Immunochemical properties of lipoprotein lipase

38

Blochimra

et Blophysicu Acta, 152 (1983) 38-45 Elsevier

BBA 51401

IMMUNOCHEMICAL DEVELOPMENT THOMAS Department (Received

OF LIPOPROTEIN

OF AN IMMUNOASSAY

OLIVECRONA of Physiological December

PROPERTIES

and GUNILLA Chemistry,

APPLICABLE

LIPASE TO SEVERAL

MAMMALIAN

SPECIES

BENGTSSON

University of UmeP, Limed (Sweden)

17th. 1982)

Key words: Immunoassay;

Lipoprotein

lipase

The reaction of bovine lipoprotein lipase with its antibodies was found to be conformation-dependent. One aspect of this was that most antisera were more reactive with denatured than with native ‘251-labeled lipoprotein lipase. Another aspect was that denatured lipase did not compete effectively with native lipase for those antibodies which caused inhibition of the enzyme’s activity. This latter observation leads to the conclusion that the inhibiting antibodies recognize conformation-dependent determinants on the native enzyme. Fab fragments prepared from an inhibiting antiserum blocked the binding of the lipase to triacylglycerol/phospholipid droplets. This suggests that the inhibition results from reaction of the antibodies with the enzyme as it exists in solution, either covering the lipid-binding site on the enzyme or making it impossible for the enzyme to go through the conformational transitions necessary for binding to lipid. Most rabbit antisera did not react with rat or mouse lipoprotein lipase, but some sera showed a weak cross-reaction. Antisera raised in hens showed a much stronger cross-reaction, enough to be useful for heterologous immunoassays. An immunoassay for the bovine lipase was developed. For reproducible results it was necessary to have tracer, standard and samples in denatured form. This was accomplished by heating them in SDS, and running the immunoreaction in a Triton X-lOO-containing medium

Introduction Lipoprotein lipase is a much studied enzyme. The reaction that it catalyzes, hydrolysis of triacylglycerols and phospholipids in lipoproteins, is physiologically important and is biochemically interesting because of its complexity and because the enzyme is activated by one of the apolipoproteins [I]. The activity of the enzyme in various tissues is under hormonal control and is important for the regulation of lipoprotein metabolism [2]. For many studies on lipoprotein lipase it would be of interest to measure not only enzyme activity but also the amount of enzyme protein. An immunoassay has been established for native and denatured avian 0005-2760/83/$03.00

0 1983 Else&r

Science Publishers

B.V.

lipoprotein lipase [3]. Jansen et al. [4] have described an antiserum against rat lipoprotein lipase and its use for immunotitration based on inhibition of enzyme activity. However, there is no immunoassay for catalytically inactive forms of a mammalian lipoprotein lipase. Bovine lipoprotein lipase can be purified relatively easily from milk in mg quantities. Rabbit antibodies against this enzyme also react with human and swine lipoprotein lipases [5,6]. We report here that by immunizing hens with the bovine lipase it is possible to obtain antisera which cross-react with rat and mouse lipoprotein lipases. We have also studied the reactions of native and denatured forms of the lipase with the antibodies, and on the basis of these

39

studies we have developed an immunoassay which can be adapted to use in several mammalian species. Materials and Methods

Lipoprotein lipase was purified from bovine milk as described (71. It was iodinated by the lactoperoxidase method [8] and repurified by chromatography on heparin/ Sepharose. Antisera were raised in rabbits and in hens by subcutaneous injection of 50-100 pg purified lipoprotein lipase in complete Freund’s adjuvant. Booster injections of 100 pg lipoprotein lipase in incomplete adjuvant were given 3 weeks later and then at intervals of about 1 month. Blood was taken 7-10 days after a booster injection. Serum was collected and heat-inactivated for 30 min at 56°C before use. To prepare Fat, fragments a yglobulin-enriched fraction was first obtained from a rabbit antiserum by precipitation with 1.75 M ammonium sulphate. It was then redissolved in (56 mg/5 ml) and dialyzed against 0.1 M sodium phosphate, pH 7.0, 0.01 M cysteine and 0.002 M Na,EDTA. 1 mg papain was added and the mixture incubated for 16 h at 37°C. The papain was inactivated by iodoacetamide (13 mM) and the products were dialyzed against 0.01 M acetate buffer, pH 5.5, and then separated by chromatography on CM-Sephadex. Elution was by a gradient of increasing concentrations of sodium acetate. The peak containing the Fab fragments was collected, dialyzed into a buffer of 0.1 M sodium borate/O.15 M NaCl, pH 8.2, and passed through a column of protein A-Sepharose to remove any undigested immunoglobulins. Lipoprotein lipase enzyme activity was measured at pH 8.5 in an assay system with the phospholipid-stabilized triacylglycerol emulsion Intralipid (containing [ 3H]oleic acid-labeled triolein, a kind gift from AB Kabi-Vitrum, Stockholm, Sweden) as substrate, human serum as source of activator protein and bovine serum albumin as fatty acid acceptor [9]. To test whether the antibodies reacted with mouse lipoprotein lipase, an extract of acetone/ diethyl ether powders from mouse hearts was used. For this mouse hearts were homogenized (1 g in 10 ml) in a buffer containing 25 mM ammonia, 5 mM

EDTA and, per ml, 40 pg heparin, 10 pg leupeptin, 1 pg pepstatin and 35 pg aprotinin. The pH was 8.2. The homogenate was added dropwise to 10 vol. of acetone chilled in dry ice. The mixture was vacuum-filtered at room temperature. The precipitate was washed with an equal volume of acetone/diethyl ether (1 : 1) and then with a small volume of diethyl ether, and was then dried in a desiccator. To prepare an extract the powder was homogenized (10 mg/ml) with a glass pestle homogenizer and then left to extract for 30 min at 4’C in the following buffer: 25 mM ammonia, 5 mM EDTA, also containing, per ml, 1 mg bovine serum albumin, 40 pg heparin, 10 pg leupeptin, 1 pg pepstatin, 35 pg aprotinin, 4 mg Triton X-100 and 0.4 mg SDS. The pH was 8.2. The suspension was centrifuged at 30000 x g for 30 min and the supernatant used for the experiments. For measurement of binding of the lipase to lipid droplets in the experiment in Table I, isolated triacylglycerol-rich particles from Intralipid [9] corresponding to 10 mg triacylglycerol were mixed in a total volume of 1.9 ml containing 0.2 mm01 NaC1/0.04 mmol Tris-HCl, pH 8.5,’ 1 mg heparin/120 mg bovine serum albumin at room temperature. 100 ~1 of the solutions of ‘251-labeled lipoprotein lipase incubated with FBb, F, or buffer as specified in the figure legend was added. Then 1 ml of the mixture was layered under 4 ml 0.1 M NaCl/O.l M Tris, pH 8.5, containing 10 mg albumin/ml in tubes for the Beckman SW50.1 rotor. The samples were centrifuged for 20 min at 25000 rpm, 15°C. The tubes were then sliced and a top fraction of about 1 ml containing the lipid layer was collected separated from the infranatant. The amounts of iz5I radioactivity in both fractions were determined and the binding was expressed as the amount of radioactivity in the top phase in percent of the total amount of radioactivity in the tube. A blank of 4% was subtracted from each top phase, representing the amount of radioactivity floating in tubes without lipid droplets. The total recovery of radioactivity after cent~fugation and slicing was better than 95%, calculated from the amount of radiactivity in 0.5 ml of the original mixture. The immunoassay shown in Fig. 6 was carried out as follows. Samples, standards and the tracer were all heated in 1% SDS in 0.02 M sodium

40

phosphate buffer, pH 7.4, at 95°C for 5-10 min. Each tube received a total of 210 ~1 of these solutions or a corresponding buffer (200 ~1 sample or standard and 10 ~1 tracer, about 4 ng, 5000 dpm). Then 1.7 ml of 0.02 M sodium phosphate/ 0.1 M NaCI, pH 7.4, containing 1 mg/ml bovine serum albumin, 5.9 mg/ml Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride and 2 mM EDTA were added, followed by 100 ~1 of the appropriate dilution of antiserum (0.25-0.40 pi/sample) in 0.02 M sodium phosphate/O. 1 M sodium chloride, pH 7.4, with 1 mg/ml bovine serum albumin. The tubes were incubated for 48 h at 4°C. Then, 50 ~1 of a goat antiserum to chicken immunoglobulins was added and the tubes incubated overnight. After centrifugation, the supernatant was decanted and the “‘1 radioactivity in the precipitate was determined in a gamma counter. Heparin and Intralipid were from AB KabiVitrum, Stockholm, Sweden, leupeptin, pepstatin, aprotinin and lactoperoxidase were from Boehringer-Mannheim, Mannheim, F.R.G. Results

Fig. I. Cross-reactivity with mouse lipoprotein lipase of antisera raised against bovine lipoprotein lipase in rabbit (A) and hen (B), respectively. An extract of acetone/diethyl ether powders from mouse hearts was prepared as described in Materials and Methods. Purified bovine milk lipoprotein lipase was diluted in buffer of the same composition to give the same enzyme activity per ml. Equal volumes of the enzyme solution and the antiserum, or a dilution of the antiserum with a corresponding nonimmune serum, were mixed and incubated at 4’C, 16 h. Then remaining enzyme activity was measured using the assay described in Materials and Methods. No detectable loss of enzyme activity occurred during a corresponding incubation with nonimmune serum. This stability is due to the presence of the detergents. A, Rabbit antilipoprotein lipase serum (TE), B, hen antilipoprotein lipase serum (HI 1). 0, Mouse lipoprotein lipase; 0, bovine lipoprotein lipase.

Antisera

The antisera selected for this study gave single precipitin lines on double diffusion against the purified bovine lipase and against bovine skim milk. They gave no precipitates against bovine plasma or bovine antithrombin. It was previously known that antisera against bovine lipoprotein lipase can be raised in rabbits and that some of these antisera cross-react with the human and the pig enzyme [5,6]. In the present study antisera from 13 rabbits were tested. Several of these inhibited not only the bovine enzyme but also the lipoprotein lipase activity in human post-heparin plasma. Most of these antisera did not inhibit rat or mouse lipoprotein lipase, but some showed a weak cross-reaction. This is illustrated in Fig. 1A for the antiserum TE. At the highest dose studied, i.e., equal volumes of antiserum and enzyme extract, this antiserum caused a 40% inhibition of lipoprotein lipase from mouse heart. This amount of antiserum was more than lOOO-fold that needed to effect the same inhibition of an equivalent amount of bovine lipoprotein lipase. In contrast to the rabbit antisera, several of the hen antisera

showed a fairly strong cross-reaction with rat and mouse lipoprotein lipase. Data for hen antiserum Hl 1 are shown in Fig. 1B. Here, only ten times more antiserum was needed to effect the same inhibition of mouse as of bovine lipoprotein lipase. Tracer

The iodinated bovine lipase used in this study had been repurified by chromatography on heparin/Sepharose; thus, it retained its ability to bind to heparin. It also retained its enzyme activity (Fig. 2) and its ability to bind to lipid (Table I and Refs. 9 and 10). The specific radioactivity was typically 1200 dpm/ng. This corresponds to approximately 0.2 mol of iodine per mol peptide chain. It should be noted that the active form of lipoprotein lipase is probably the dimer [ 11,121. Attempts to prepare tracer with higher specific activity gave preparations with high non-specific binding and reduced reactivity with the antibodies. With the preparations used more than 90% was precipitated by the antibodies (compare Figs. 2, 4

41

TABLE

I

OF ANTIBODIES EFFECT OF Fat, FRAGMENTS AGAINST LIPOPROTEIN LIPASE ON THE BINDING OF THE ENZYME TO LIPID DROPLETS 0.35 pg ‘251-labeled lipoprotein lipase was diluted in 370 pl buffer, pH 7.4, containing 0.5 mg heparin/ml and 10 mg bovine serum albumin/ml. 125 ~11Fab fragments, F, fragments or buffer only were added. After 20 min in an ice bath IOO-~1 aliquots were taken for assay of remaining enzyme activity, using IntraIipid as substrate and human serum as source of activator. The incubation was for 1 h at 37°C. Other ahquots were used to determine binding of the lipase to the lipid particles as described in Materials and Methods. The binding studies were carried out in duplicate. Addition

Lipoprotein lipase activity ( n mol fatty acid released/ml per h)

Fab fragments F, fragments Buffer

0.05 6.16 4.62

Binding (5%)

2.7, 3. I 6663 62,63

and 5). When the tracer was run on polyacrylamide gel electrophoresis in the presence of SDS, it gave a single radioactive band in the position expected for lipoprotein iipase (see Fig. 1 in Ref. 10). After reduction, faint radioactive bands were also seen in a position corresponding to about half the molecular size of the intact peptide chain. These bands probably correspond to enzyme nicked by proteolysis f lo].

Fig. 2. Comparison of the inhibition of enzyme activity and the immunoprecipitation of freshly prepared ‘*sI-labeled lipoprotein lipase. 38 ng ‘251-labeled lipoprotein lipase were diluted into 60 pl buffer, pH 7.4, at 4°C with the follovv?ng final concentrations: sodium phosphate, 20 mM; sodium chloride, 150 mM; heparin, 0.5 mg/ml; Triton X-100,0.5%; SDS, 0.1%; EDTA, 2 mM; phenylmethylsulfonylfluoride, 0.1 mM. 40 ~1 of antiserum or a logarithmic dilution of antiserum with nonimmune serum was added and the mixture incubated at 4’C for 16 h. Then 50 pl of the mixture was taken to assay the remaining enzyme activity, as described in Materials and Methods. Duplicate samples of 20 ~1 were diluted to 2 ml with the above-described buffer and 100 ~1 of a goat antiserum to rabbit or hen immunoglobulins added. 6 h later these samples were separated by centrifugation and radioactivity in the immunoprecipitate and in the supematant was determined. The % of total radioactivity in the precipitate is plotted. In parallel incubations with nonimmune serum (‘nonspecific binding‘) about 6X of the radioactivity was in the precipitate. This is subtracted. The enzyme activity is expressed as % of that in the incubation with nonimmune serum. No significant loss of activity occurred in this sample during the 16 h incubation. The activity was 1.63pmol fatty acid released in 2 h. 0, Lipoprotein lipase activity; 0, radioactivity. Three different antisera were used, as indicated in the figure.

Relation between reaction of tracer with antibody and inhibition of enzyme activity

Fig. 2 compares the inhibition of enzyme activity to the immunoprecipitation of the radioactive tracer. In all instances when this experiment was run with freshly repurified tracer the two parameters paralleled each other closely. This was true over the entire range, i.e., from more than 95% inhibition/immunoprecipitation to less than 10%. This is shown in Fig. 2 for three different antisera, namely the rabbit antiserum TE, which was later found to react more strongly with the denatured than with the native form of the enzyme, the rabbit antiserum A30, which was found to react more strongly with the native than the denatured form, and the hen antiserum H 11, which was used for the immunoassay shown in Fig. 6, These data

furnish strong evidence that the tracer was indeed pure lipoprotein lipase. In buffer solution lipoprotein lipase rapidly loses its catalytic activity [13-151, i.e., it is conformationally unstable. Special precautions have to be taken to avoid this loss of activity. Here we used a detergent mixture of Triton X-100 and SDS. This is based on previous observations that anionic detergents bind to and stabilize lipoprotein lipase 113-151. Whereas there was a close correspondence between the formation of antigen-antibody complexes and inhibition of enzyme activity for the native form of the enzyme, denatured enzyme did not compete effectively with the native enzyme for

42

ments (Table I). Thus, the inhibition was a direct consequence of the reaction between antigen and antibody and was not contingent upon immunoprecipitation. The data in Table I demonstrate that reaction with the Fat, fragments abolished the binding of the enzyme to triacylglycerol droplets, providing a plausible explanation for the inhibition of enzyme activity. The activity against the soluble substrate pnitrophenylbutyrate was not inhibited (data not shown). 30

loo

300

1000

30

100

300

1000

ng LipaseAdded

Fig. 3. Inhibition of lipoprotein lipase activity by antisera. Effect of different amounts of native or heat-inactivated lipase. Purified bovine milk hpase was diluted in the buffer described in Materials and Methods for extraction of mouse heart acetone/diethyl ether powders. Lipase was also denatured by heating in buffer with 1% SDS at 95°C for 5 min and then diluted with buffer and Triton X-100 to yield an equivalent concentration of lipase protein in the same buffer as for the native hpase. These stock solutions were then diluted at 4°C with buffer to yield solutions containing 30-1230 ng active lipase in 50 ~1 or 30 ng active lipase plus 0- 1200 ng denatured lipase. These solutions were then mixed with 50 pl of the same buffer without detergents but with either 0.40 ~1 antiserum TE or 1.60 gl antiserum 005. After 16 h at 4°C the remaining enzyme activity was determined. For each point a corresponding incubation with nonimmune serum was carried out. 0, Native

lipoprotein

lipase,

0, denatured

lipoprotein

Reactivity of native and denatured forms of the enzyme For these experiments a portion of the tracer was denatured by treatment with 6 M guanidinium chloride. This completely abolished the enzymatic activity and had a profound effect on the reaction with the antisera. With the rabbit antiserum TE

F

* 30

lipase.

the antibodies that cause inhibition of enzyme activity. This is illustrated in Fig. 3. For this experiment we selected an amount of antiserum which caused more than 90% inhibition of 30 ng lipase. When the amount of active lipase was increased the percent inhibition decreased, and at 1230 ng lipase there was less than 10% inhibition. In contrast, when heat-inactivated lipase was added to the active lipase there was no significant decrease in inhibition. With 30 ng active lipase and 1200 ng heat-inactivated lipase, inhibition was still more than 80%. This was true for both rabbit and hen antisera. It is also true for a goat antiserum to guinea pig lipoprotein lipase (Wallinder, L., personal communication). Mechanism of enzyme inhibition The enzyme activity was inhibited not only by reaction with whole antiserum but with Fab frag-

1

16

266

Fig. 4. Effect of denaturation on the reaction of lipoprotein lipase with three different antisera. For this an aliquot of the lipase was dialyzed stock solution of ‘25 I-labeled lipoprotein against 6 M guanidinium chloride at room temperature overnight, and then further dialyzed against 10 mM Tris/lS M sodium chloride, pH 7.4, to remove all the guanidinium chloride. This solution was then diluted into the buffer described in Fig. I to give 5000 cpm (10 ng) in I.9 ml. Native ‘251-labeled enzyme was also diluted in the buffer at 4’C to give the same concentration. These mixtures were aliquoted in test tubes and 10 ~1 antiserum or logarithmic dilutions of antiserum in nonimmune serum was added. After 48 h goat antiserum to rabbit or to hen immunoglobulins, respectively, was added and I6 h later the samples were separated by centrifugation. Radioactivity in the immunoprecipitate and in the supernatant was determined. 0, Native lipoprotein lipase; 0, denatured lipoprotein lipase. Note that the amounts of labeled lipase and of antiserum as well as the total volume differ from those in Fig. 1. As a consequence, the precipitation curves for native lipase are here shifted to the left.

43

(Fig. 4a) about 330 nl was required for 50% reaction with the native form of the tracer, but this amount decreased to only 20 nl when the tracer was denatured. Although such increased reactivity was found with most of the antisera, there were also antisera against which the native form was more reactive. This is illustrated in Fig. 4b with rabbit antiserum A30. With the hen antisera tested the denatured form was more reactive but the difference was not as marked as with the rabbit antiserum TE. Fig. 4c shows results with the hen antiserum Hll. Competition experiments Fig. 5 shows the displacement of ‘251-labeled lipoprotein lipase by unlabeled lipase. For this particular experiment a 1 : 6500 final dilution of hen antiserum HI 1 was used. This precipitated more than 95% of the tracer. A sigmoidal displacement curve was obtained over the range of 1- 1000

80

I

I

I

I

I

I

.l

1

10

100

1000

ng lipase added. The most sensitive portion of the curve corresponded to the addition of 5-200 ng lipase. Radioimmunoassay standard curve It was clear from the different reactivity of the native and denatured forms of the enzyme that in an immunoassay of lipoprotein lipase in biological material, the samples as well as the standards and the tracer should all be denatured. To accomplish this the materials were treated with SDS. Since the amounts of SDS required were not compatible with the immune reaction, their effect was neutralized by addition of sufficient amounts or Triton X-100 to form mixed micelles. At a weight ratio of Triton to SDS of 5 : 1 the immunoreactivity was almost as high as in the absence of SDS. The final conditions were set up to be 0.5% Triton and 0.1% SDS. This allowed inclusion of samples of up to 200 pl 1% SDS in a 2 ml assay system. The detergent mix was also required to decrease the non-specific binding, which in the absence of detergent was quite high. Fig. 6 shows the results of an assay run under these conditions and evaluated by the logit-log transformation. A linear standard curve was obtained over the range of 2-200 ng lipase. Values of assays on six identical samples differed from the mean value by 3.7 it 3.1% (mean f S.D.), thus providing a measure of intraassay variation. For one sample assayed in six consecu-

1

loo00

ng Lipase Added lipase from Fig. 5. Displacement of lz5 I-labeled lipoprotein antilipoprotein lipase antibodies by increasing amounts of unlab&d lipoprotein lipase. For this both the tracer and the unlabeled hpase were denatured as described in the legend to Fig. 3. The composition of the buffer was atso the same and 5 pl of hen antiserum HI1 was added. The samples were then incubated 48 h at 4°C and the second antibody then added. After further incubation overnight the samples were separated by centrifugation. Nonspecific binding was about 6%.

4

10

20

40

loo

,

ml

I

i

4cm

ml0

ng Lipase Added

Fig. 6. Radioimmunoassay was carried out as described

standard curve. This experiment in Materials and Methods.

44

tive assays the values differed 5.7 & 3.3% (mean + SD.).

from the mean

by

Discussion This paper describes some characteristics of the reaction of bovine lipoprotein lipase with its antibodies and how an immunoassay can be set up based on these experiences. The basic requirement for such an assay is to have either a mono-specific antibody or a sufficiently pure tracer which reacts in parallel with the unlabeled protein. It would seem that in the present system both these requirements are fullfilled. Several groups have found that antisera against lipoprotein lipase inhibit the enzyme’s activity [3-61. In our experiments denatured lipoprotein lipase did not compete effectively with the native enzyme for the antibodies responsible for inhibition of enzyme activity. Thus, these antibodies were directed against conformation-dependent determinants on the native enzyme. Recently, Shirai et al. [6] reported that monovalent Fat, fragments retain the ability to inhibit. They noted that preincubation of the enzyme with lipid substrates decreased inhibition by antibody [6]. We demonstrate here that Fab fragments abolish binding of the enzyme to triacylglycerol/phospholipid droplets and inhibit activity against these droplets, but not against soluble substrates. These observations indicate that the inhibiting antibodies interact with the enzyme as it exists in solution but do not directly affect the active site. The antibodies may cover the lipid-binding site on the enzyme. Another possibility is that the antibody-enzyme interaction impedes the conformational flexibility of the enzyme molecule, making it more difficult or impossible for it to go through the conformational transitions necessary for binding to lipid. That such conformational transitions take place has been shown for several other lipid-binding proteins [ 161, including some lipases [ 171. Another demonstration that the reaction of lipoprotein lipase with its antibodies is conformation-dependent was provided by the large differences in reactivity between native and denatured ‘251-labeled lipase protein. With most of our antisera, the denatured enzyme was more reactive, and in some cases the difference was quite large.

For instance, in Fig. 4 more than 15 times as much of the rabbit antiserum TE was needed to precipitate the same amount of native as of denatured tracer. We have not pursued these studies in detail now, but it is evident that the differing reactivity of different forms invites immunochemical studies of conformational transitions in the enzyme molecule. A main incentive for the present study was to develop an immunoassay for lipoprotein lipases. The purpose of such an assay would be to detect and quantitate inactive or subactive forms of the enzyme. The fully active form can be measured readily by its catalytic activity, and an immunoassay would probably not increase the sensitivity much. Our demonstration here that the immunoreactivity of active and inactive forms of the enzyme can be quite different, shows that it is necessary to ensure that all materials, samples, standard, as well as the tracer are in the same form. Here we chose to denature them by heating in SDS. This approach has the additional advantage that it will dissociate and bring into solution lipase associated with cell membranes, lipoproteins or otherwise complexed in biological samples. The deleterious effects of SDS on the reaction of antibody with antigen could be overcome by including Triton in the system. With this approach it was possible to find conditions where the bovine enzyme could accurately and readily be measured. Rabbit antisera showed only a weak cross-reaction, if any, with rat or mouse lipoprotein lipase. To obtain antisera with useful cross-reaction we had to use an animal other than the rabbit. Our choice of the hen was based on the presumption that an animal with a more distant evolutionary relation to mice, rats and cows would be more likely to detect immunological determinants common for these animals. With this approach, several useful antisera were obtained. Preliminary experiments indicate that it will be possible to develop immunoassays for rat, mouse and human lipoprotein lipases based on the hen anti-bovine antibody and the bovine tracer. However, the displacement curves are not parallel to that for the bovine lipase but are shallower. Thus, it will be necessary to relate the measurements to some arbitrary standard unit for each species. Furthermore, each ap-

45

plication will require careful consideration of how the samples are to be prepared. Nonetheless, the present methodology should make it possible to detect and quantitate inactive forms of lipoprotein lipase in studies of the regulation of its activity in experimental animals. Acknowledgements We thank Mrs. Asa Lundsten for her skilful technical assistance and Mrs. Marianne Lundberg for preparing the manuscript. This research was supported by the Swedish Medical Research Council (13X-00727), the Medical Faculty in Umea and the Swedish Margarine Industry Fund for Research on Nutrition. References Posner, I. (1982) in Atherosclerosis Reviews (Gotto, A.M. and Paoletti, R., eds.), pp. 123-156, Raven Press, New York Cryer, A. (1981) Int. J. Biochem. 13, 525-541 Cheung, A.H., Bensadoun, A. and Cheng, C.F. (1979) Anal. B&hem. 94, 346-357

4 Jansen, H., Garfinkel, AS., Twu, J.S., Nikazy, J. and Schotz, M.C. (1978) Biochim. Biophys. Acta 531, 109-114 5 Hernell, O., Egelrud, T. and Ohvecrona, T. (1975) Biochim. Biophys. Acta 381, 233-241 6 Shirai, K., Wisner, D.A., Johnson, J.D., Srivastava, L.S. and Jackson, R.L. (1982) Biochim. Biophys. Acta 712, IO-20 7 Bengtsson, G. and Olivecrona, T. (1977) Biochem. J. 167, 109-119 8 David, G.S. and Reisfeld, R.A. (1974) Biochemistry 13, 1014-1021 9 Bengtsson, G. and Olivecrona, T. (1980) Eur. J. Biochem. 106, 549-555 10 Bengtsson, G. and Olivecrona, T. (1981) Eur. J. Biochem. 113, 547-554 11 Iverius, P.-H. and Gstlund-Lindqvist, A.-M. (1976) J. Biol. Chem. 251, 7791-7795 12 Olivecrona, T., Bengtsson, G. and Osborne, J.C. (1982) Eur. J. Biochem. 124, 629-633 13 Fielding, C.J. (1968) Biochim. Biophys. Acta 159, 94-102 14 Baginsky, M.L. and Brown, W.V. (1977) J. Lipid Res. 18, 423-437 15 Bengtsson, G. and Olivecrona, T. (1979) B&him. Biophys. Acta 575, 471-474 16 Morrisett, J.P., Jackson, R.L. and Gotto, A.M. Jr. (1977) Biochim. Biophys. Acta 472, 93- 133 17 Verger, R. (1980) Methods Enzymol. 64, 340-392