Vox Sang. 22: 420-431 (1972)

Anti-P-Lipoprotein Activity of Human Monoclonal Immunoglobulins

W. RIESEN, G. NOSEDA and R. BUTLER

Institute for Clinical and Experimental Cancer Research and Institute for Clinical Pro- tein Research of the University of Bern, and Central Laboratory of the Blood Transfusion

Service of the Swiss Red Cross, Bern

Abstract. Human sera containing monoclonal immunoglobulins were screened for antibody-like activity against lipoproteins by immunodiffusion tests and the passive hemagglutination of low-density lipoprotein (LDL) coated red cells. Weak positive reactions with human LDL were observed in about 10% of fresh sera. All sera reacted with the patient’s own LDL and did not display any specificity against the 8 iso-antigens of the Ag-system. No reaction with human high-density lipoproteins (HDL) and with LDL of animals could be detected. In the case of IgA and IgG myeloma proteins the binding activity was due to the M-component. The Fab fragment obtained after enzymatic digestion of myeloma IgG with papain could be shown to be the reactive part of the immunoglobulin molecule. The described antibody-like activity was associated with hypolipidemia.

I t is now well established that myeloma proteins (M-components) may display antibody-like activity [reviewed in 171. Such monoclonal immuno- globulins are of great potential value in studying antibody functions and the nature of the binding site. Furthermore, the existence of antigen-binding M-components provides a potential source of information concerning the pathogenesis of benign and malignant clones of immunoglobulin-producing cells.

The purpose of this investigation was to search among human sera containing monoclonal immunoglobulins for antibody-like activity against lipoproteins, an antigenic substance which occurs as well in the plasma as in the membrane of cells and mitochondria.

Immunological characterization of intact lipoproteins indicate that low- density lipoproteins (LDL) are antigenically distinct from the high-density

~~ ~ ~

Received: September 27, 1971; accepted: November 9, 1971.

M-Components Reacting with 8-Lipoproteins 421

lipoproteins (HDL) [l, lo]. On the other hand, immunological identity within the low-density and very low-density lipoprotein (VLDL) class has been reported [ 1, 9, 201.

At the present time at least two genetically independent LDL-polymor- phisms are known to exist in humans, namely the Lp-system which may be demonstrated by heteroimmune sera and the Ag-system which is revealed by precipitating and non-precipitating human iso-antibodies. The formation of anti-Ag antibodies occurs in practically all cases by isoimmunization after multiple blood transfusions as they have been found almost without exception in patients having received multiple transfusions.

Two IgA and one IgG myeloma proteins which form complexes with lipoproteins have been described earlier by BEAUMONT [2]. These myeloma proteins appear to react with LDL and HDL, a fact which is not observed with authentic antibodies to LDL.

Materials and Methods Screening Procedures Two different screening methods for the detection of binding activity against lipo-

proteins were used: The double diffusion in agar and agarose (well diameter 3.5 mm, distance 4 mm,

buffer 0.05 M Verona], pH 8.6) and the passive hemagglutination of lipoprotein coated red cells according to Butler IS].

For this purpose human red cells of blood group 0 were coated by means of bis- diazotized benzidine with the following lipoproteins :

(1) LDL from three single donors (LDL 30, 35 and 26), covering all the presently- known 8 factors of the Ag-system (see table I).

(2) LDL isolated from the patients’ own serum. (3) HDL from the same three single donors. 0.1 rnl of dilution series (1 :4,1:8,1:16,1:32) of the myeloma sera which had previously

been inactivated at 56°C for 30 min were incubated with 0.05 ml of the coated red cells at 0°C. The first reading was done after 2 h, the second after 20 h. Simultaneously, thefollow- ing controls were set up:

(1) Negative controls: (a) an inactivated normal serum pool incubated with LDL- coated human red cells; (b) the inactivated myeloma sera, incubated with uncoated human red cells.

(2) Positive controls: (a) rabbit anti-LDL antiserum (Behringwerke Marburg, Lahn), which was pre-absorbed with human red cells, incubated with LDL coated human red cells; (b) rabbit anti-HDL antiserum (Behringwerke) pre-absorbed with human red cells, in- cubated with HDLcoated human red cells, and (c) human anti-LDL (anti-Ag (al, g, x), incubated with LDLcoated human red cells.

Passive Hemagglutinationllnhibition for Studies on the Specifcity against &-Factors The specificity of the myeloma proteins against the 8 iso-antigens of the Ag-system

was studied by the inhibition of the passive hemagglutination of LDLcoated human red

422 RIESEN/NOSEDA/BUTLER

cells according to BUTLER [S]. As inhibitors the LDL of 6 single donors with known Ag- factors (listed in table I) and LDL of animals were used. The same positive and negative controls as described above were set up.

Isolation of Lipoproteins The different lipoprotein fractions were isolated by repeated ultracentrifugation (in

a Christ ultracentrifuge, Type Omega 11, Rotor 9530) at 0°C as follows: Chylomicrons at 23,000 rpm (26 x 1,000 g) for 30 min; VLDL (d = 0.93-1.019 g/ml) at d = 1.019, 45,000 rpm (100 x 1,OOO g) for 20 h; LDL (d = 1.019-1.064 g/ml) at d = 1.063, 45,000 rpm (100 x 1,000 g) for 20 h, or by precipitation with heparine in the presence of MgCl2 and sucrose and subsequent

HDL (d = 1.063-1.21 g/ml) at d = 1.21, 45,000 rpm (100 x 1,000g) for 20 h. The isolated lipoproteins were subsequently dialyzed against saline at 0°C. Purity

was assessed by immuno-electrophoresis using rabbit antisera against whole normal human serum and specific antisera against a-lipoproteins and p-lipoproteins (Behringwerke).

The labeling of the LDL with lZzI was performed by the iodine monochloride tech- nique according to MCFARLANE [12].

ultracentrifugation as described by BURSTEIN [4] ;

Isolation of Myeloma Proteins The IgA myeloma protein (LW) was isolated from the serum of L. W. by ion-exchange

chromatography on QAE-Sephadex (A-50) in EDTA pH 7, with a linear gradient of ionic strength from 0.01 to 0.1 M. Fractions containing IgA were then further purified by filtration through Sephadex G-200. Purity of the isolated IgA was assessed by immuno- electrophoresis using a rabbit antiserum to whole normal human serum and a mono- specific anti-IgA antiserum. No protein other than IgA was detected at a concentration of 20 mg/ml.

The IgG myeloma proteins (E.S. and M.M.) were isolated either by zone electro- phoresis on polyvinylchloride Pevikon C-870 (Kenia Nord) or by ionexchange chromato-

Table I . Ag-factors of single donors LDL used in passive hemagglutination and hemagglu- tination/inhibition studies

LDL a1 C d g t X Y Z

- + +

- 10 + + + + + - + + + 21

- + + + + 26 + 30 + + + + + + + + 3s + + + + + + +

+ + 38 + + 39

- - - - -

- - - - - +

- - + - + + - - - 209 + - + + + +

The numbers correspond to the single donors.

M-Components Reacting with &Lipoproteins 423

graphy on QAE-Sephadex (A-50) in EDTA (0,Ol M, pH 7). No protein other than IgG was detected by immuno-electrophoresis at a concentration of 40 mg/ml using an anti- serum to whole normal human serum and a monospecific anti IgG antiserum.

Preparation of Proteolytic Fragments Pepsin Digestion of Monoclonal IgG (E.S.) and Isolation of the Fragment F(ab)'z 40 mg IgG (E.S.) were dialyzed against 0.1 M acetate buffer pH 4.6. Pepsin digestion

was performed at 37°C for 16 h using an enzyme: protein ratio of 2:100. The reaction mixture was subsequently filtrated through a column of Sephadex G-150 in 0.1 M tris pH 7.5. Protein fractions appearing under the second peak only contained F(ab)'z and Fab', but no intact IgG and no Fc', as detected by immuno-electrophoresis.

Papain Digestion of Monoclonal IgG (E.S.) and IgG (M.M.) and Isolation of the Corresponding Fab- and Fc-Fragments

The method described by PORTER [16] was employed. 100 mg protein were dissolved in 6 ml phosphate buffer, 0.1 M pH 7.5, made 0.003 M in EDTA and 0.01 M in cysteine. Crystalline papain (Worthington Biochem. Corp.) 1.5 mg was added and digestion was carried out for 4 h at 37°C. The reaction was stopped by the addition of 0.6 ml 0.1 M N-ethylmaleinimide. In order to separate the intact IgG from the fragments the reaction mixture was then filtrated through Sephadex G-100 in 0.1 M tris pH 7.5 at a flow rate of 2 ml/h. The fragments Fab and Fc were subsequently separated by zone electrophoresis on Munktell's cellulose powder on a column electrophoresis apparatus (LKB).

Determination of Serum Lipids Blood samples were drawn after a 12-hour fast. Lipoprotein-electrophoresis was perform-

ed on agarose plates in Verona1 buffer (0.025 M, pH 8.6), containing 0.3% EDTA and 0.5% HSA in a Gelman apparatus. The agarose plates were stained with oil red and evaluated densitometrically .

Chemical lipid determinations: Total lipids as described by ZOLLNER and KIRSCH [21]; triglycerides as described by ECGSTEIN and KREUTZ [7]; total cholesterol as described by RICHTERICH and LAUBER [18]; phospholipids as described by LOWRY [l 11 and BLOOR 131.

Delipidation of LDL The procedure outlined by GOT~O et at. [S] was followed. LDL was delipidated by

shaking with a mixture of ether/ethanol 3:l for 20 h at 0°C. The ether/ethanol solution was repeatedly removed. The dry residue of apoLDL was dissolved by incubation for 16 h at 37°C with 0.15 tris buffer pH 8.5 (0.01% EDTA), containing 60 mM sodium dode- cylsulfate. The preparation was then dialyzed against the same buffer at a 2 - m ~ concentra- tion of sodium dodecylsulfate.

Results

Screening for Binding Activity against Lipoproteins The results of the different screening methods are summarized in tables IIa

and b, the distribution of binding activity among IgG subclasses and light

424 RIESEN/NOSEDA/BUTLER

Table IIa. Reaction of stored myeloma sera with lipoproteins

M-protein class Number of cases Immunodiffusion Hemagglutination analyzed binding of binding of

LDL LDL

n Yo n %

183 32 49

I5 41 20 11 1 1 34 3 9 13 26 7 14

Total 264 99 37 30 1 1

Table IIb. Reaction of fresh myeloma sera with lipoproteins

M-protein class Number of cases Immunodiffusion Hemagglutination analyzed binding of binding of

LDL LDL

n Yo n Yo

10 11 7 8 3 10 2 I 4 20 5 24

Total 138 17 12 14 10

chain types in table 111. The sera which were found to react with all the 3 LDL of single donors were also tested for binding activity against VLDL and the patient’s own LDL; all of them reacted. On the other hand no reaction could be detected when these sera were tested with HDL from the mentioned three single donors or with their own HDL.

A marked difference between results from the immunodiffusion tests and the passive hemagglutination appears, especially with stored myeloma sera, whereas no differences between the precipitation in agar and agarose could be observed.

Table IV summarizes the titers found with the passive hemagglutination and their relation to precipitability. The data indicate that precipitation

M-Components Reacting with /?-Lipoproteins 425

Table ZZZ. Distribution of binding activity among IgG subclasses and light chain types ~

IgG subclass Number of cases Positive reaction against analyzed LDL (tested by passive hemagglutination

n %

GI G2 G3 G4

164 16 10 11

Light chain type K 125 L 76

16 5 1 2

12 10

10 31 10 18

10 13

Table ZV. Hemagglutination titers and relation to precipitation

Patient M-protein class Total protein M-gradient Hemaggluti- Precipita- g % % nation titer tion

U. D. R. L. M. W. A. P. G. G. 1. L. A. Z. E. S. w. s. L. w. M. M. C. B. A. K.

9.4 10.7 9.4 6.3

10.6 12.2 6.2 7.6 7.1

10.0 7.1 9.0

13.2

31 50 41 22 38 54 26 21 18 44 38 43 51

1 :32 1 :32 1 :32 1 :64 1 :64 1 :64 1 :128 1 :128 1 :256 1 :320 1512 1 : 1,024 1:1,024

occurs with hemagglutination titers exceeding 1 :128 when the M-protein belongs to the immunoglobulin classes IgG and IgA. In the IgM class precipitation seems to be facilitated and occurs already with lower titers such as 1:32. On the other hand precipitation could also be observed in some cases where the passive hemagglutination was negative.

426 RIESEN/NOSEDA/BUTLER

In addition, 200 normal sera were screened for binding activity against the mentioned three different LDL and the corresponding HDL. Among these sera no one could be shown to react with lipoproteins.

Localisation of Binding Activity In order to establish whether the binding activity against 8-lipoproteins

was associated with the monoclonal immunoglobulins or with an other serum protein, the M-gradients of three different sera IgG (E.S.), IgG (M.M.) and IgA (L.W.) were isolated. All sera precipitated LDL and VLDL and agglutinated human erythrocytes coated with LDL. The hemagglutination titers were 1 :128, 1 :512 and 1 :320, respectively. In all three cases the isolated M-protein showed the same reactions as was observed with the whole serum.

Evidence that the binding activity is due to the M-component was also obtained from double diffusion experiments, done with lZ5 I-labeled LDL followed by autoradiography (fig. 1). In the case of IgA and IgG myeloma sera radioactivity was found in the precipitation lines, formed by the M- components and the corresponding antisera. The same experiments done with sera of patients with macroglobulinemia Waldenstrom showed no radioactivity in the precipitation line of IgM and the corresponding anti- serum, but in the precipitation of BIC and its antiserum.

In addition most of the macroglobulinemic sera reacting with p-lipopro- teins contained cryoglobulins. Further investigations of these cryoprecipitates are presently undertaken.

Studies on the localization of the binding site on the IgG myeloma pro- teins were done after enzymatic cleavage of the immunoglobulin molecules with papain in the case of IgG (M.M.) and with pepsin and papain in the case of IgG (E.S.). Both Fab-fragments isolated after papain digestion inhibited the passive hemagglutination of LDL coated red cells. With the Fc-fragments no inhibition could be observed. The Fab-fragments alone did not agglutinate the LDL coated red cells, addition of an anti-light chain antiserum, however,

Fig. 1. Autoradiograph of double diffusion experiment. a IgA myeloma serum L.W. incubated with *251-labeled LDL. b Anti-/%lipoprotein antiserum. c Anti-IgG antiserum. d Anti-IgA antiserum. e Anti-BIC antiserum. !Anti-IgM antiserum.

M-Components Reacting with p-Lipoproteins 421

caused agglutination. On the other hand, fragment Fc incubated with LDL coated red cells failed to give an agglutination after addition of an anti-IgG Fc antiserum. The bivalent fragment F(ab)’, obtained after enzymatic clea- vage of IgG (E.S.) with pepsin still agglutinated the LDL sensitized red cells.

The reactive part of the lipoprotein could be shown to be the protein moiety as the mentioned isolated myeloma proteins (IgA [L.W.], IgG [E.S.] and IgG [M.M.]) precipitated with delipidated human LDL (apo LDL) in agar. Normal human sera, used as a control, did not precipitate with this apo LDL.

Studies on the Specijicity of M-Proteins against Ag-Factors and /?-Lipo- proteins of Animals Informations about the specificity of IgA (L.W.) and ZgG (E.S.) against

the 8 presently known iso-antigens of the Ag-system obtained by the passive hemagglutination/inhibition technique are shown in table V. The hemagglu- tination of the LDL coated red cells could be inhibited by the patient’s own LDL and by every LDL listed in table I. In none of the investigated cases a

Table Y. Inhibition of the passive hemagglutination of LDL coated red cells with isolated IgA (L.W.) and IgG (E.S.)

Inhibitor Inhibition of passive hemagglutination

IgA (L.W.) IgG (E.S.)

Human LDL1 10 21 26 30 35 38 39

209 Autologous LDL Pig LDL Rabbit LDL Human HDL Pig HDL

The numbers correspond to the single donors.

428

rng'l. 1,400

1,300

1,000

800

600

400

200

a Nr 20 38

RIESEN/NOSEDA/B~~TLER

Binding activity between paraprotein and LDL-VLDL

0 No binding activity

Normal range

rn g *I.

400

C

23 38 b

16 36

T

d e f 20 37 16 36 16 37

Mal lipids Lipid content Cholesterol Triglycerids Lipid Phospholipids of LDL and content VLDL of HDL

Fig. 2. Serum lipids in patients with paraproteinaemia.

specificity against a single Ag-factor could be observed. In addition all positive sera were tested for specificity against /?-lipoproteins of animals such as dog, horse, calf, pig and rabbit. For this purpose red cells were coated with LDL from the mentioned animals. No agglutination was caused by anyone of the sera which previously had been shown to react with human LDL. Moreover animal LDL was not able to inhibit the agglutination of red cells coated with human LDL.

Relation between Binding Activity and Serum Lipid Concentration The existence of auto-antibody-like activity against LDL in myeloma

sera raises important questions with respect to the lipid concentrations in these sera. Accordingly the lipid concentrations in 61 sera were determined. 23 reacted with /?-lipoprotein, 38 did not. figure 2 illustrates the obtained results :

The mean values of the total lipids, the total cholesterol and the lipid content of the /?-lipoproteins of the sera (calculated as the product of the percentage of /?-lipoprotein in the lipoprotein-electrophoresis and the total li- pids) reacting with /Mipoproteins were below normal values and significantly

M-Components Reacting with /?-Lipoproteins 429

lower than in non-reacting sera. The mean values of the lipid content of the a-lipoproteins, however, and the phospholipids, which are the main lipids in the a-lipoproteins, were in both groups within the normal range and not significantly different.

Discussion

Human antibodies against LDL described up to the present time have been found almost without exception in patients having received multiple transfusions and are exclusively isoimmune antibodies. The frequencies studied by various authors vary between 12-30% in patients suffering from Cooley anemia treated with multiple transfusions and 1 4 % in patients with multiple transfusions because of other clinical entities [6]. Most patients from our panel, however, suffering from multiple myeloma or macroglobu- linemia Waldenstrom have not received multiple transfusions. The different M-proteins did not show any specificity against the 8 iso-antigens of the Ag- system and they reacted with the patients own p-lipoproteins-like auto- antibodies. The restriction of the binding site to the Fab fragment of the immunoglobulin molecule which is an important criterion for antibody-like activity could be shown with two IgG myeloma proteins.

Hemagglutination titers were found between 1 :32 and 1 :1,024 indicating that these reactions are rather weak, a fact which is common to almost all M-proteins displaying antibody-like activity.

A marked discrepancy was observed between results obtained with the passive hemagglutination and results obtained from precipitation reactions. Using the precipitin technique artefacts due to unspecific reaction of LDL with the agar gel have to be counted with. In our experiments, however, the precipitations could be reproduced in agarose gels. An explanation for these results might be the fact that unspecific precipitation is facilitated by aggrega- tion of the M-components. This assumption is supported by the fact that in stored sera (3-6 years old) the frequency of precipitation was increased markedly (30-40%). The frequency of 12% positive reactions against LDL obtained with the passive hemagglutination technique, however, is independ- ently found with stored and fresh myeloma sera.

The observed cross-reaction with LDL and VLDL indicates that the described binding activity is directed against the protein moiety of these lipoprotein fractions, the apoprotein. VLDL and LDL have been shown to possess an apoprotein in common and different from the apoprotein of HDL [8 a]. In fact, precipitation with the delipidated LDL could be observed

430 RIESEN/NOSEDA/BUTLER

with the sera showing a positive reaction with LDL. The M-proteins reacting both with LDL and HDL described by BEAUMONT [2], on the other hand appear to be directed against the lipid haptens and not the protein moiety. In addition these authors found that the binding activity was associated with hyperlipidemia. This hyperlipidemia was explained with a reduced catabolism of the lipoprotein-paraprotein-complexes formed in the patient’s serum. However, most antigen-antibody complexes have a faster catabolism than the unbound antigen and hypolipidemia as a consequence seems more plausible. The assumption of a faster catabolism leading to a hypolipidemia is supported by the fact that in one case a complete disappearance of the myeloma protein - which displayed auto-antibody-like activity against @-lipoproteins - was accompanied by a distinct increase of the $-lipoproteins [ 13, 191. In addition, turnover studies with radioiodinated LDL performed in a case of polyclonal gammopathy where the patient’s IgG reacted with his own LDL indeed showed a faster catabolism of the LDL [14].

The finding of antibody-like activity among monoclonal immunoglobulins raises fundamental questions with regard to the pathogenesis of some proli- ferating lymphoreticular disorders which lead to the synthesis of these immunoglobulins. The cells undergoing neoplastic transformation must have previously been committed to the production of specific antibodies. This committment may be due to an antigenic stimulation or not. The clinical reports of chronic infections preceeding the monoclonal proliferation support the theory of a repeated stimulation by antigen [15]. Most inter- esting in this connexion is the fact that the observed immune phenomena are frequently autoimmune in character.

Acknowledgements

The authors want to thank Dr. F. SKVARIL for the determination of the subclasses,

This investigation was supported by the Swiss National Foundation for Scientific Miss E. SCHLUMPF and Miss E. BRUNNER for excellent technical assistance.

Research.

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Authors' addresses: Dr. W. RIESEN, Institute for Clinical and Experimental Cancer Research of the University of Berne, Tiefenau, 3004 Berne: Dr. R. BUTLER, Central Laboratory of the Blood Transfusion Service of the Swiss Red Cross, 3000 Berne; Dr. G. NOSEDA, Institute for Clinical Protein Research of the University of Bern, Tiefenau-Hospital, 3004 Berne (Switzerland)