WO2007058588A1

WO2007058588A1 - Monoclonal antibodies against apolipoprotein m

Info

Publication number: WO2007058588A1
Application number: PCT/SE2006/001291
Authority: WO
Inventors: Björn DAHLBÄCK
Original assignee: T.A.C Thrombosis And Coagulation Aktiebolag
Priority date: 2005-11-18
Filing date: 2006-11-15
Publication date: 2007-05-24

Abstract

The invention describes monoclonal antibodies that have the capacity to immunoreact with human apolipoprotein M (apoM). Such antibodies can be used in methods for determining apoM and also in methods for purification of apoM or enrichment of apoM-containing lipoprotein particles.

Description

Monoclonal antibodies against apolipoprotein M

Technical Field

The present invention is concerned with apolipoproteins and more specifically with apolipoprotein M and methods to determine apolipoprotein M in a sample or to purify and/or recover this protein e.g. from blood or plasma.

The present invention is also concerned with monoclonal antibodies (MAbs) raised specifically against apolipoprotein M (apoM). Such MAbs can be used in methods devised to determine apolipoprotein M in a sample. Such MAbs could also be used to purify and/or recover plasma-derived or recombinant apoM.

Moreover, the present invention is related to use of the test results from an assay devised to determine, e.g. to detect and quantify, apoM in the prognosis for, or for prediction of, individuals at risk for atherosclerosis or other diseases, such as diabetes, cardiovascular diseases, hyperlipidemia, metabolic syndrome, obesity, hypertension etc.

Background

The modern western diet is rich in lipids, which provides a rich energy source. In situations with high demand of calories, such as during strenuous physical work, this is physiologically appropriate. However, during modern life, a high lipid intake is converted to a risk factor for diseases such as atherosclerosis, obesity, metabolic syndrome, hypertension and diabetes.

After uptake of dietary lipids they are transported in blood in lipoproteins, which are particles of variable size formed by different combinations of apolipoproteins phospholipids, cholesterol and triglycerides. The lipoproteins are crucial for the transport in blood of the hydrophobic lipids as well as lipid-soluble compounds, such as vitamins. In blood, the major lipids, i.e. cholesterol and triglycerides (TG), are transported in such lipoproteins. The lipoprotein particles vary considerably in size and composition but in principle they have the same basic architecture. The surface is composed of single layer phospholipid and different apolipoproteins. The centre contains the hydrophobic lipids triglycerides and cholesterol- esters. In accordance with the densities at which they are isolated, the five major lipoprotein classes are defined as high-density lipoproteins (HDL), low-density lipoproteins (LDL), intermediate- density lipoproteins (DDL), very low-density lipoproteins (VLDL), and chylomicrons. HDL and LDL are rich in cholesterol, whereas TG constitutes the major lipid in the remaining subclasses. The specialized apolipoproteins play major roles in the structural organisation and intravascular metabolism of the lipoproteins. The apolipoproteins are mainly synthesized in the liver and in the intestine. There are several different apolipoproteins that all fulfil important functions. They are involved in specific binding of lipoproteins to cellular receptors, in the regulation of lipolytic enzymes and in the process of lipid exchange and transfer. The apolipoproteins are denoted with letters A, B, C etc. There are five distinct but related apolipoprotein A variants denoted apoAI-V, two variants of apolipoprotein B (apoB 10 and apoB48), three alipoprotein C variants denoted apoCI-CIII. Before apolipoprotein M was discovered, the last apolipoprotein to be identified was apolipoprotein L.

Plasma lipoproteins play essential roles in formation of atherosclerotic lesions. It is widely accepted that whereas high plasma levels of apolipoprotein B-containing lipoproteins, i.e. VLDL, IDL, LDL, and lipoprotein(a), promote atherosclerosis, high plasma concentrations of apoAI-containing HDL protects against atherosclerosis. The apoB-containing lipoproteins synthesized in the intestine transport TG and cholesterol to the liver whereas apoM-containing lipoproteins made in the liver transport the lipids from the liver to peripheral tissues. In contrast, HDL is important for reverse cholesterol transport, i.e. the transport of cholesterol from periferal cells to the liver. ApoAI has a key role in this reverse cholesterol transport, since it activates lecithin-cholesterol acyl transferase (LCAT) and interacts with the ATP- binding cassette transporter Al (ABCAl). HDL particles may contain other apolipoproteins, e.g. apoAII, apoAIV, apoAV, apoCI-III, and apoE and the protein composition of HDL in plasma is highly heterogeneous. The other apolipoproteins serve different functions. For instance, apoAV and apoCII stimulate lipoprotein lipase, and apoE interacts with lipoprotein receptors.

The present invention is related to apolipoprotein M (apoM). This protein was first reported in 1999 (Ref. 1). Apolipoprotein M (apoM) is a plasma protein predominantly associated with high-density lipoproteins (HDL). To a minor extent, it is also present in triglyceride (TG)-rich lipoproteins, such as chylomicrons and very low-density lipoproteins (VLDL), and in low- density lipoproteins (LDL). The mature human apoM protein contains 188 amino acid residues, predicting a molecular weight of 21 kDa. The majority of the apoM protein present in human plasma contains an N-linked carbohydrate side chain. Based on estimated values for the plasma concentration of apoM, it can be concluded that, in plasma, apoM is present only in a subpopulation of HDL particles. However, the physiological significance of apoM being present in a minor fraction of the HDL particles is unknown.

An unusual property of apoM is that its signal peptide is retained in the mature circulating protein. During synthesis of secreted proteins, the signal peptide directs the nascent peptide chain through the phospholipid of the endoplasmic reticulum (ER). hi almost all proteins, the signal peptide is cleaved off by a signal peptidase that reacts with a conserved recognition site at the end of the signal peptide, hi contrast, the signal peptidase cleavage site is lacking in apoM and, therefore, the signal peptide remains present in the mature protein. It is likely that the retained signal peptide acts as a hydrophobic anchor, which serves to localize apoM to the single phospholipid layer of the lipoproteins.

The physiological relevance of apoM is demonstrated by the observed increased risk of atherosclerosis and diabetes among individuals having reduced levels of apoM in blood and plasma. Assays for measuring blood or plasma levels of apoM could, thus, become an important tool for the clinician. For instance, the outcome of such assays could be used to screen individuals at risk of developing atherosclerosis or diabetes. ApoM is in fact regarded as an anti-atherosclerotic protein.

Previous methods, e.g. based on immunob lotting (Western blotting) using a standard control serum (Ref. 2), for measuring the levels of apoM in blood, plasma or serum are tedious and not easily automated and do not easily provide accurate and reliable results. In the described assays, samples containing apoM have been subjected to SDS-polyacrylamide gel electrophoresis, the proteins then after the electrophoresis being electrophoretically transferred to a membrane, which has been incubated with polyclonal anti-peptide antibodies made in rabbits. The immunereactive bands have then been estimated and used for calculation of the apoM level in the original sample. Western blot techniques are not suitable for quantification of proteins due to the number of steps and the inherent problems with variability in each of these steps. Dot-blotting have also been used in efforts to quantify the apoM concentration in plasma. In this technique, the apoM-containing samples are directly applied to membranes and the amount of immobilized apoM has been estimated with polyclonal anti-peptide antibodies raised in rabbits. The dot-blot technique is even more unreliable than the western blot technique, in particular as apoM is only a minor component of the sample and there is no specificity in the immobilization of apoM to the membrane. The amount of immobilized apoM varies with the protein content of the samples as the immobilization process is indiscriminating and all proteins bind, but not quantitatively. Thus from a plasma sample mainly albumin and immunoglobulins are immobilized on the membrane.

Accordingly, there is a need for methods that could be used to measure by routine the level of apoM in a sample or to purify and/or recover apoM from a fluid sample. There is also a need for antibodies, preferably monoclonal antibodies (MAbs), which have the capacity to immunoreact specifically with apoM and, thus, could be used in such methods that comprise immunological apoM assays.

Brief Summary of the Invention

According to one embodiment, the present invention is directed to monoclonal antibodies (MAbs) that have the capacity to immunoreact specifically with apoM. Suitably, such MAbs have been raised against apoM using a recombinant truncated human apoM that lacks the signal peptide, as antigen in mice to provide MAbs that have the capacity to immunoreact with human apoM.

Such MAbs can be used to devise immunological assays for the detection and quantification of apoM in plasma. These MAbs can also be used for other purposes, e.g. to purify plasma- derived or recombinant apoM.

Accordingly, the present invention is also directed to a method for detection and quantification of apoM in an assay sample comprising a biological fluid, such as blood, plasma or serum. According to a further aspect, the present invention is directed to a method to purify plasma-derived or recombinant apoM to provide apoM that is free from other proteins.

An additional embodiment of the present invention is directed to use of an apoM value obtained with the present assay method in a screening method based on the anti-athero- sclerotic activity of apoM, which indicates that a reduced level thereof would predict an increased risk for an individual to be afflicted by atherosclerosis. A reduced apoM value could also predict that the individual is at risk for diabetes.

The present invention also relates to hybridomas producing MAbs having the capacity to immunoreact with human apoM and to a selective adsorbent for human apoM comprising an insoluble solid carrier and a MAb of the present invention operatively linked thereto.

Brief Description of the Drawings

Figure 1 illustrates the results of Western blot analysis of apoM in isolated human HDL. HDL was isolated from human plasma using traditional ultracentrifugation and applied (1 μl in each lane) to a Western blotting procedure both at non-reducing (lanes marked 1) and at reducing (lanes marked 2) conditions. The presence of apoM in the HDL is demonstrated with the three monoclonal antibodies apoM23, apoM42, and apoM58 of the present invention. The apoM bands in the non-reducing lanes are highlighted with asterisks, whereas the arrowheads point at reduced apoM. These three monoclonal antibodies reacted strongly with the non- reduced apoM but only faintly with the reduced protein.

Figure 2 illustrates the results of ELISA for assaying apoM. During the development of an ELISA method, various monoclonal anti-apoM antibodies of the present invention were immobilized at the bottom of microtiter plates; different dilutions of human plasma were added; and apoM present in the samples was bound to said antibodies. Bound apoM was detected with a second biotinylated monoclonal antibody against apoM. The combination of immobilizing apoM58 and using biotinylated apoM42 and then streptavidin-HRP was found to give a useful dose-response curve. In contrast, apoM23 was found not to be effective if used in combination with apoM58.

Figure 3 illustrates results obtained with purified apoM-containing lipoprotein particles and isolated apoM protein from human plasma. The two lanes to the left demonstrate apoM- containing particles, which have been isolated from human plasma with a combination of ion exchange chromatography and affinity chromatography on a Hi-trap with coupled apoM23. The apoM bands are highlighted with asterisks, whereas apoAI is indicated with an arrowhead. The other weak bands are other apolipoproteins present in the apoM-containing lipoprotein particle. The sample to the left is analyzed under non-reducing conditions, whereas the sample to the right is reduced. The two samples to the right represent the apoM protein obtained from the Hi-trap column after washing the column with detergent (Triton X- 100, 1% in the buffer) before eluting the bound apoM with low pH. The protein is analyzed under non-reducing conditions (left) and after reduction of disulfide bridges (right). The asterisks indicate the apoM-specific bands.

Detailed Description of the Invention

A. Monoclonal Antibodies

According to one embodiment, the present invention is related to monoclonal antibodies that have the capacity to immunoreact specifically with human apoM.

A further embodiment of the present invention is concerned with such monoclonal anti-apoM antibodies that have the capacity to immunoreact specifically with human apoM and comprise monoclonal antibodies that have been raised in mice using apoM, suitably truncated recombinant apoM that lacks the signal peptide, as antigen.

A suitable embodiment of the present invention is directed to a monoclonal antibody that immunoreacts specifically with human apoM and has been raised against recombinant, truncated apoM lacking the signal peptide comprising the N-terminal amino acid residues 1- 20 of apoM but comprising amino acid residues 22-188 of mature apoM.

The monoclonal antibodies of the present invention are of murine origin and, more specifically, they are derived from mice. According to a suitable embodiment of the present invention, the MAbs have the capacity to immunoreact both with apoM associated with lipoproteins and with lipoprotein-free apoM.

A suitable embodiment of the invention is concerned with monoclonal antibodies that have been isolated and characterized as disclosed below. Specifically, three monoclonal antibodies designated apoM23, apoM42, and apoM58, respectively, have been produced and the corresponding hybridomas are available. Accordingly, the present invention is also concerned with hybridomas producing MAbs of the present invention, and specifically with the hybridomas that are available as stated above.

B. Assay Methods

The MAbs of the present invention have been used to develop assay methods for the detection and quantification of human apoM in a biological fluid, such as blood, plasma, or serum, or in a tissue sample.

Accordingly, the present invention is concerned with assay methods for determining the presence and amount of lipid-free apoM or the presence and amount of apoM associated with lipoprotein particles in an assay sample comprising a biological fluid, such as blood, plasma, or serum, using a first polyclonal, or suitably monoclonal, antibody of the present invention as a catcher antibody to form an immunoreaction product, the amount of which is related, either directly or indirectly, to the amount of apoM in said sample and using a second antibody, suitably a monoclonal antibody of the present invention to detect and determine apoM. Such methods can be non-competitive or competitive and at least one MAb of the present invention is used as antibody in said method. Suitably, at least the catcher antibody is a MAb of the present invention. The Mabs can also be used to determine the presence and amount of apoM in tissue samples using immune-histochemical techniques or extracts of the tissue.

(1) Capture Immunoassay

According to one embodiment, the present invention is related to a method for determining the level of human apolipoprotein M (apoM) in an assay sample comprising a biological fluid, such as blood, plasma, or serum, said method comprising the steps of

(a) contacting a first (catcher) antibody, which immunoreacts with human apoM, with said assay sample to form a first immunoreaction mixture comprising at least a liquid phase,

(b) maintaining said first immunoreaction mixture for a time period sufficient for human apoM in said assay sample to bind to said first antibody thereby to form a first immunoreaction product comprising apoM, (c) contacting said first immunoreaction product formed in step (b) with an indicating means- containing second (detecting) antibody that has the capacity to immunoreact with said first immunoreaction product comprising human apoM and form a second immunoreaction mixture,

(d) maintaining said second immunoreaction mixture for a time period sufficient for said second antibody to immunoreact with said first immunoreaction product of step (b) and form a second immunoreaction product, and

(e) determining the level of said indicating-means containing second antibody present in said second immunoreaction product formed in step (d), and thereby the level of apoM in said assay sample, and

wherein at least one of said first and second antibodies is a monoclonal antibody having the capacity to immunoreact with human apoM.

According to a suitable embodiment, said first antibody of step (a) is operatively linked to a solid carrier; said first immunoreaction mixture formed in step (b) is comprised of a liquid phase and a solid phase; and said first immunoreaction product formed in step (b) is contained in the solid phase which is linked to the carrier.

A further aspect of the present invention is related to a method wherein said indicating means is operatively linked to or incorporated in said second antibody and is capable of producing, directly or indirectly, a detectable signal at the formation of said second immunoreaction product in step (d), and wherein said second immunoreaction product formed in step (d) comprises said indicating means and the determination in step (e) comprises measuring the amount of indicating means in said second immunoreaction product. Suitably, said indicating means-containing second antibody comprises an enzyme, a luminescent substance or a radioisotope as indicating means.

A further aspect of the present invention is directed to the present method wherein said indicating means-containing second antibody and the assay sample are simultaneously contacted with said first antibody which is bound to an insoluble solid carrier. Suitably, said first antibody is a monoclonal antibody and, preferably, also said second antibody is a monoclonal antibody. In accordance with a suitable embodiment, both said first and said second antibody is a monoclonal antibody selected from the group consisting of monoclonal antibodies designated apoM 58, apoM 42, and apoM 23 and produced by hybridomas having the accession numbers respectively, provided that said first and said second monoclonal antibodies are comprised of different monoclonal antibodies selected from the above-mentioned group of monoclonal antibodies.

A further aspect of the invention is related to the present methods wherein, in a pre-step, detergent is added to the assay sample to release apoM contained in the assay sample from lipoprotein.

Another possible embodiment is to measure the amount of lipoproteins containing a combination of apoM and another apolipoprotein, e.g. apoAI, apoB, apoC etc. In such an assay, the Mabs against apoM can be used to catch lipoproteins containing apoM and then imune-reagents against one of the other apolipoproteins can be used, e.g. anti-apoAI, ant- apoB etc. The quantitative measurement of such apoM containing lipoproteins may prove to have value in the diagnosis and or handling of different diseases.

(2) Competition Immunoassay

A further embodiment of the present invention is related to assaying the amount of apoM in an assay sample comprising a biological fluid, such as blood, plasma, or serum, utilizing a competition reaction in which an anti-apoM antibody of the present invention is present as an immunochemical reagent immobilized in a solid phase and labelled apoM is present in solution in the liquid phase. A fluid assay sample is contacted with the solid phase reagent to form a competition immunoreaction mixture and the resulting amount of label in the solid phase is proportional, either directly or indirectly, to the amount of apoM in the fluid assay sample.

Accordingly, the present invention is related to a method for determining the level of human apolipoprotein M (apoM) in an assay sample comprising a biological fluid, such as blood, plasma, or serum, said method comprising the steps of (a) bringing said assay sample into contact with an antibody, which is operatively linked to a solid carrier and has the capacity to immunoreact with human apoM, and with labelled human apoM to form a competition immunoreaction mixture comprising a liquid phase and a solid phase,

(b) maintaining said competition immunoreaction mixture for a time period sufficient for human apoM and labelled human apoM present in the liquid phase to compete for immunoreaction with said solid phase antibody to form an immunoreaction product containing labelled apoM in the solid phase, and

(c) determining the level of labelled apoM present in said immunoreaction product formed in step (b) and thereby the level of apoM in said assay sample, and

wherein said antibody of step (a) is a monoclonal antibody.

A suitable embodiment of the present invention is related to the above method wherein said monoclonal antibody is selected from the group consisting of monoclonal antibodies designated apoM58, apoM42, and apoM23 and produced by hybridomas having the accession numbers and wherein further labelled apoM contains a radioisotope label.

C. Immunoaffinity Purification

Since the monoclonal antibodies of the present invention immunoreact specifically with human apoM, such antibodies can be used as a reagent for purifying apoM from a complex biological fluid, such as blood, plasma, or serum, or other sources of apoM. In addition, such antibodies can be used to purify apoM from expression systems for the production of apoM using recombinant DNA methods for expression of apoM-encoding genes.

In a similar manner, the present monoclonal antibodies can be used for purification and enrichment of apoM-containing lipoproteins. Whereas the method for purification of lipoprotein-free apoM from a biological fluid requires a step comprising addition of detergent to said fluid before or after immunoreaction of said fluid with the first antibody used in the present methods, enrichment of apoM-containing lipoproteins is obtained in absence of detergent. Accordingly, the present invention is also related to a method for purifying and recovering human apoM from an aqueous solution that contains human apoM, which method comprises

(a) contacting said solution with a monoclonal antibody of the present invention that specifically immunoreacts with human apoM to form an immunoreaction mixture comprising at least a liquid phase,

(b) maintaining said immunoreaction mixture for a time period sufficient for said antibody to immunoreact with human apoM and form an immunoreaction product, and

(c) recovering the immunoreaction product formed in step (b) from the immunoreaction mixture, thereby producing purified human apoM.

According to a suitable embodiment of the present invention, said antibody is operatively linked to a solid carrier such that the immunoreaction mixture has a liquid phase and a solid phase and the imunoreacton product formed is contained in the solid phase, and said recovering in step (c) comprises eluting human apoM contained in the solid phase immunoreaction product off of the solid phase and into the liquid phase using an elution buffer or chaotropic salts, optionally after washing the solid phase containing said immunoreaction product with detergent.

D. Reagents and Compositions

The present invention is also related to a reagent system for immunological determination of human apoM in an assay sample comprising

a) a primary catcher antibody bound to an insoluble solid carrier, and

b) an indicating means-containing secondary detecting antibody,

wherein one of or both the primary and secondary antibodies is (are) monoclonal antibodies of the present invention. Suitably, said secondary detecting antibody is a monoclonal antibody containing an indicating means which is an enzyme, a fluorescent group, a luminescent substance or a radioisotope. Optionally, the first and also the second antibody can be bound to a solid phase such as a latex particle or other type of particle, which can also be magnetic.

According to a suitable embodiment of the present invention, said insoluble solid carrier is a plastic receptacle, a microtiter plate, plastic beads, glass beads or metal particles.

The present invention is also related to an immunoaffmity composition for purifying human lipid-free apoM or apoM-containing lipoproteins from an aqueous solution, said composition comprising an insoluble solid carrier and a monoclonal antibody of the present invention operatively linked to said carrier. Suitably, said solid carrier is a carrier as described above.

E. Diagnostic Use

As stated in the Background Section, the level of apoM in blood or plasma is relevant to assessment of the risk of an individual to be afflicted by atherosclerosis and/or diabetes, e.g. M0DY3 (Ref. 2). The levels of other apolipoproteins, such as apoAl and apoB, are also relevant and the level of such proteins can be measured individually with commercially available techniques. More specifically, ApoAl correlates well with the HDL cholesterol level, whereas apoB correlates with LDL cholesterol. Thus, it might be useful to make comparisons between the level of apoM and the levels of other apolipoproteins or cholesterol fractions in the analysis of patient samples. For instance, such comparisons could comprise calculating the ratios between apoM and apoAl or apoB. According to the present invention, a relatively high correlation between HDL cholesterol and apoM has been found. Since a high HDL cholesterol level is considered to be a marker for protection against atherosclerosis, measurements of apoM and calculations of ratios (e.g. apoM/apoAl; apoM/apoB; apoM/HDL chol; apoM/LDLchol) can prove to be highly useful for the prediction of risk for atherosclerosis or other diseases. It might also be useful to measure the amount of complexes between apoM and the other apolipoproteins, i.e. the amount of lipoprotein particles carrying both apoM and another apolipoprotein, e.g. an apoA variant, apoB or another apolipoprotein. Examples

In the following description, particular embodiments of the present invention are disclosed in detail. However, this description is only illustrative and should not be construed as limiting the invention.

Unless otherwise specified, all references to apoM in the Examples refer to human apoM.

Example 1. Production of MAbs

A) Production of Recombinant apoM for Use as Immunogen

ApoM in human plasma is tightly bound to the lipoprotein particles and it is challenging to purify the apoM free of other proteins. Therefore, recombinant techniques were used to produce apoM that was used for the production of poly- and monoclonal antibodies. The isolation of cDNA clones for human apoM has been described previously (Ref. 1). The cDNA was used to construct an expression vector for prokaryotic expression. As the signal peptide (amino acids 1-20 in apoM) is very hydrophobic, this part of apoM was deleted from the expression construct. Thus, the truncated apoM corresponding to amino acids 22-188 were included in the construct. The cDNA corresponding to these amino acids was introduced in a pET-30 Xa/Lic vector from Novagen. This vector contains an N-terminal His-tag, useful for the purification of the protein, followed by an S-tag, which is useful for the immune-detection of the recombinant protein. Between the His- and S-tags, there is a thrombin-cleavage site and after the S-tag, an FXa-cleavage site is located just before the apoM sequence. The amino acid sequence of the construct is presented in Ref. 1, which sequence has been deduced from the nucleotide sequence NCBI accession number AFl 18393.

This cDNA construct was used to transform bacteria and to express the recombinant apoM, as detailed in Example 6. Thereafter, the apoM was recovered from the inclusion bodies, purified on a Ni-column and refolded, also as detailed in Example 6.

B) Production of MAbs specific for human apoM The refolded recombinant human apoM was used to raise monoclonal antibodies using standard hybridoma technologies. In brief, two BALB/c mice were immunized with the recombinant human apoM through subcutaneous injections received repeatedly over several months (initial immunization and subsequent boostering). Blood was drawn from the mice and tested for antibody titer against the recombinant apoM protein using a microtiter-based assay. In this assay, recombinant apoM was immobilized in the microtiter plates by normal coating procedure, the plates were quenched according to standard procedures and the serum from the mice were added in different dilutions to the wells.

The bound antibodies were detected with an enzyme-labelled secondary antibody using standard procedures. The mouse giving the best antibody titer was selected for fusion with a myeloma cell line (SP/20-Agl4) and monoclonal antibody production using standard procedures. The clones were screened with the same microtiter plate assay that was used for the serum antibody titration, i.e. with immobilized recombinant apoM. Positive clones were recloned once or twice and when the cell lines were stable, the clones were expanded and the monoclonal cell line was cultured to collect medium for purification of the antibodies.

Three hybridomas producing antibodies reacting on Western blotting with both the recombinant protein and with apoM present in human HDL were identified and the antibodies produced by these hybridomas were further characterized as disclosed in Example 2. These antibodies that were named apoM23, apoM42 and apoM58 were produced and purified using standard procedures. The purification was done on commercially available (from Amersham) columns with protein A and protein G. As stated in the detailed description of the invention, these hybridomas are available.

Example 2. Characterization of monoclonal antibodies

The three antibodies produced in Example 1 all reacted well with recombinant and plasma- derived apoM on Western blotting (Fig. 1). The reactivity was primarily against unreduced apoM, whereas apoM was hardly recognized after reduction of disulfide bridges, suggesting that the epitopes for all three antibodies were conformation dependent. The three antibodies did not react with apoM in mouse plasma suggesting specificity for the human apoM. The three antibodies also reacted well with immobilized apoM in a microtiter-based analysis. In this assay, recombinant apoM (10 μg/ml) was used to coat microtiter plates using standard procedures and the diluted biotinylated monoclonal antibodies were added to the wells. After a couple of hours of incubation, unbound monoclonal antibodies were washed off and the bound antibodies were detected with streptavidin-HRP (horse radish peroxidase) technique using standard methods.

In an effort to devise an ELISA method for human apoM, the competition for apoM between the three antibodies was tested (Fig. 2). In this assay, the three antibodies were individually immobilized in microtiter plates using standard procedures.

Diluted plasma was added as a source of apoM. The buffer used was 50 mM Tris-HCL, 0.15 M NaCl, pH 7.5 containing 1% bovine serum albumin (BSA) and 1% Triton X-100. The purpose of using Triton was to solubilize the lipoprotein particles in order to liberate the bound apoM. The plasma dilutions were incubated in the microtiter plates over night at room temperature and then the individual biotinylated MAbs were added. In this way, each antibody was tested as catcher antibody and as detecting antibody. The bound biotinylated antibodies were detected with streptavidin-HRP conjugate.

It was found that the MAb designated apoM58 was superior as catcher and could only be combined with the biotinylated monoclonal antibody apoM42, whereas the apoM23 antibody was unable to detect the bound apoM, presumably due to overlapping epitopes between apoM58 and apoM23.

The use of detergent (Triton X-100) in the dilution buffer was found to be optional as essentially similar dose-response curves were obtained in the absence and presence of the detergent. This suggests that apoM is available for the antibodies also when being a constituent of the lipoprotein particle.

Example 3. Enzyme Linked Immuno Sorbent Assay for human apoM

One of the monoclonal antibodies (preferably apoM58) was immobilized in microtiter plates using standard procedures. The diluted plasma samples and a standard derived from pooled normal plasma were added to the different wells and after incubation and washing, the second monoclonal antibody (preferably apoM42-biotinylated) and the corresponding detection system (streptavidin-HRP) was used to detect the amount of bound apoM.

The standard curve was derived from diluted pooled normal plasma (Fig. 2). The dilutions are between 1/500 and 1/8000. The plasma to be tested was diluted 1/500, 1/100 or 1/2000. The standard curve is sigmoidal and a four-parameter curve fit (least square) was made. When samples from human blood were tested, the samples were provided in the form of lithium- heparin plasma, EDTA plasma or serum, which gave similar results. Citrated plasma gave somewhat lower values, which was due to the dilution provided by the fluid citrate.

Assay range: The standard dilution of 1/2000 yields a test range of 25-200%. Values outside this range can be retested after appropriate dilution.

Specificity of ELISA: Both monoclonal antibodies (apoM58 and apoM42) are specific for human apoM and reacts specifically with apoM on Western blotting. The antibodies have been raised with recombinant apoM as antigen. This ELISA was used to analyze plasma before and after ultracentrifugation. The apoM was mainly recovered in HDL (density 1.063- 1.21) and to a minor fraction in the other lipoprotein classes. The lipid-free plasma remaining after the last centrifugation was completely depleted of apoM suggesting that no other plasma component apart from the lipoprotein-associated apoM is measured.

Recovery of added apoM: When the truncated, recombinantly expressed apoM was added to plasma to a final concentration of 10 μg/ml (mg/L), the total apoM level increased with around 80%. Moreover, use of highly purified apoM from human plasma, the concentration of which had been determined with amino acid analysis, suggested the plasma concentration to be around 20 μg/ml (mg/L).

Accuracy: Intra-assay CV at 100% was 4.9. Inter-assay CV at 100% was 5.0.

In Example 4, a detailed protocol for one embodiment of the present ELISA procedure is described.

Exempel 4 ApoM Sandwich ELISA Protocol

1. Microtiter plates were coated with 10 μg / ml of monoclonal antibody M58, 100 μl / well.

2. Quenching buffer, 200 μl / well, of 2 % BSA in TBS (50 mM Tris - HCl, 0.15 M NaCl, pH 7.4) was added and incubated for at least 1 h at RT or over night at +4⁰C. 3. For each plate, a standard curve was made in 1% BSA in TBS + 1% Triton X-100.

4. Samples were diluted (e.g. 1 :2000) in the same buffer as for the standard curve and 100 μl / well were added and incubated at room temperature (RT), e.g. over night.

5. After washing, biotinylated monoclonal antibody M42, 100 μl of 1 μg/ml in 1% BSA in TBS + 0.1 % Triton X - 100 was added and incubated at RT for 2 hours. 6. After washing, 100 μl/well of Streptavidin complex with HRP (horse radish peroxidase) diluted in 1% BSA in TBS + 0.1 % Triton X - 100 was added and incubated at RT for 30 min.

7. After washing, 100 μ 1/ well of substrate (OPD ortho phenylene diamine) was added and after appropriate color development (6 min), the reaction was stopped by addition of 100 μl Of I M H₂SO₄

The absorbance at 490 nm was measured.

Example 5. Purification of human apoM

The antibodies produced in Example 1 were also used to develop a purification procedure of apoM from human plasma. In this case, the individual monoclonal antibodies were coupled to Hi-trap columns (Amersham) at a density of approximately 10 mg antibody per ml gel. The columns were able to bind apoM-containing particles present in plasma. Plasma or serum was applied directly to the columns or the apoM-containing lipoproteins were partially purified prior to application to the affinity column. In this case, the different lipoproteins could be isolated from human plasma by ultracentrifugation at different densities. To isolate the different lipoprotein classes, such as chylomicrons, VLDL, LDL, and HDL, is a standard procedure. Alternatively, the plasma can be fractionated using other chromatography methods, such as ion exchange chromatography or gel filtration chromatography. Regardless of the initial fractionation procedure for the lipoprotein containing apoM, the affinity columns comprising monoclonal antibodies against apoM were able to catch the apoM- containing lipoproteins. The bound proteins were eluted with buffers giving low pH values (e.g. pH 2.7) or with chaotropic salts (e.g. guanidine). When the procedure was performed in the absence of detergents, the affinity columns enriched apoM-containing lipoproteins. These purified lipoproteins contained other apolipoproteins such as apoAI, apoAII, apoCl-III, apoJ etc. Thus, these particles were similar to other HDL particles. If the affinity columns were washed with detergent before elution of the bound apoM, the purified apoM was devoid of other apolipoproteins.

Accordingly, the affinity columns containing the monoclonal antibodies apoM23, apoM42, or apoM58 were able to yield highly purified apoM-containing particles or highly purified apoM protein, depending on whether or not detergent is used to wash away other lipoprotein constituents such as lipids and other apolipoproteins. The protein content of the isolated apoM particle and the isolated apoM protein are illustrated in Fig. 3.

Example 6. Protocol for Preparation of Recombinant apoM

A. Transformation of BL21 (DE3) chemically competent E. coli for protein expression and purification

Reagents: BL21 (DE3) Competent Cells, catalogue #200131, STRATAGENE

DNA construct

LB-antibiotic agar plates (RT)

Falcon 2059 polypropylene tubes Protocol: 1. Gently thaw E. coli on ice. Add 100 μl to a pre-chilled Falcon tube.

2. Transfer DNA to competent cells (50-100 ng/ml). Swirl gently to mix and incubate on ice for 30 minutes. Not by pipetting or vortexing. (Remaining ligation reaction can be stored at -20°C)

3. Heat pulse the transformation reaction for 5 minutes at 37°C (water bath; do not mix or shake) and then place the reaction on ice for 2 minutes.

4. Add 1.0 ml LB and incubate the transformation reaction at 37°C for 1 hour with shaking at 225-250 rpm to allow the production of resistance proteins. 5. Immediately plate the transformation reaction onto LB agar plates supplemented with selective antibiotics (e.g. Ampicillin 10 μg/ml). Plates should have been dried and pre-heated at 37⁰C before use.

6. Incubate at 37°C for >16 hours (Turn Petri dish upside down to avoid condensation on agar.)

7. Pick colonies from each plate and transfer each colony to 3 ml LB medium supplemented with selective antibiotics (in sterile Falcon tubes) respectively.

8. Inoculate at 37°C, 225-240 rpm for 12-16 hours.

B. Expression and purification of recombinant human apoM

9. Inoculate 1 colony (monoclone) in 5 ml LB + ampicillin (final 100 μg/ml) medium at 37⁰C, 240 rpm, for about 6-8 hours. Then transfer 1 ml of this cell suspension in 50 ml medium under the same conditions O/N. 10. Transfer 10 ml O/N culture to 1 L fresh medium, and inoculate at above conditions until cell log phase (absorbance 600 nm between 0.6-0.8, about 2.5 h). 11. Induce the expression of the recombinant protein by 1 mM IPTG at 37°C for 4 hours.

12. Centrifuge the culture at 5000 rpm, 4⁰C for 20 minutes and discard the supernatant.

13. Wash the pellet twice in less than totally 200 ml cold TBS. Keep the pellet on ice all the time.

14. Transfer the solution to sterile Falcon tubes and centrifuge again as above. Discard the supernatant.

15. Store washed pellet at -2O⁰C O/N (over night).

16. Re-suspend the pellet in approximately 40 ml TBS.

17. Add lysozyme (final 100 μg/ml) and 0.5M Benzamidin. Benzamidin inhibits enzyme degradation of the proteins. 18. Leave samples at RT for 30 minutes to 1 hour to allow breakdown of bacterial cell walls.

19. Sonicate samples on ice to a clear, non- viscous solution (about 30 minutes, 20 sec on, 5 sec off, level 4.5 per aliquot). 20. Centrifuge at 5000 rpm for 15 minutes at 4°C - and separate supernatant (soluble fraction) and pellet (insoluble fraction; i.e. inclusion bodies).

21. Wash the pellet in TBS twice, centrifuge as above.

22. Solubilize inclusion bodies in denaturing extraction buffer (6 M Guanidine-HCl, 20 mM Tris (pH 8), 10 mM reduced Glutathione) by shaking ON at RT.

23. Centrifuge the "clear" solution at 12000 g for 90 minutes. No sonication step is needed at this step of purification. The pellet obtained after centrifugation represents cell-debris whereas the "clear" supernatant contains soluble recombinant mouse/human apoM. To avoid a loss of protein, do not sterile filtrate the material before loading it on the Nickel column.

24. Equilibrate a 25 ml Ni-NTA Superlow Qiagen using the buffer mentioned above and after loading, wash with 6 M guanidine-HCl, 20 mM Tris-HCl (pH 8), 10 mM reduced Gluthatione.

25. Eluate the protein with an Imidazol gradient (0-500 mM Imidazol) 2x130 ml in the same buffer.

26. Determine the fractions to use for refolding by using dot blotting and the apoM affinity column-purified polyclonal antibody against truncated mouse/human apoM (10 μg/ml).

27. Dilute the pool in buffer 1 specified below to 100 μg/ml to avoid precipitation. 28. Refold the eluted protein by dialysis against the following buffers:

Buffer 1 : 3 M Guanidine-HCl, 20 mM Tris-HCl (pH 8), 4 mM reduced

Glutathione, 0,4 mM oxidized Glutathione, 10 % Glycerol 2-3 days at 4⁰C (40 mg/3L)

Buffer 2: 20 mM Tris-HCl (pH 8), 4 mM reduced Glutathione, 0,4 mM oxidized Glutathione, 10 % Glycerol O/N at 4°C, adding iodoacetamid (final 5 mM) (when dialysis is ready). (Not less than 5L)

Buffer 3 : 20 mM Tris-HCl (pH 8), 10 % Glycerol O/N at 4°C. (Not less than 5L) Change the dialysis fluid every morning and afternoon for buffers 2 and 3. 29. Measure the conductivity against the dialysis fluid. ^' 30. After refolding, measure absorbance at 280 nm.

31. Use the samples for Silver-stain and Western-blot analysis. C. Purification of refolded human recombinant apoM using an ion exchange chromatography 1. Equilibrate the 100 ml column (Q Sepharose Fast Flow Amersham) with buffer, 20 rtiM Tris (pH 8,0) 10% Glycerol. (Equilibrate with at least 5 times the volume of the column.)

2. Centrifuge the sample at 12000 rpm for 35 minutes.

3. Load the sample and collect the flow through. 4. Wash the column with buffer, 20 mM Tris (pH 8.0) 10 % Glycerol. Collect the wash.

(Wash with at least 5 times the volume of the column.)

5. Run a 0-0.3 M 2x250 ml NaCl gradient and collect fractions. Begin with 5 ml fractions and change to 2 ml fractions when the absorbance starts to increase. When the peak has passed, increase the fraction size to 5 ml again. 6. When the entire gradient has passed, wash the column with 0.5 and then 1 M NaCl until all protein is eliminated from the column.

7. Wash with 20 mM Tris (pH 8.0) O/N.

8. Wash the column with Na-azide, 0.02% in 20 mM Tris, pH 8.0.

9. Run a silver stain to evaluate the purification.

References

1. Xu N, Dahlback B. A novel human apolipoprotein (apoM). J. Biol. Chem. 1999;274:31286-90.

2. Richter S, Shih DQ, Pearson ER, et al. Regulation of apolipoprotein M gene expression by MODY3 gene hepatocyte nuclear factor- 1 alpha: haploinsufficiency is associated with reduced serum apolipoprotein M levels. Diabetes 2003;52:2989-95

Claims

I) A method for determining the level of human apolipoprotein M (apoM) in an assay sample comprising a biological fluid, such as blood, plasma, or serum, said method comprising the steps of

(b) maintaining said first immunoreaction mixture for a time period sufficient for human apoM in said assay sample to bind to said first antibody thereby to form an immunoreaction product comprising apoM,

(c) contacting said first immunoreaction product formed in step (b) with an indicating means- containing second detecting antibody that has the capacity to immunoreact with said first immunoreaction product comprising human apoM and form a second immunoreaction mixture,

2) The method of claim 1 wherein said first antibody of step (a) is operatively linked to a solid carrier; said first immunoreaction mixture formed in step (b) is comprised of a liquid phase and a solid phase; and said first immunoreaction product formed in step (b) is contained in the solid phase which is linked to the carrier. 3) The method of claim 1 or 2 wherein said indicating means is operatively linked to or incorporated in said second antibody and is capable of producing, directly or indirectly, a detectable signal at the formation of said second immunoreaction product in step (d), and wherein said second immunoreaction product formed in step (d) comprises said indicating means and the determination in step (e) comprises measuring the amount of indicating means in said second immunoreaction product.

4) The method of claim 1, 2 or 3 wherein said indicating means-containing second antibody comprises an enzyme, a luminescent substance or a radioisotope as indicating means, or is fluorescence labeled or labeled with use of fluorescence resonance energy transfer (FRET),

5) The method of anyone of claims 1-4 wherein said indicating means-containing second antibody and the assay sample are simultaneously contacted with said first antibody which is bound to an insoluble solid carrier.

6) The method of anyone of claims 1-5 wherein said first antibody is a monoclonal antibody.

7) The method of anyone of claims 1-5 wherein said second antibody is a monoclonal antibody.

8) The method of anyone of claims 1-5 wherein said first antibody is a monoclonal antibody and said second antibody is a polyclonal antibody.

9) The method of anyone of claims 1 -5 wherein said first antibody is a polyclonal antibody and said second antibody is a monoclonal antibody.

10) The method of anyone of claims 1-5 wherein both said first and said second antibody is a monoclonal antibody.

11) The method of anyone of claims 1-10 wherein said monoclonal antibodies are selected from the group consisting of monoclonal antibodies designated apoM 58, apoM 42, and apoM

23 and produced by hybridomas having the accession numbers respectively and provided that if both said first and said second antibody comprises a monoclonal antibody, said first and said second antibody are comprised of different monoclonal antibodies.

12) The method of claim 1 wherein, in a pre-step, detergent is added to the assay sample to release apoM contained in the assay sample from lipoprotein.

13) A method for determining the level of human apolipoprotein M (apoM) in an assay sample comprising a biological fluid, such as blood, plasma, or serum, said method comprising the steps of

(a) bringing said assay sample into contact with an antibody, which is operatively linked to a solid carrier and has the capacity to immunoreact with human apoM, and with labelled human apoM to form a competition immunoreaction mixture comprising a liquid phase and a solid phase,

wherein said antibody of step (a) is a monoclonal antibody.

14) The method of claim 13 wherein said monoclonal antibody is selected from the group consisting of monoclonal antibodies designated apoM 58, apoM 42, and apoM 23 and produced by hybridomas having the accession numbers and wherein labelled apoM contains a radioisotope label.

15) A monoclonal antibody that has the capacity to immunoreact specifically with human apoM and has been raised against recombinant, truncated apoM lacking the signal peptide comprising the N-terminal amino acid residues 1-20 of apoM but comprising amino acid residues 22-188 of mature apoM which antibody does not comprise an antibody that has been raised in rabbits against an apoM derived peptide containing amino acid residues 140-159 of apoM.

16) The monoclonal antibody of claim 15 that has the capacity to immunoreact with human apoM bound to lipoprotein particles and derived from human plasma.

17) The monoclonal antibody of claiml5 that has the capacity to immunoreact with human apoM which has been released from lipoprotein particles associated with apoM of human plasma.

18) The monoclonal antibody of claiml5, which is selected from the group consisting of monoclonal antibodies designated apoM 58, apoM 42 and apoM 23 and produced by hybridomas having the accession number

19) A hybridoma that produces a monoclonal antibody of anyone of claims 15-18.

20) A hybridoma, which produces an antibody selected from the group consisting of monoclonal antibodies designated apoM 58, apoM 42 and apoM 23 and has the accession number

21) A method for purifying and recovering human apoM from an aqueous solution that contains human apoM, which method comprises

(a) contacting said solution with an antibody of claim 15 that specifically immunoreacts with human apoM to form an immunoreaction mixture comprising at least a liquid phase,

(c) recovering the immunoreaction product formed in step (b) from the immunoreaction mixture, thereby producing purified human apoM,

in which method the antibody used in step (a) is a monoclonal antibody. 22) The method of claim 21 wherein said antibody is operatively linked to a solid carrier such that the immunoreaction mixture has a liquid phase and a solid phase and the imunoreacton product formed is contained in the solid phase, and

wherein said recovering in step (c) comprises eluting human apoM contained in the solid phase immunoreaction product off of the solid phase and into the liquid phase using an elution buffer or chaotropic salts, optionally after washing the solid phase containing said immunoreaction product with detergent.

23) A selective adsorbent for human apoM comprising an insoluble solid carrier and the monoclonal antibody of claim 15 operatively linked thereto.

24) The adsorbent of claim 23 wherein the insoluble solid carrier is a plastic receptacle, a microtiter plate, plastic beads, glass beads or metal particles.

25) The method of claim 1 wherein steps (a)-(d) are performed simultaneously in an assay medium comprising a dispersion such as a latex comprising a liquid and a solid phase; said first and said second antibodies are operatively linked to said solid phase which is comprised of solid particles of said dispersion, and each antibody-binding particle binds either said first or said second antibody.