intravenous) boost of 20 /xg were given to those mice thatshowed anti-Lp(a) antibody production as verified by anELISA assay of diluted mouse plasma taken from the plex-us retro-orbitalis. The mice were killed 4 days after the finalboost, and the spleens were removed aseptically.

Splenic lymphocytes were fused with Ag8 mouse mye-loma cells and were distributed into 24-well plates (con-taining 1 ml/well RPMI-HAT medium and mouse peritonealmacrophages as feeder cells). Screening was begun whenthe colonies were 3 to 5 mm in diameter.

Screening by ELISA and Immunoblottlng

For an ELISA assay, 100 /xl of Lp(a) antigen, togetherwith antimouse immunoglobulins at 2 to 5 /ig/ml in PBS(pH 7.4), was added to each of the 96-well Dynatech Im-mulon II ELISA plates. These were incubated overnight at4°C. Before use, the plates were blocked (PBS, pH 7.4,1 %bovine serum albumin, 0.05% Tween 20, and 1 mg/mldextran sulfate) for 30 minutes at room temperature.

Supernatants (100 /JL\) from the growing culture wellswere transferred by pipette into duplicate wells and wereallowed to react overnight at room temperature. Plateswere washed thrice quickly with water, once with PBS+ 0.05% Tween 20 for 10 minutes at 37°C, and againthrice with water. To each well was added 100 /J of goatantimouse immunoglobulins conjugated to alkaline phos-phatase (Dakkopatts, Denmark) and diluted 1:1000 inPBTD. This was incubated for 5 hours at 37°C. Plates werewashed as explained above, and 100 /xl of p-nitrophenylphosphate (Sigma, FRG) in 0.05 M Na2CO3 (pH 9.6) wasadded. After 30 minutes at 37°C, the plates were readvisually and the positives were noted.

Positive wells were cloned by limiting dilution in HT-RPMI medium (without aminopterin). These secondarycultures were screened again by ELISA and cloned bylimiting dilution. Recloned cultures were screened, select-ed, and grown as before in T25 and T75 flasks (Falcon,USA) when appropriate.

Mass Production and Purification of MonoclonalAntibody

Hybridomas were injected intraperitoneally in 12-week-old BALB/c mice that had been primed intraperitoneally7 days earlier with 0.5 ml Pristane (Sigma, FRG). After 10to 20 days, their asdtes fluid was obtained and the cellswere removed by centrifugation at 400 g. The IgG fractionwas purified from the mouse ascites fluid by using fastperformance liquid chromatography (Pharmacia, Swe-den). Ascites (up to a protein amount of 50 mg) was ap-plied undiluted, but filtered through a 0.2 /xm membranefilter, on a Mono Q 5/5 anion exchange chromatographycolumn after dialysis against 10 mM Tris (pH 7.4). IgG wasobtained by elution with a 20 ml, 0 to 500 mM NaCI gradi-ent in 10 mM Tris (pH 7.4). Purity was judged on the basisof 10% SDS-PAGE according to the method of Neville.17

Characterization of Antibody

Antibody specificity was characterized by Western blot-ting of the purified Lp(a)-specific glycoprotein exactly asdescribed above for the serum samples.

Further characterization was done by radioimmunoas-say of the radiolabeled antigen. Lp(a) antigen was labeledwith 12SI by the chloramine T method. The labeled antigen(about 200 000 cpm) was incubated with 200 AII of diluted

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214 ARTERIOSCLEROSIS VOL 8, No 3, MAY/JUNE 1988

culture supernatant (or ascites or purified antibody) for2 hours at 37°C, then incubated with the second antibody(antimouse immunoglobulins) for 2 hours at 37°C, followedby an incubation with Protein-A Sepharose (Pharmacia,Sweden) for 30 minutes at room temperature. After a5-minute centrifugation, the pellet was extensively washedwith 10 mM Tris (pH 7.4) containing sodium desoxychc-late, Triton X-100, SDS, and MgCI2, and this was countedin a gamma counter (LKB, Sweden).

Subisotyping was done with an ELISA kit from AmericanQualex. This assay consisted of a sandwich ELISA proce-dure, where the plate was coated with a capture antibody(goat antimouse immunoglobulins), followed by the un-known antibody sample (culture supernatant), followed byrabbit antisera against the various immunoglobulin sub-classes, finally followed by a goat antirabbit, enzyme-

Blot A

2 3 4 5 6 7 8 9 10 11 12 13 14 15

Blot B

Figure 1. Samples from 15 different subjects were applied intwo series on one gel as described under Methods. After theelectrophoretic run, the gel was divided and the two parts wereelectroblotted. Blot A was developed by using the polyclonal anti-body against Lp(a). Blot B was developed with the monoclonalantibody. The following Lp(a) phenotypes can be seen on theblots in the respective lanes: Lane 1, S1 + S2; Lane 2, B; Lane 3,S1; Lane 4, S1 +S2; Lane 5, S2; Lane 6, S2 + S3; Lane 7, S2;Lane 8, S1 + S3; Lane 9, S4; Lane 10, S4; Lane 11, S4; Lane 12,S1 +S2; Lane 13, B; Lane 14, B + S4; and Lane 15, F + S4.

conjugated, detection system. All incubation steps hadsufficient washing between procedures as previouslydescribed.

Results

Lp(a) phenotypes were identified in total plasma or se-rum by SDS-PAGE in horizontal slab gels followed by im-munoblotting. Figure 1 shows two representative Lp(a) im-munoblots: the upper one has been incubated with apolyclonal anti-Lp(a) antibody and the lower one, with ourmonoclonal antibody, MA 5', which came out to be an IgG1 antibody by subisotyping. Samples from 15 different indi-viduals were applied twice in two series on one gel. Afterthe electrophoretic run, the gel was divided and the twoparts were blotted as described. Comparing the two blots,it can be seen that the monoclonal antibody recognizes thesame phenotypes as the polyclonal one does. Four differ-ent single-band phenotypes, as well as four different dou-ble-band phenotypes, are shown on the blots. The respec-tive phenotypes are designated according to their relativeelectrophoretic mobility compared with that of apo B-100("F means faster than apo B-100, "B" means equal to apoB-100, and "S1-S4" are all slower than apo B-100 to differ-ent degrees). The position of apo B-100 was determinedby using a specific anti-apo B antiserum as the first anti-body in the immunoblot procedure (not shown). The "P-phenotype has so far been seen only in another group(patients of the local lipid clinic).

By using the new procedure, the Lp(a) phenotypes of194 healthy individuals who served as blood donors weredetermined. The group consisted of 98 women (18 to 71years old, mean age = 38.6, SD = 12.7) and 96 men (20 to65 years old, mean age = 38.4, SD = 12.2). Their Lp(a)plasma concentrations were measured with an electroim-munoassay as described. The frequencies of the differentphenotypes in this group are shown in Table 1. The tablegives also the Lp(a) plasma concentrations in the respec-tive, single-band phenotypes. The nonparametric Kruskal-Wallis test showed a highly significant (p<0.001) differ-ence between the Lp(a) lipoprotein concentrations of therespective Lp(a) phenotypes, thus confirming the recentlyfound association of Lp(a) lipoprotein concentration withLp(a) glycoprotein phenotype.16

We assumed that Lp(a) phenotypes are controlled byfive alleles that code for the B, S1, S2, S3, and S4 forms ofLp(a) glycoprotein and by a "0" allele,16 and we calculatedthe allele frequencies (Table 2). The introduction of a nullallele is necessary to explain the occurrence of subjectswith no detectable Lp(a), as well as the transmission ofLp(a) types in the family studies.16

Discussion

The described method easily enables Lp(a) phenotypingof up to 40 samples on one gel, in contrast to our hithertoused vertical gel system16 which allowed only 13 sampleson one gel. The new method is, therefore, ideally suited forscreening large populations. A second great advantage isthe simple casting procedure. In the vertical gel system,the soft, stacking gel gave rise to problems of leakagebetween the sample wells, which frequently resulted in

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LP(a) PHENOTYPING Kraft et al. 215

Table 1. Lp(a) Phenotype Frequencies and Lp(a)Lipoprotein Concentration In Tyrollan Blood Donors

Lp(a)pheno-type

BS1S2S3S4"0"B + S1B + S2B + S3B + S4S1+S2S1+S3S1+S4S2 + S3S2 + S4S3 + S4Totals

Table 2

Allele

Lp(a)B

Lp(a)s1

Lp(a)82

Lp(a)53

Lp(a)54

Lp(a)°

M i l

Observed

38

28254273

1000031712

194

mD©rI Expected

3.07.6

26.824.14169.7

0.20.40.40.71.31.21.83.86.35.7

194

. Frequencies of

Frequencyobserved

1.64.2

14.412.921.637.6

0.500001.60.53.60.51.0

100

Lp(a) lipoproteinconcentration

Mean±SD (mg/dl)

61.7 ±7.823.5±1926.3±2111.3 + 7.110.2±6.14.4 ±4.9

Lp(a) Alleles

Frequency (%)

0.0130.0320.1060.0960.1560.600

These were calculated according to the method of Wiener etal.23 and the correction formula of Dobson and Ikin.24

smearing the Lp(a) bands on the blots. These problems inthe vertical system could be overcome only by laborioustricks, e.g., treating the upper part of the glass plates (towhich the stacking gel adheres) with Bind-Silane fromLKB, Sweden (Menzel HJ, unpublished data). These prob-lems do not exist in the horizontal system.

The same phenotypes were demonstrated with the con-ventional polyclonal rabbit antisera, as well as with themonoclonal antibody MA 5' produced against the purifiedLp(a)-specific glycoprotein, proving that high molecularweight bands are, indeed, Lp(a) glycoprotein. All geneticLp(a) species were recognized equally well by the mono-clonal antibody, MA 5'. This is in contrast to the reactivity ofthe monoclonal antibody, LHLP-1, developed by Duvic etal.25 who used purified lipoprotein Lp(a) as the immuno-gen, which did not react with the reduced glycoprotein.

Our family studies showed that Lp(a) glycoprotein phe-notypes are genetically controlled.16 The frequency distri-bution for Lp(a) phenotypes determined here in a group ofnormal, unrelated blood donors is in keeping with thishypothesis. The Lp(a) allele frequencies that we calculat-ed here are similar to those in a first group16 with onenotable exception. The frequency of the S4 allele, which isthe one that is associated with the lowest Lp(a) plasmaconcentration, increased from 0.076 to 0.156 at the ex-

pense of the Lp(a)° allele, which declined from 0.70 in theprevious study to 0.60. This is due to the increased sensi-tivity of our new procedure. But still, in 37.6% of ourprobes, we found no detectable Lp(a) bands on the im-munoblots. We do not know whether these are true nega-tives or whether they have small, though undetectable,amounts of Lp(a) in plasma. It is also possible that we lostsome of the heterozygotes, especially when a faint S4band was associated with a strong S1 or S2 band. Moresensitive assays will be needed to answer these ques-tions. Also, the association of the respective single-bandLp(a) phenotypes with Lp(a) plasma concentration, whichwas established in the previous study, was confirmed herein an unrelated group of subjects. The large variability ofLp(a) concentration within a °single-band phenotype,"which is expressed in the standard deviation given in Table1, is partly due to the fact that these groups consist ofhomozogytes for the respective allele, as well as of hetero-zygotes with the null allele.

The method described in this paper will be helpful inperforming large-scale genetic studies. Such studies areneeded to confirm the genetic hypothesis, to determineLp(a) glycoprotein phenotype frequencies in different pop-ulations, and to prove the postulated association of thegenetic Lp(a) trait with atherosclerosis.

An association of Lp(a) lipoprotein concentration withmyocardial infarction has been demonstrated in severalcase-control studies.6"11 However, although under stronggenetic control and, in general, rather constant individual-ly, Lp(a) lipoprotein levels are known to be influenced byhormones, drugs, and disease states.26"29 Hence, it hasnot yet been rigorously ruled out that elevated Lp(a) levelsin myocardial infarction patients are the result, rather thanthe cause, of the disease. Prospective studies are neededto answer that question definitively. Our finding that thegenetic Lp(a) protein type is associated with Lp(a) concen-trations may allow us to circumvent this problem by com-paring Lp(a) glycoprotein phenotypes in patients and con-trols. Moreover, our observation raises the challengingpossibility that Lp(a) type, rather than concentration, is arisk factor for premature atherosclerosis. Studies on theseaspects of Lp(a) glycoprotein polymorphism are now inprogress in our laboratory, and we are using the techniquedescribed in this paper.

AcknowledgmentsThe authors appreciate the expert technical assistance of Ulrike

Keller, Linda Fineder, and Artur Friedl. We are also thankful toHans Reissigl, Head of the Blood Bank of the University of Inns-bruck, for providing blood from his donors.

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11. Annstrong VW, Cremer P, Eberle E, et al. The associationbetween serum Lp(a) concentrations and angtographicallyassessed coronary atherosclerosis — Dependence on serumLDL levels. Atherosclerosis 1986:62549-257

12. Utermann G, Weber W. Protein composition of Lp(a) lipopro-tein from human plasma. FEBS-Let 1983:154:357-361

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Index Terms: Lp(a) lipoproteinmonoclonal antibodies

Lp(a) glycoprotein • Lp(a) immunoblotting • Lp(a) phenotypes

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H G Kraft, H Dieplinger, E Hoye and G UtermannLp(a) phenotyping by immunoblotting with polyclonal and monoclonal antibodies.

Print ISSN: 1079-5642. Online ISSN: 1524-4636 Copyright © 1988 American Heart Association, Inc. All rights reserved.

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