NO894709L - DIAGNOSIS PROCEDURES AND SYSTEMS FOR QUANTIFYING AVAPO AI. - Google Patents

Description

Technical area

The present invention relates to antibodies and polypeptides which can be used for immunological determination of the amount of Apo AI in a vascular fluid sample.

Background

Lipoproteins are the main carriers of plasma cholesterol. They are micellar lipid-protein complexes (particles) with a surface film, consisting of one or more proteins associated with polar lipids, surrounding a cholesterol-containing core. Lipoproteins were originally classified on the basis of their buoyancy densities measured by ultracentrifugation. Accordingly, four main density classes have been recognized: chylomicrons, very low-density lipoproteins (VLDL), low-density lipoproteins LDL, and "high-density lipoproteins" (HDL).

Many studies have now established an inverse correlation between plasma HDL cholesterol levels and risk of coronary artery disease (CAD). That is, elevated levels of plasma cholesterol found in HDL particles correlate with a reduced risk of CAD.

Likewise, many investigations have now shown that plasma levels of apolipoprotein AI (Apo AI), the main protein component of HDL, are also inversely related to the risk of CAD. In addition, Weisweiler et al., Clin. Chem., 27:348

(1981) reported that knowledge of levels of Apo AI can

increase the predictive value for HDL cholesterol.

Because of its inverse correlation with CAD, has

there has been extensive research into the structure and effect of Apo AI in lipid metabolism. Functionally, Apo AI is now assumed

to mediate the removal of cholesterol from tissues and activate

LCAT.

Structurally, Apo AI is purified. described as containing a large proportion (55%) of alpha helices, increasing to 70%

when associated with phospholipids, such as in the HDL particle. The lipid-binding properties of Apo AI appear to be a function of a series of interspersed, repeating segments of 22 amino acid residues mostly interrupted by proline residues that are alpha-helical and amphophilic.

The amino acid residue sequence of Apo AI determined by Edman digestion of cyanogen bromide and trypsin fragments of intact Apo AI has been described by Brewer et al., Biochem. Biophys. Res. Comm., 80:623-630 (1978). According to Brewer et al. Cyanogen bromide cleavage (CNBr cleavage) of Apo AI yielded four major fragments designated CNBrl, CNBr2, CNBr3 and CNBr4, in the order they appear along the Apo AI sequence from amino terminus to carboxyl terminus.

It should be noted that CNBrl, CNBr2, CNBr3 and CNBr4 are polypeptides with homoserine lactone at their carboxyl termini as a result of the methionine residue at that position being degraded during the CNBr cleavage process.

Immunochemical characterization of naturally occurring Apo AI, i.e. Apo AI as found on HDL particles, has been problematic because it is antigenically heterogeneous and unstable. The antigenic heterogeneity of Apo AI appears to be the result of some epitopes being masked by lipids in the intact HDL, or the antibody-binding ability of some epitopes being dependent on conformations of Apo AI influenced by lipids or other HDL -associated proteins. The antigenic instability of Apo AI, as manifested by its changing immunoreactivity over time with certain antisera, appears to be due to such phenomena as self-binding and deamidation, both of which have been shown to occur in vitro.

The antigenic heterogeneity and instability of Apo AI has made it difficult to produce analytical systems for the quantification of Apo AI in vascular fluid samples from patients. This is due, among other things, to the fact that such systems require a reference material (a standard) with an immunoreactivity for the system's primary anti-Apo AI antibody that is at least uniform, and preferably equivalent to the immunoreactivity of the Apo AI that is in the patient's sample.

More recently, efforts to overcome problems associated with the antigenic heterogeneity and instability of Apo AI have focused on using monoclonal antibodies (MABs) to identify epitopes on naturally occurring Apo AI whose expression is uniform or "conserved". under specific isolation and storage conditions. Such epitopes, herein referred to as "conserved naturally occurring epitopes", are further defined as Apo AI epitopes whose expression on HDL is not significantly affected, i.e. not significantly increased or decreased, as a result of processing or storage that resulting in deamidation.

An exemplary conserved naturally occurring Apo AI epitope, designated epitope A, has been defined by Milthorpe et al., Arterio., 6:285-296 (1986), as being the portion of Apo AI CNBrl that immunoreacts with MAB 4H1 . According to Milthorpe et al. the expression of epitope A remains constant over time in patient serum samples stored at temperatures ranging from 4°C to -80°C. This is in contrast to epitopes designated C, C and C", all located in the CNBr2 region of Apo AI, and all of which were found to be "non-conserved" epitopes, i.e. epitopes whose expression was significantly increased or decreased after storage at a similar temperature range.

Brief summary of the invention

The present invention relates to an Apo AI polypeptide which essentially consists of no more than approx. 40 amino acid residues and, as part of the amino acid residue sequence, has a sequence of the formula

Also encompassed is a monoclonal antibody containing anti-Apo AI antibody molecules that react immunologically with:

(a) Apo AI/HDL

(b) isolated Apo AI

(c) Apo AI CNBrl and

(d) the polypeptide DEPPQSPWDRVKDLA,

but which do not react immunologically with:

(e) Apo AI CNBr2,

(f) Apo AI CNBr3,

(g) Apo AI CNBr4,

(h) the polypeptide DEPPQSPWDR and

(i) the polypeptide QSPWDRVKDLA.

In another embodiment, the present invention relates to a diagnostic system in kit form which comprises, in a sufficient amount to perform at least one analysis, an Apo

.AI polypeptide consisting mainly of no more than approx. 40 amino acid residues and which, as part of the amino acid residue sequence, has a sequence with the formula:

A diagnostic system in kit form comprising, in an amount sufficient to perform at least one assay, a monoclonal antibody containing anti-Apo AI antibody molecules that immunologically reacts with:

(a) Apo AI/HDL

(b) isolated Apo AI

(c) Apo AI CNBrl and

(d) the polypeptide DEPPQSPWDRVKDLA,

but which do not react immunologically with:

(e) Apo AI CNBr2,

(f) Apo AI CNBr3,

(g) Apo AI CNBr4,

(h) the polypeptide DEPPQSPWDR and

(i) the polypeptide QSPWDRVKDLA,

is also covered.

Still further encompassed is a method for analyzing the amount of Apo AI in a vascular fluid sample comprising the following steps: (1) forming an immunoreaction mixture by mixing a vascular fluid sample with: (a) an anti-Apo AI monoclonal antibody with antibody molecules that react immunologically with:

(i) Apo AI/HDL

(ii) isolated Apo AI

(iii) Apo AI CNBrl and

(iv) the polypeptide DEPPQSPWDRVKDLA,

but which do not react immunologically with:

(v) Apo AI CNBr 2

(vi) Apo AI CNBr 3

(vii) Apo AI CNBr 4

(viii) the polypeptide DEPPQSPWDR and

(ix) the polypeptide QSPWDRVKDLA and

(b) an Apo AI polypeptide consisting essentially of no more than about 40 amino acid residues and which has, as part of the amino acid residue sequence, a sequence with the formula:

(2) maintaining the immunoreaction mixture i

a time period sufficient to form an Apo AI-containing immunoreaction product, and

(3) determination of the amount of product formed in step (2), and thereby the amount of Apo AI in the vessel sample. In preferred assay methods, the anti-Apo AI-MAB according to step (1) (a) is MAB AI-16 and the polypeptide according to step (1) (b) is a polypeptide selected from the group consisting of:

(i) DEPPQSPWDRVKDLA and

(ii) DEPPQSPWDRVKDLATVYVDV.

Brief description of the drawings

The drawings forming part of this specification show: Figure 1 the amino acid residue sequence of Apo AI CNBrl, as reported by Brewer et al., Biochem. Biophys. Res. Comm., 80:623-630 (1978), from and including residues 1 to 86.

Apo AI CNBrl, which is formed by cleavage in the methionine residue (M) located in position 86, corresponds in sequence to positions from and including 1 to 86 as the carboxyl-end methionine is converted to homoserine lactone. The positions of the four fragments produced after trypsin cleavage of CNBrl, denoted T1 - T4, and the position of the one fragment produced after cleavage of fragment T3 with BNPS-skatole, denoted S1, are also indicated.

Figure 2 shows the ability of Apo AI/HDL, HDL present in fresh plasma and polypeptide AIl-15 to competitively inhibit MAB AI-16 binding to Apo AI/HDL. Protein concentration was determined according to the method of Markwell et al., Anal. Biochem., 87:206-220 (1978). The log-transformed data for Apo AI (A), plasma (0) and polypeptide AIl-15 (#) gave slopes of -1.96, -2.47 and -2.60, respectively.

Figure 3 shows the immunoreactivity [molar (M) amount of peptide required for 50% inhibition (ID^q) of antibody binding in the solid-phase competitive RIA described in Example 6] to each of the peptides AIl-10, AIl-15, AI1- 21 and AI5-15 for MAB AI-16. The IDs were obtained after linear regression analysis of the logarithmically transformed curves for competition and are shown in the upper part of the figure. The affinity of MAB AI-16 for peptides AI1-15 and AI1-21 was determined to be essentially identical as the slopes of the logarithmically transformed lines produced using these peptides were -2.60 and -2.57, respectively. The concentration of peptide competitor is shown in moles per liters (MM) determined as described in Figure 2.

Detailed description of the invention

A. Definitions

Amino acid: All amino acid residues identified here are in the natural L configuration. Adhering to standard polypeptide nomenclature, J. Biol. Chem., 243: 3557-59, (1969), the abbreviations for amino acid residues are as shown in the following correspondence table:

It should be noted that all amino acid residue sequences are shown here using formulas with a left-to-right orientation which is in the usual direction from amino end to carboxyl end. Furthermore, it should be noted that a dot at the beginning or end of an amino acid residue sequence indicates a bond to a further sequence with one or more amino acid residues up to a total of approx. 50 residues in the polypeptide chain.

Apo AI/ HDL: Denotes Apo AI when present on HDL particles.

Delipidated Apo AI: Refers to the Apo AI that is in it

substantially free of associated lipids.

Isolated Apo AI: Denotes Apo AI which is essentially free of both associated lipids and other proteins, such as e.g. those, such as Apo All, which are usually found on

HDL in addition to Apo AI.

Polypeptide and peptide: Polypeptide and peptide are terms used here interchangeably to denote a straight-chain series of no more than approx. 40 amino acid residues where one is bound to the next by means of peptide bonds between alpha-amino and carboxyl groups in neighboring residues.

Protein: Protein is an expression used here to denote a straight-chain series of more than approx. 50 amino acid residues where one is linked to the next as in a polypeptide.

B. Polypeptides

As used herein, the term "Apo AI polypeptide" refers to a polypeptide having an amino acid residue sequence that is homologous (similar in structure) to a portion of the Apo AI molecule.

In one embodiment, an Apo AI polypeptide according to the present invention mainly consists of at least approx. 15 and no more than approx. 40, and preferably no more than approx. 25, amino acid residues, and has as part of its sequence, a sequence with the formula:

In another embodiment, an Apo AI polypeptide according to the invention mainly consists of at least approx. 15 and no more than approx. 25, preferably no more than approx. 21 amino acid residues, and has as part of its sequence, a sequence with the formula:

Preferred Apo AI polypeptides are shown in Table 1.

Preferably, an Apo AI polypeptide according to the invention is further characterized by its ability to immunologically mimic an epitope (antigenic determinant) expressed by Apo AI on essentially all HDL.

An -Apo AI polypeptide of the present invention, herein also referred to as a subject polypeptide, can be synthesized by any of the techniques known to those skilled in the polypeptide art, including recombinant DNA techniques. Synthetic chemistry techniques, such as a Merrifield-type solid-phase synthesis, are preferred for reasons such as purity, antigenic specificity, freedom from unwanted byproducts, "ease of manufacture, and the like. An excellent summary of the many techniques available,

can be found in J.M. Steward and J.D. Young, "Solid Phase Peptide Synthesis", W.H. Freeman Co., San Francisco, 1969;

M. Bodanszky et al., "Peptide Synthesis", John Wiley&Sons, 2nd ed., 1976 and J. Meienhofer, "Hormonal Proteins and Peptides", vol. 2, p. 46, Academic Press (New York), 1983

for solid-phase peptide synthesis, and E. Schroder and K. Kubke, "The Peptides", vol. 1, Academic Press (New York) 1965 for classical solution synthesis. Suitable protecting groups which can be used in such syntheses are described in the above-mentioned literature and in J.F.W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press, New York, 1973.

In general, they include solid-phase synthesis methods

as contemplated herein, the sequential addition of one or more amino acid residues or suitable protective amino acid residues to a growing peptide chain. Normally, either the amino or carboxyl group in the first amino acid residue is protected

by means of a suitable, selectively removable protecting group. A different, selectively removable protective group is used for amino acids that contain a reactive side group, such as lysine.

When a solid phase synthesis is used as an example, the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group. The protecting group on the amino or carboxyl group is then selectively removed and the next amino acid in the sequence where the complementary (amino or carboxyl) group is suitably protected is added and reacted under conditions suitable for the formation of the amide bond with the residue already attached to it permanent carrier. The protecting group on the amino or carboxyl group is then removed

from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, etc. After all the desired amino acids have been bound in the correct order, any remaining end and side group protecting groups (and fixed support) are removed sequentially or simultaneously -early, whereby the final polypeptide is obtained.

It should be understood that a subject polypeptide need not be identical to the amino acid residue sequence of Apo AI, as long as it comprises the required sequence and is capable of immunologically reacting with antibodies that immunologically react with Apo AI. Substitutions of one amino acid for another, whether conservative or non-conservative, when such changes provide certain advantages in use, are covered. Conservative substitutions are those where one amino acid residue is replaced by another, biologically similar residue. Examples of conservative substitutions include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine, for another, or the substitution of a polar residue for another, such as between arginine and lysine, between glutamic and aspartic acid or between glutamine and asparagin, and the like. The term "conservative substitution" also encompasses the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that such polypeptide also exhibits the required binding activity.

When a polypeptide according to the present invention

has a sequence that is not identical to the sequence of an Apo AI because one or more conservative or non-conservative substitutions have been made, is usually not more than 30% by number, more usually not more than 20% by number and preferably not more than 10% by number, of the amino acid residues substituted, except that the proline residue in position 99 cannot be substituted or removed when further residues have been added at one of the ends for the purpose of providing a "linker" by which the polypeptides of the invention can be suitably attached to a marker or solid matrix, or carrier, the linker residues do not form Apo AI epitopes, i.e. are not similar in structure to Apo AI. Markers, solid matrices and carriers that can be used together with the polypeptides according to the invention are described below.

Amino acid residue linkers are usually at least one residue and can be 40 or more residues, more often 1-10 residues, but do not form Apo AI epitopes. Typical amino acid residues used for binding are tyrosine, cysteine, lysine, glutamine-

and aspartic acid, or the like. In addition, a subject polypeptide may differ, unless otherwise indicated, from the native sequence of Apo AI in that the sequence has been modified by terminal N1^ acylation, e.g. acetylation, or thioglycolic acid amidation, by terminal carboxyl amidation, e.g. with ammonia, methylamine and the like.

When linked to a carrier to form what is known in the art as a carrier-hapten conjugate, an Apo AI polypeptide of the present invention can induce antibodies that immunologically react with Apo AI, preferably Apo AI when it is part of an HDL particle (Apo AI/HDL). Based on the well-established principle of immunological reactivity, the present invention therefore includes antigenically related variants of the polypeptides in table 1. An "antigenically related variant" is a subject polypeptide that contains at least approx. 15, and no more than approx. 40, amino acid residues, including the amino acid residue sequence DEPPQSPWDRVKDLA, and which can induce antibody molecules that react immunologically with a polypeptide from Table 1 and

Apo AI.

C. Antibodies and monoclonal antibodies

The term "antibody" in its various grammatical forms is used herein as a common noun referring to a population of immunoglobulin molecules and/or immunologically active parts of immunoglobulin molecules, i.e. molecules containing an antibody-binding site or a paratope.

An "antibody binding site" is the structural part of an antibody molecule consisting of heavy and light chain variable and hyper-variable regions that specifically bind antigen.

The term "antibody molecule" in its various grammatical forms includes, as used here, both an intact immunoglobulin and an immunologically active part of an immunoglobulin molecule1.

Examples of antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those parts of a globulin molecule containing the paratope, including those parts known in the art as Fab, Fab<1>, F(ab')2 and F(v).

Fab and F (ab')2 parts of the antibody are prepared by means of the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibodies by means of methods which are well known. See e.g. US Patent No. 4,342,566. Fab<1->antibody moieties are also well known and are prepared from F(ab 1)2~ moieties followed by reduction of the disulfide bonds that bind together the two heavy chain moieties, e.g. mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with such a reagent as iodoacetamide. An antibody containing intact antibody molecules is preferred, and is used here as an illustration.

A polyclonal antibody according to the present invention is characterized by its ability to react immunologically with 1) a subject polypeptide containing no more than 25 amino acid residues, and 2) Apo AI/HDL. A polyclonal antibody according to the present invention is further characterized in that it is essentially free of antibody molecules that react immunologically with Apo AI CNBr2, CNBr3 and CNBr4.

The term "monoclonal antibody" in its various grammatical forms refers to a population of antibody molecules containing only one species of antibody-binding site capable of immunologically reacting with a particular antigen. Thus, a monoclonal antibody typically exhibits a single binding affinity to any antigen with which it reacts immunologically. A monoclonal antibody can therefore contain an antibody molecule with several antibody-binding sites, each of which is immunologically specific for a different antigen, e.g. a bispecific monoclonal antibody.

A "monoclonal antibody (MAB) according to the present invention (subject MAB) is characterized in that it reacts immunologically with:

(a) Apo AI/HDL,

(b) isolated Apo AI

(c) Apo AI CNBrl and

(d) polypeptide AIl-15;

but that it does not react immunologically with:

(e) Apo AI CNBr2,

(f) Apo AI CNBr3,

(g) Apo AI CNBr4,

(h) polypeptide AIl-10 and

(i) polypeptide AI5-15.

A preferred MAB according to the present invention reacts immunologically with Apo AI/HDL, and thus has a ratio between immunoreactivities for Apo AI/HDL and polypeptide AIl-15 in the range from approx. 1:5 to approx. 5:1, preferably approx. 1:2.5 to approx. 2.5:1, and preferably approx. 1.5:1 to approx. 1:15.

As used herein, the term "immunological reactivity" in its various grammatical forms refers to the concentration of antigen required to achieve a 50% inhibition of the immunological reaction between a specified amount of the antibody and a specified amount of Apo AI/HDL . That is, immunological reactivity is the concentration of antigen required to achieve a B/BQ value of 0.5 when Bo is the maximum amount of antibody bound in the absence of competing antigen, and it is the amount of antibody bound in presence of competing antigen, and both Bq and B have been adjusted for background value. See Rodbard, Clin. Chem., 20:1255-1270 (1974).

Preferably, a monoclonal antibody according to the present invention has identical (indistinguishable) affinities for naturally occurring Apo AI/HDL and polypeptide AI1-15. That is, a preferred monoclonal antibody has an affinity for polypeptide AI1-15 which, when determined separately, is not distinguishable (equivalent to) by statistical analysis within a confidence limit of p<0.1, preferably p< 0.05, preferably p<0.01.

Methods for determining the affinity of a monoclonal antibody for an antigen and for comparing these affinities with respect to equivalents are well known in the art. See e.g. Muller, J. Immunol. Meth., 34:345-352 (1980) and Sokal et al., Biometry, W.H. Freeman & Co., (1981). A preferred method of determination

of affinity of monoclonal antibody, is by equilibrium competitive inhibition assay. In that method, the ability of Apo AI/HDL and polypeptide AIl-15 to compete with Apo AI/HDL for binding to the monoclonal antibody being characterized is determined separately and compared for equivalents. See Tsao et al.,

J. Biol. Chem., 257:15222-15228 (1982).

E.g. determination of whether the affinities exhibited by the monoclonal antibody with respect to Apo AI/HDL and polypeptide AIl-15 are identical (indistinguishable) can be performed as follows: (a) The percentage of a known amount of the monoclonal antibody bound to solid phase Apo AI /HDL in the presence of polypeptide AIl-15 present as a liquid phase competitor is determined at various known competitor concentrations. The logarithmic transformation of each determination of percent bound is then plotted against concentration of competitor (liquid phase polypeptide). [Logarithm (Y)=log (Y/1-Y) , where

Y is percent binding of monoclonal antibody in the presence of

a certain amount of competitor].

(b) Using the same amount of monoclonal antibody as in step (a), determine the percentage of antibody bound to solid phase Apo AI/HDL in the presence of Apo AI/HDL present as a liquid phase competitor, at the same concentrations as the competitor in step (a). The logarithmic transformation of each percent bound is then plotted against concentration of competitor (liquid phase Apo AI/HDL). (c) Linear regression analysis is performed on each of the plots obtained in steps (a) and (b) to arrive at their respective slopes. (d) The slopes obtained for Apo AI/HDL and the slope obtained for polypeptide AIl-15 are then compared using a test for equality of slopes, such as that described by Sokal et al., supra, p. 485, route 14.5.

A subject monoclonal antibody, usually containing whole antibody molecules, can be prepared using the polypeptide-induced hybridoma technique described by Niman et al., Proe. Nati. Sci., U.S.A., 80:4949-4953 (1983). In order to form the hybridoma from which the mixture of monoclonal antibody is prepared, a myeloma or other self-perpetuating cell line is briefly fused with lymphocytes obtained from the spleen of a mammal newly immunized with a polypeptide according to the invention.

It is preferred that the myeloma cell line is from the same species as the lymphocytes. Generally, a mouse of strain 129 GlX<+> is the preferred mammal. Suitable mouse myelomas for use in the present invention include the hypoxanthine-aminopterin-thymidine-sensitive (HAT) cell lines P3X63-Ag8.653 and Sp2/0-Agl4 which are available from the American Type Culture Collection, Rockville, MD, under the names respectively CRL 1580 and CRL 1581.

Splenoocytes are usually fused with myeloma cells using polyethylene glycol (PEG) 1500. Fusion hybrids are selected for their sensitivity to HAT. Hybridomas that produce a monoclonal antibody according to the invention are identified using the radioimmunological method

analysis (RIA) described in Example 6.

A monoclonal antibody according to the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma secreting antibody molecules with the appropriate polypeptide specificity. The culture is maintained under conditions and for a period of time sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated using well-known techniques.

Usable media for the preparation of these mixtures are both well known in the art and commercially available, and include synthetic culture media, inbred mice and the like. An example of a synthetic medium is Dulbecco's "minimal essential medium" (DMEM; Dulbecco et al., Virol., 8:396 (1959)) supplemented with 4.5 g/liter glucose, 20 mM glutamine and 20% fetal calf serum. An example of an inbred mouse strain is Balb/c.

The monoclonal antibody produced using the above method can e.g. is used in diagnostic and therapeutic applications where the formation of an Apo AI-containing immunoreaction product is desired.

A hybridoma that can be used in the production of a subject monoclonal antibody, i.e., MAB AI-16, is hybridoma H131E4, which has been deposited under Budapest Convention requirements at the American Type Culture Collection (ATCC), Rockville, MD 20852, U.S.A., March 29, 1988 and given the ATCC designation HB 9677. It should be noted that hybridoma ATCC

HB 9677 can be used, as is well known in the art, to produce other immortal cell lines that produce a subject monoclonal antibody, and thus production of a subject monoclonal antibody is not dependent on growing hybridomas using ATCC HB 9677 per se.

D. Diagnostic systems

A diagnostic system in kit form according to the present invention comprises, in an amount sufficient for at least one analysis, an Apo AI subject polypeptide and/or a monoclonal subject antibody, as well as separately packaged immunochemical reagents. Instructions for use of a packaged immunochemical reagent are also usually included.

As used here, the term "packaging" refers to a solid matrix or such a material as glass, plastic, paper, foil and the like which can keep a polypeptide, polyclonal antibody or monoclonal antibody according to the present invention within certain limits. Thus, a seal can e.g. be a glass ampoule used to contain milligram amounts of a subject polypeptide, or it may be a microtiter plate well in which microgram amounts of a subject polypeptide have been operatively fixed, i.e. bound so that it is capable of being immunologically bound by an antibody.

"Instructions for use" usually include a concrete indication that describes the reagent concentration or at least one analytical procedure parameter, such as the relative amounts of reagent and sample to be mixed together, storage time for reagent/sample mixtures, temperature, buffer conditions and the like.

In preferred embodiments, a diagnostic system according to the present invention further comprises a marker or indicator which can signal the formation of a complex containing a polypeptide or antibody molecule according to the present invention.

The word "complex" as used herein refers to the product of a specific binding reaction, such as an antibody/antigen or receptor/ligand reaction. Examples of complexes are immunoreaction products.

As used herein, the terms "marker" and "indicator" in their various grammatical forms refer to individual atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex. Any marker or indicator may be bound to or incorporated into an expressed protein, polypeptide or antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention, or used separately, and these atoms or molecules may be used alone or along with additional reagent. Such markers are in themselves well known in clinical diagnostic chemistry and form part of the invention only to the extent that they are used together with otherwise new proteins, methods and/or systems.

The marker may be a fluorescent marker

which binds chemically to antibodies or antigens without denaturing them so that a fluorochrome (dye) is formed

which is an effective immunofluorescent indicator. Suitable fluorescent markers are fluorochromes, such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), 5-dimethylamine-l-naphthalene sulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200 sulfonyl chloride (RB 200 SC) and the like. A description of immunofluorescence analysis techniques can be found in DeLuca, "Immuno-fluorescence Analysis", in Antibody As a Tool. Marchalonis et al., ed. John Wiley & Sons, Ltd. 189-231 (1982).

In preferred embodiments, the indicator group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase or the like. In such cases where the main indicator group is an enzyme, such as HRP or glucose oxidase, an additional reagent is required to visualize the fact that a receptor/ligand complex (immunoreactant) has been formed. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor, such as diaminobenzidine. An additional reagent that can be used in conjunction with glucose oxidase is 2,2'-azino-di-(3-ethy1-benzthiazoline-G-sulfonic acid) (ABTS).

Radioactive elements are also effective tracers and are used here as an illustration. An example of a radioactive tracer is a radioactive element that emits gamma rays. Elements which themselves emit gamma-.... ,124T 125T 128T 132T 51„ ..^ rays, such as I, I, I, I and Cr, constitute a class of gamma-ray emitting indicator groups

125

with radioactive element. Particularly preferred is I. Another group of effective marker agents are those elements, such as <11>C, 18 F, 150 and <13>N, which themselves emit positrons. The positrons thus emitted produce gamma rays

after encountering electrons present in the animal's body. Also effective is a beta emitter, such as "'""'"^indium, of<3>H.

The binding of markers, i.e. labeling of polypeptides and proteins, is well known in the art. E.g. antibody molecules produced by means of a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component of the culture medium. See e.g. Galfre et al., Meth. Enzymol., 73:3-46 (1981). The techniques of protein conjugation or coupling through activated fused groups are particularly applicable. See e.g. Aurameas et al., Scand. J. Immunol., vol. 8, Suppl. 7:7-23 (1978), Rodwell

et al., Biotech., 3:889-894 (1984) and US Patent No. 4,493,795.

The diagnostic systems may also include, preferably as

a separate seal, a specific binding agent. A "specific binding agent" is a molecular unit which can bind selectively a reagent species according to the present invention or a complex containing such a species, but which is not itself a polypeptide or an antibody molecule mixture according to the present invention. Examples of specific binding agents are secondary antibody molecules, complement proteins or fragments thereof, S. aureus protein A and the like. Preferably, the specific binding agent binds to the reagent species when the species is present as part of a complex.

In preferred embodiments, the specific binding agent is labeled. However, when the diagnostic system includes a specific binding agent that is not labeled, the agent is usually used as an amplification agent or reagent. In these embodiments, the labeled specific binding agent can bind specifically to the enhancing agent when the enhancing agent is bound to a reagent species-containing complex.

The diagnostic kits according to the present invention can be used in an "ELISA" format to detect the amount of Apo AI

in a vascular fluid sample, such as blood, serum or plasma. "ELISA" refers to an enzyme-linked immunosorbent assay where an antibody or antigen bound to a solid phase and an enzyme/antigen or enzyme/antibody con-

jugat to detect and quantify the amount of an antigen present in a sample. A description of the ELISA technique can be found in Chapter 22 of the 4th edition of Basic and Clinical Immunology by D.P. Sites et al., published by Lange Medical Publications of Los Altos, CA in 1982 and in US Patent Nos. 3,654,090, 3,850,752 and 4,016,043.

In preferred embodiments, an Apo AI polypeptide or a monoclonal antibody according to the present invention can thus be attached to a solid matrix, whereby a solid carrier is formed which constitutes a package in the present diagnostic systems.

A reagent is usually attached to a solid matrix by adsorption from an aqueous medium, although other means of attachment which are useful proteins and polypeptides, and which are well known to those skilled in the art, may be used.

Applicable solid matrices are also well known in the art. Such materials are water insoluble and include the cross-linked dextran available under the trade name Sephadex<®>; agarose, balls of polystyrene from approx. 1 micrometer to approx. 5 mm in diameter; polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose or nylon-based fabrics, such as sheets, strips or paddles; or tubes, plates, or the wells of a microtiter plate, such as those made of polystyrene or polyvinyl chloride.

The reagent species, labeled specific binding agent or amplification reagent in each diagnostic system described herein may be provided in solution, as a liquid dispersion or as a substantially dry powder, e.g. in lyophilized form. When the indicator agents are an enzyme, the enzyme's substrate can also be provided in a separate package in a system. A solid carrier, such as the previously described microtiter plate, and one or more buffers can also be included as separate packing elements in this diagnostic analysis system.

The packing materials that are discussed here in connection with diagnostic systems are those that are usually used in diagnostic systems. Such materials include glass and plastic (e.g. polyethylene, polypropylene and polycarbonate) bottles, ampoules,

plastic and plastic foil laminated sleeves and the like.

E. Analytical procedures

The present invention relates to various immunological analysis methods for determining the amount of Apo AI in a biological fluid sample by using a polypeptide, a polyclonal antibody or monoclonal antibody according to the invention as an immunochemical reagent for the formation of an immunological reaction product in an amount that is in proportion, either directly or indirectly, to the amount of Apo AI in the sample. Those skilled in the art will understand that there are several well-known clinical-diagnostic chemical methods where an immunochemical reagent according to the invention can be used to form an immunological reaction product in an amount commensurate with the amount of Apo AI present in a body sample. Although exemplary analysis methods have been described here, the invention is thus not limited to these.

Various heterogeneous and homogeneous methods, either competitive or non-competitive, can be used when carrying out the analysis method according to the invention. E.g. the present invention comprises a competitive method for analyzing the amount of Apo AI in a vascular fluid sample comprising the following steps: (a) Formation of an immunological reaction mixture by mixing a vascular fluid sample with: (i) a monoclonal antibody according to the present invention, preferably AI -16, and (ii) an Apo AI polypeptide according to the present invention, preferably AI1-15 or AI1-21.

The vascular fluid sample is preferably provided as a known amount of blood, or a blood-derived product, such as serum or plasma. Regardless of the type of sample used, it is preferably provided from a person who has fasted for at least approx. 12 hours, which is known in the art. Such a test is referred to as a "fixed" test. It is also noted that when serum or plasma is used as the sample, that sample need not be subjected to denaturing or chaotropic agent treatment for the purpose of altering the expression of the Apo AI epitope being analyzed.

Preferably, the amount of monoclonal antibody that is added is known. Further preferred are embodiments where the monoclonal antibody is labeled, i.e. operatively bound to an indicator agent, such as an enzyme, radionuclide and the like.

Preferably, the Apo AI polypeptide is present as part of a solid carrier, i.e. operatively bound to a solid matrix, so that the immunological reaction mixture that is formed has a solid and a liquid phase. Further preferred are embodiments wherein the amount of polypeptide present in the immunological reaction mixture is a sufficient amount to form an excess of epitopes relative to the number of antibody-binding sites present in the immunological reaction mixture capable of react immunologically with these epitopes. (b) The immunological reaction mixture is kept under biological assay conditions for a predetermined period of time, such as approx. 10 minutes to approx. 16 - 20 hours, at a temperature of approx. 4°C to approx. 45°C, this time being sufficient for the Apo AI present in the sample to react immunologically with (bind immunologically to) part of the anti-Apo AI antibody-binding sites present on the monoclonal antibody , whereby an Apo AI-containing immunological reaction product is formed.

Biological analysis conditions are those which maintain the biological activity of the immunochemical reagents according to the invention and the Apo AI that is sought to be analysed. These conditions include a temperature range from approx. 4°C to approx. 45°C, a pH value range of approx. 5 to approx. 9, and an ionic strength that varies from the ionic strength of distilled water to the ionic strength of approx. 1 molar sodium chloride. Methods for optimizing such conditions are well known in the art. (c) The amount of Apo AI-containing immunological reaction product formed is determined, and thereby the amount of Apo AI in the sample.

Determination of the amount of the Apo AI-containing immunological reaction product, either directly or indirectly, can be performed using analytical techniques well known in the art, and usually depends on the type of indicator used.

In preferred competitive assay methods, the amount of product determined in step (c) is related to the amount of immunological reaction product formed in a similar manner and determined using a control sample in place of the vascular fluid sample where the control sample contains a known amount of a subject polypeptide, preferably AIl-15 or AI1 - 21.

In another embodiment, the present invention relates to a double antibody or "sandwich" immunoassay which comprises the following steps: (a) Formation of a first immunological reaction mixture by mixing a vascular fluid sample with a first antibody, preferably a monoclonal antibody, where the antibody and Apo AI/HDL present in the sample is capable of forming a first immunological reaction product that can react immunologically with a subject monoclonal antibody, preferably MAB AI-16. Preferably, the first antibody is operatively bound to a solid matrix. (b) Maintaining the first immunological reaction mixture thus formed under biological assay conditions for a period of time sufficient to form the first immunological reaction product. Preferably, the first immunological reaction product is then separated from the sample. (c) Formation of a second immunological reaction mixture by mixing the first immunological reaction product with: (i) a monoclonal antibody according to the present invention, preferably MAB AI-16, and (ii) an Apo AI polypeptide according to the present invention, preferably AIl- 15 or AI1-21. Preferably, step (ii) is carried out before step (i) or essentially simultaneously with it, i.e. within approx. 5-10 minutes, preferably within approx. 1-2 minutes.

(d) Maintenance of the thus formed, other immuno-

logical reaction mixture under biological assay conditions for a precise period of time sufficient to form the second or "sandwich" immunological reaction product. (e) Determination of the amount of second immunological reaction product formed, and thereby the amount of Apo AI in the sample.

Preferably, the subject monoclonal antibody according to step (c) (i) is labeled, preferably with an enzyme, and the second immunological reaction product that is formed is a labeled product.

In preferred double antibody assay methods, the amount of immunological reaction product determined in step (e) of the double antibody method is related to the amount of immunological reaction product formed in a similar manner and determined using a control sample in place of the vascular fluid sample, the control sample containing a known amount of a subject polypeptide, preferably AIl-15 or AIl-21.

Examples

The following examples illustrate, but do not limit, the present invention.

1. Production of antigens

A. Polypeptides

Polypeptides AI1-10, AI1-15, AI1-21 and AI5-15 were synthesized using the classic solid phase technique described by Merrifield, Adv. Enzymol., 32:221-96 (1969) adapted for use with a Model 430A Automated Peptide Synthesizer. Polypeptide resins were cleaved with hydrogen fluoride, extracted and analyzed for purity by high performance liquid chromatography using a reversed phase C18 column.

The amino acid residue sequences of the polypeptides AI1-15 and AI1-21 have been shown above in Table 1. The sequences of the polypeptides AI1-10 and AI5-15 are shown in Table 2 below.

B. Preparation of Apo AI/HDL

HDL was isolated from plasma obtained by plasmaphoresis of blood from a normal fasting donor at the local blood bank (San Diego Plasma Center, San Diego, CA). For that purpose, the plasma thus obtained was adjusted so that it contained a final concentration of 5 millimoles (mM) benzamidine, 1 mM di-isopropylfluorophosphate, 10 mM ethylenediaminetetraacetic acid (EDTA), 10 milligrams per milliliter (mg/ml) soybean trypsin inhibitor and 10,000 units per ml aprotinin. The HDL was then isolated from this adjusted plasma by sequential ultracentrifugation using solid potassium bromide (KBr) for density control.

First, the regulated plasma was centrifuged at approx. 2,000,000 x g for 18-24 hours and the bottom layer of the resulting supernatant was recovered. Solid KBr was mixed into the bottom layer until the density was greater than 1.063 grams per; milliliters (g/ml). The resulting mixture was then placed in a layer under a 0.1% EDTA solution containing KBr at a density of 1.063 g/ml and centrifuged at 200,000 x g for more than 48 hours. The bottom layer was extracted again and solid KBr was mixed into it until the density was regulated to more than 1.21 g/ml. The regulated layer was placed under a 0.1% EDTA solution containing KBr at a density of 1.21 g/ml, and was centrifuged at 200,000 x g for more than 48 hours.

The top layer was then recovered and solid KBr was added until the density was greater than 1.063 g/ml. The regulated top layer was placed under a 0.1% EDTA solution containing KBr at a density of 1.063 g/ml, and further centrifuged at 200,000 x g for more than 48 hours.

The middle layer was recovered and solid KBr mixed into it until the density was adjusted to greater than 1.21 g/ml. The regulated middle layer was placed under a 0.1% EDTA solution containing KBr at a density of 1.21 g/ml and centrifuged at 300,000 x g for more than 48 hours. The resulting HDL-containing top layer, with a density equal to 1.063 - 1.21 g/ml, was recovered. The extracted HDL was dialyzed against lipoprotein buffer (LLB; water containing 0.15 mM NaCl, 0.3 mM EDTA and 0.005% alpha-tocopherol) and the resulting Apo AI/HDL was stored under sterile conditions and used within 3 days.

C. Preparation of delipidated Apo AI

Delipidated Apo AI was prepared by organically extracting the lipids from Apo AI/HDL. A sample of Apo AI/HDL prepared in Example IB was first dialyzed against 0.01% EDTA at a pH of 7.5 overnight (about 18 hours), then dialyzed against 0.003 percent EDTA for about 12 hours, and then lyophilized at 10 - 20 mg protein per tube. 35 ml of absolute ethanol:anhydrous ether (1:1) at 4°C was added to each tube. After mixing, the solution was kept for 20 minutes at -20°C. The solution was then centrifuged for 30 minutes at 1000 x g at 0°C, the supernatant was discarded and the Apo AI-containing pellet was retained.

Ethanol ether extraction as described above was carried out twice more for a total of three extractions. Then 35 ml of anhydrous ether at 4°C was mixed with the sample. The mixture was kept for 30 minutes at -20°C, centrifuged at 1000 x g for 30 minutes at -20°C, and the Apo AI-containing pellet was recovered and dried using nitrogen gas, whereby delipidated Apo AI was formed. It should be noted that delipidated Apo AI contains not only Apo AI, but also other proteins associated with HDL, so that Apo AI.

D. Preparation of Isolated Apo AI

Apo AI was isolated from delipidated Apo AI by size fractionation using high pressure liquid chromatography (HPLC) following the methods of Kinoshita et al.,

J. Biochem., 94:615-617 (1983). About. 300 mg of delipidated Apo AI was dissolved in 200 microliters (ul) of 0.1% sodium dodecyl sulfate

(SDS), 0.1 M sodium phosphate (pH 7.0) and size fractionated on Spherogel - TSK 3000 SW HPLC columns. Fractions containing the isolated Apo AI were stored at -20°C. Apo AI was also isolated by delipidation and electrophoresis on polyacrylamide as described here.

E. Preparation of polyacrylamide-HDL

HDL was immobilized in polyacrylamide by mixing the following indicated amounts of separately prepared solutions, whereby a cross-linking reaction mixture was formed:

(a) 4.3 ml LLB containing 50 mg HDL,

(b) 1.25 ml of water containing 28% (w/v) acrylamide, (c) 2.5 ml of water containing 2% (w/v) N,N'-methylene-bis-acrylamide,

(d) 1.25 mL LLB and

(e) 1.2 ml of water containing 1% (w/v) ammonium persulfate.

The cross-linking reaction was allowed to continue for approx. 16 hours at 37°C. As cross-linking did not occur, TEMED (N,N,N',N,1-tetramethyl-ethylene-diamine) was then added, which cross-linked within approx. 90 minutes at 37°C.

The resulting polyacrylic mass was homogenized mechanically

in the presence of 20 ml of LLB and then washed with LLB by centrifugal filtration, whereby polyacrylamide-HDL was formed.

2. Production of monoclonal antibodies

Balb/c ByJ mice were immunized intraperitoneally (i.p.) with 50 pg polyacrylamide-HDL as immunogen in complete Freund's adjuvant (CFA) and 500 units of interferon-gamma followed by a second and a third immunization, each approx. three weeks apart, in incomplete Freund's adjuvant (IFA) without interferon. Approximately nine months after the last adjuvant immunization, the mice received a booster injection of 50 µg naturally occurring HDL intravenously (i.v.) in normal saline 4 days before perfusion and a second equal perfusion boost one day later.

The animals thus treated were killed and the spleen of each mouse removed. A spleen cell suspension was then prepared. Spleen cells were then extracted from the spleen cell suspension by centrifugation for approx. 10 minutes at 1000 rpm, at 23°C. After removal of supernatant, the cell pellet was resuspended in 5 ml of cold NH^Cl lysis buffer, and was incubated for approx. 10 minutes.

To the lysed cell suspension, 10 ml of Dulbecco's modified Eagle's medium (DMEM) and HEPES [4-(2-hydroxyethyl)-1-piperidine ethanesulfonic acid] were mixed, and that mixture was centrifuged for approx. 10 minutes at 1000 rpm at 23°C.

The supernatant was decanted, the pellet resuspended in 15 ml of DMEM and HEPES, and was centrifuged for approx. 10 minutes at 1000 rpm at 23°C. The above procedure was repeated.

The pellet was then resuspended in 5 ml of DMEM and HEPES. An aliquot of the spleen cell suspension was then removed for counting. Fusions were performed in the following manner using the non-secreting mouse myeloma cell line P3X63Ag8.653.1, a subclone of line P3X63Ag 8.653 (ATCC 1580). By using a ratio between myeloma and spleen cells of approx. 1-10 or approx. 1-5, a sufficient amount of myeloma cells was centrifuged into a pellet, washed twice in 15 ml of DMEM and HEPES, and centrifuged for 10 minutes at 1000 rpm at 23°C.

Spleen cells and myeloma cells were pooled in round-bottomed 15 ml tubes. The cell mixture was centrifuged for 10 minutes at 1000 rpm at 23°C and the supernatant removed by aspiration. Next, 200 µl of 50% (weight per volume) aqueous polyethylene glycol of molecular weight 4000 (PEG; ATCC Baltimore, MD) at approx. 37°C mixed using a 1 ml pipette with vigorous agitation to break up the pellet, and the cells were gently mixed for approx. 15-30 seconds. The cell mixture was centrifuged for 4 min at 700 rpm.

At approx. 8 min before the time of PEG addition, 5 ml of DMEM plus HEPES buffer was slowly mixed with the pellet without breaking up the cells. After 1 minute, the resulting mixture was broken up with a 1 ml pipette and was incubated for another 4 minutes. This mixture was centrifuged for 7 minutes at 1000 rpm. The supernatant was decanted, 5 ml of HT medium (hypoxanthine/thymidine) was slowly mixed with the pellet and the mixture kept undisturbed for 5 minutes. The pellet was then broken up into large slices, and the final cell suspension placed in T75 flasks (2.5 ml per flask) in which 7.5 ml of HT medium had been previously placed. The resulting cell suspension was incubated at 37°C for

to culture the fused cells. After 245 hours, 10 ml of HT medium was added to the flasks, followed 6 hours later by the addition of 0.3 ml of 0.04 mM aminopterin. 48 hours after fusion, 10 ml of HAT medium (hypoxanthine/aminopterin/thymidine) was added to the flasks.

Three days after fusion, viable cells were plated in 96-well tissue culture plates at approx. 2 x 10 4 viable cells per well (a total of 7 68 wells) in HAT buffer medium as described in Rennett et al., Curr. Top. Microbiol. Immunol., 81:77 (1978). The cells were fed HAT medium 7 days after fusion and then fed HT medium at approximately 4-5 day intervals as needed. The growth was followed microscopically and culture supernatants were collected approx. 2 weeks later, and analyzed for the presence of HDL-specific antibody by solid phase radioimmunoassay (RIA) essentially as described in Curtiss and Edgington J. Biol. Chem., 257: 15213-15221

(1982).

Briefly, 50 µl PBS containing 5 µg/ml Apo AI/HDL was added to the wells of microtiter plates. The plates were kept overnight (about 16 hours) at 4°C to allow Apo AI/HDL to adhere to the well walls. After washing the wells four times with SPRIA buffer (2.68 mM KCl, 1.47 mM KH2PO4, 137 mM NaCl, 8.03 mM Na2HP04, 0.05% Tween<®->20, 0.1 KIU/ml

'Traysol", 0.1% BSA, 0.015% NaN^) 200 µl SPRIA buffer containing 3% normal goat serum (NGS) and 3% bovine serum albumin (BSA) was added to each well to block excess protein binding sites .The plates were kept for 30 minutes at 20°C, the wells were emptied by shaking and dried using blotting paper, whereby a solid

carrier, i.e. a solid matrix to which Apo AI/HDL was effectively attached.

To each well, 50 µl hybridoma tissue culture supernatant was then added, whereby an immunological reaction mixture with solid and liquid phase was formed. The mixture was kept for 2 hours at 37°C to allow formation of solid phase immunological reaction products to occur. After washing well-125

one as previously described, 50 µl of I-labeled goat anti-mouse IgG was added at 0.25 µg protein per ml to each well, thereby forming a marker reaction mixture. This mixture was kept for 1 hour at 37°C to let

125

formation of I-labeled immunological reaction products x solid phase occurs. After washing the wells as previously be-125

written, the amount of I-labeled product bound to each well was determined by gamma scintillation.

Hybridoma AI-16 was selected from approx. 16 hybridoma cultures secreted anti-HDL antibodies in their culture media. Hybridoma AI-16 was determined to have an IgG~ z a heavy immunoglobulin chain and was further characterized as described here.

3. Purification of a monoclonal antibody preparation

Ascites fluids were obtained from 10-week-old Balb/c mice that had been pretreated with 0.3 ml of mineral oil and injected intraperitoneally with 5 x 10 5 hybridoma cells. The average time for the development of ascites was 9 days. After clarification by centrifugation at 15,000 x g for 15 minutes at 23°C, ascitic fluids produced by hybridoma H135D3 were pooled and stored frozen at -20°C.

Purified AI-16 monoclonal antibody from each of five hybridomas was prepared by fast protein liquid chromatography (FPLC) using a Pharmacia Mono Q HR5/5 anion exchange column using a 0 - 0.5 mol (M) NaCl gradient in 10 mM Tris, pH 8.0, following the guidelines supplied with the column. Purified Mabs were concentrated using an Amicon stirred ultrafiltration cell (PM 30 membrane) to a concentration of

1 mg/ml, dialyzed into PBS (phosphate buffered saline,

pH 7.2) and stored at -70°C.

4. Radioiodination

Radioiodination of HDL, Apo AI and immunochemically purified goat anti-mouse Ig was performed enzymatically using the "Enzymo-bead" iodination procedure and "Enzymobeads". "Enzymobeads" iodination was used to characterize the antigens and antibodies for the solid phase radioimmunoassay as discussed below.

5. Apo AI-cyanobromide fragment-specificity

The apo AI-CNBr fragment specificity of MAB AI-16 was determined by Western blot analysis according to the method of Curtiss et al., Proceeding of the Workshop on Lipo-protein Heterogeneity, ed. by Lippel, NIH Publication No. 87-2646, pp. 363-377 (1987). Briefly, CNBr fragmentation was performed on isolated Apo AI dissolved in 90% formic acid. CNBr was added in a 13,000 molar excess and the reaction mixture was kept approx. 15 hours at approx. 20°C. After lyophilization, the resulting CNBr fragments were solubilized in 1% SDS, 0.01 m Tris, pH 8.2, and subjected to isoelectric focusing in 6% polyacrylamide plate gels containing 8 M urea and 2% ampholine (pH 4 - pH 6) as described by Curtiss et al., J. Biol. Chem., 260: 2982-93 (1985). Electrophoretically separated proteins were transferred to nitrocellulose for immunological reaction with MAB AI-16. Production of immunological reaction products was detected using radioiodinated goat anti-mouse Ig followed by autoradiography.

The results of these investigations indicate that MAB AI-16 does not immunologically react with the Apo AI-CNBr fragments CNBr2, CNBr3 and CNBr4, but does immunologically react with CNBrl. It should also be noted that these results indicate that MAB AI-16 reacts immunologically with isolated Apo AI.

6. MAB AI-16 immunoreactivity

The immunoreactivity of MAB AI-16 for naturally occurring Apo AI/HDL, deamidated Apo AI/HDL and various polypeptides was examined by a competitive RIA performed as follows: 100 µl PBS (0.15 M NaCl, 0.01 M NaPC >4, pH 7.2) containing 10 µg/ml Apo AI/HDL was added to the wells in microtiter plates. The plates were kept for 1 hour at 20°C on a rotating platform to allow Apo AI/HDL to attach to the wells and form solid supports. After aspirating excess liquid from the wells, 200 µl blocking solution (3% BSA, 3% NGS in PBS) was added to each well and the wells were kept for 30 minutes at 20°C on a rotating platform. The blocking solution was then removed by aspiration and the wells were washed three times with SPRIA buffer. ■

To each well, first 50 µl of PBS containing 3% BSA and different concentrations of competitor antigen, i.e. Apo AI/HDL, or peptide, and then 50 µl of MAB AI-16 in the form of clarified ascites diluted 1:1 were added .25x105 in PBS containing 3% BSA, whereby competitive immunoreaction mixtures were formed. In control wells, either competing antigen or antibody was replaced with PBS containing 3% BSA.

The immunoreaction mixtures were kept approx. 16 hours at 4°C on a rotating platform to allow formation of solid-phase immunoreaction products to occur. After washing off

125

the wells as previously described were 100 µl I labeled

125 goat anti-mouse Ig (I goat anti-mouse Ig diluted to 2 x 10 5 trichloroacetic acid-precipitable degradations per minute per 100 µl in PBS containing 3% BSA) added to each well. The resulting marker-immunoreaction mixtures were kept for 4 hours at 4°C on a rotating platform. The wells were then washed with SPRIA as previously described, and

125

the amount of I-labeled solid-phase immunoreaction product formed determined.

The ability of MAB AI-16 to immunoreact with Apo AI/HDL, fresh plasma HDL, and the polypeptides AI1-15 was compared using each as a competitor in the RIA described above. The results of this investigation are shown in Figure 2. The slopes of the Apo AI/HDL, plasma HDL and polypeptide AI1-15 logarithmically transformed data were found to be -1.96, -2.42 and -2.60. MAB AI-16 exhibited essentially identical affinity for plasma HDL and peptide AI1-15, indicating that expression of the epitope recognized by MAB AI-16 on plasma HDL and the peptide is similar,

if not identical. However, the affinity of MAB AI-16 for delipidated Apo AI was significantly less, indicating

that the amino end part in Apo AI is changed by delipidation.

To compare the immunoreactivity of MAB AI-16

for the polypeptides shown in Tables 1 and 2, a standard solution of each was prepared at a concentration of 10 µg/ml. Fifty microliters of the standard or 2-fold serial dilutions of the standard were used as a competitor in the RIA described in this example. The results of this investigation, graphically illustrated in Figure 3, indicate that the peptides AIl-10 and AIl-15 were unable to inhibit the binding of MAB ÅI-

16 for solid phase Apo AI/HDL. Both peptides AIl-15 and AIl-

However, 21 were good competitors. Moreover, slope analysis of the logarithmically transformed curves indicated that the affinity of MAB AI-16 for peptides AI1-15 and AI1-21 was the same.

The above description, including the specific embodiments and examples, is intended to be illustrative of the present invention and should not be taken as limiting.

A large number of other variations and modifications can be made without departing from the same spirit and scope of the present invention.

Claims (15)

1. Apo AI polypeptide, characterized in that it consists mainly of no more than 40 amino acid residues and has as part of its amino acid residue sequence, a sequence with the formula 2. Polypeptide according to claim 1, characterized in that the polypeptide has an amino acid residue sequence with a formula selected from the groups consisting of: a) DEPPQSPWDRVKDLA and b) DEPPQSPWDRVKDLATVYVDV. 3. Monoclonal antibody, characterized in that it contains anti-Apo AI antibody molecules that react immunologically with: (a) Apo AI/HDL (b) isolated Apo AI (c) Apo AI CNBrl and (d) the polypeptide DEPPQSPWDRVKDLA, but does not react immunologically with: (e) Apo AI CNBr2, (f) Apo AI CNBr3, (g) Apo AI CNBr4, (h) the polypeptide DEPPQSPWDR and (i) the polypeptide QSPWDRVKDLA. 4. Monoclonal antibody according to claim 3, characterized in that the antibody molecules are those produced by the hybridoma with the ATCC designation HB 9677. 5. Diagnostic system in kit form, characterized in that it comprises, in an amount sufficient to perform at least one analysis, an Apo AI polypeptide with a formula selected from the group consisting of: (a) DEPPQSPWDRVKDLA and (b) DEPPQSPWDRVKDLATVYVDV. 6. Diagnostic system according to claim 5, characterized in that the polypeptide is operatively bound to a solid matrix. 7. Diagnostic system according to claim 5, characterized in that it further 'comprises, in an amount sufficient to perform at least one assay, a monoclonal antibody containing anti-Apo AI antibody molecules that immunologically reacts with: (a) Apo AI/HDL (b) isolated Apo AI (c) Apo AI CNBrl and (d) the polypeptide DEPPQSPWDRVKDLA, but which do not react immunologically with: (e) Apo AI CNBr2, (f) Apo AI CNBr3, (g) Apo AI CNBr4, (h) the polypeptide DEPPQSPWDR and (i) the polypeptide QSPWDRVKDLA. 8. Diagnostic system according to claim 7, characterized in that the antibody molecules are those produced by the hybridoma with the ATCC designation HB 9677. 9. Diagnostic system according to claim 7, characterized in that the antibody molecules are operatively bound to an enzyme indicator. 10. Diagnostic system in kit form, characterized in that it comprises, in an amount sufficient to perform at least one analysis, a monoclonal antibody containing anti-Apo AI antibody molecules that react immunologically with: (a) Apo AI/HDL (b) isolated Apo AI (c) Apo AI CNBrl and (d) the polypeptide DEPPQSPWDRVKDLA, but which do not react immunologically with: (e) Apo AI CNBr2, (f) Apo AI CNBr3, (g) Apo AI CNBr4, (h) the polypeptide DEPPQSPWDR and (i) the polypeptide QSPWDRVKDLA. 11. Diagnostic system according to claim 10, characterized in that the antibody molecules are those that can be produced using the hybridoma with the ATCC designation HB 9677. 12. Diagnostic system according to claim 11, characterized in that the antibody molecules are operatively bound to an enzyme indicator. 13. Method for analyzing the amount of Apo AI in a vascular fluid sample, characterized by the following steps: (a) forming an immunoreaction mixture by mixing a vascular fluid sample with: (i) an anti-Apo AI monoclonal antibody produced using the hybridoma with the ATCC designation HB 9677, and (ii) an Apo AI polypeptide selected from the group consisting of: a) DEPPQSPWDRVKDLA and b) DEPPQSPWDRVKDLATVYVDV, (b) maintaining the immunoreaction mixture for a period of time sufficient to form an Apo AI-containing immunoreaction product, and (c) determining the amount of product formed in step (b), and thereby the amount of Apo AI in the vascular fluid sample. 14. Method according to claim 13, characterized in that the polypeptide is operatively bound to a solid matrix, the antibody is operatively bound to an enzyme marker and the product formed in step (b) is a labeled immunoreaction product. 15. Hybridoma, characterized in that it produces antibody molecules that can react immunologically with Apo AI/HDL and the polypeptide DEPPQSPWDRVKDLA, and has the ATCC designation HB 9677.