DIAGNOSTIC METHODS AND SYSTEMS FOR QUANTIFYING APO AI

Abstract: The present invention describes a monoclonal antibody that is directed against an Apo AI/HDL epitope whose expression is substantially unaffected by deamidation. Also disclosed is a polypeptide capable of immunologically mimicking an Apo AI/HDL epitope whose expression is substantially unaffected by deamidation. Diagnostic systems and methods for determining the amount of Apo AI in a vascular fluid sample using a disclosed monoclonal antibody and/or a disclosed polypeptide are also described.

Description: DIAGNOSTIC METHODS AND SYSTEMS FOR QUANTIFYING APO Al Description Technical Field The present invention relates to antibodies and polypeptides useful for immunologically determining the amount of Apo Al in a vascular fluid sample. Background Lipoproteins are the primary carriers of plasma cholesterol. They are micellar lipid-protein complexes (particles) having a surface film, comprised of one or more proteins associated with polar lipids, that surrounds a cholesterol-containing core. Lipoproteins were originally classified based on their buoyant densities as measured by ultracentrifugation. Accordingly, four major 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. Similarly, many studies have now shown that plasma levels of apolipoprotein Al (Apo Al) , the major protein component of HDL, are also inversely related to the risk of CAD. In addition, eisweiler et al., Clin. Chem.. 27:348 (1981) have reported that knowledge of Apo Al levels may add to the predictive valve of HDL cholesterol. Because of its inverse correlation with CAD, there has been an extensive amount of research into the structure and function of Apo Al in lipid metabolism. Functionally, Apo Al is now believed to mediate the removal of cholesterol from tissues and to activate LCAT. Structurally, purified Apo Al has been described as containing a high proportion (55%) of alpha-helix, which increases to 70% when it is associated with phospholipids as in the HDL particle. The lipid binding properties of Apo Al appear to be a function of a series of tandemly repeated segments of 22 amino acid residues punctuated mostly by proline residues that are alpha-helical and amphophilic. The amino acid residue sequence of Apo Al, determined by Edman degradation of cyanogen bromide- and trypsin-fragments of intact Apo Al, has been described by Brewer et al. , Biochem. Biophvs. Res. Comm.. 80:623-630 (1978). According to Brewer et al., cyanogen bromide (CNBr) cleavage of Apo Al produced four major fragments, designated CNBrl, CNBr2, CNBr3 and CNBr4, in order of their occurrence along the Apo Al sequence from amino-terminus to carboxy-terminus. It should be noted that CNBrl, CNBr2, CNBr3 and CNBr4, are polypeptides having homoserine lactone at their carboxy termini as a result of the methione residue at that position being degraded during the CNBr cleavage process. Immunochemical characterization of native Apo Al, i.e., Apo Al as it is found on HDL particles, has been problematical because it is antigenically heterogeneous and unstable. The antigenic heterogeneity of Apo Al 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 Al as affected by lipids or other HDL associated proteins. The antigenic instability of Apo Al, as manifest by its changing immunoreactivity over time with defined antisera, appears to be due to such phenomena as self association and deamidation, both of which have been shown to occur in vitro. The antigenic heterogeneity and instability of Apo Al has made it difficult to produce assay systems for quantifying Apo Al in patient vascular fluid samples. This is because, inter alia, such systems require a reference material (standard) whose immunoreactivity for the system's primary anti-Apo Al antibody is consistent, at the very least, and preferably equivalent to that of the Apo Al in the patient's sample. Recently, efforts at overcoming problems associated with the antigenic heterogenicity and instability of Apo Al have focused on using monoclonal antibodies (MAB) to identify epitopes on native Apo Al whose expression is consistent or "conserved" under specific isolation and storage conditions. Such epitopes, referred to herein as "conserved native epitopes", are further defined as Apo Al epitopes whose expression on HDL is not significantly affected, i.e., not significantly increased or decreased, as a result of processing or storage that results in deamidation. An exemplary conserved native Apo Al epitope, designated epitope A, has been defined by Milthorpe et al., Arterio.. 6:285-296 (1986) as being that portion of Apo Al CNBrl that im unoreacts with MAB 4H1. According to Milthorp et al., the expression of epitope A remains constant overtime in patient serum samples stored at temperatures ranging from 4 degrees C to -80 degrees C. This was in contrast to epitopes designated C, C and C", all located in the CNBr2 region of Apo Al, and all of " which were found to be "nonconserved" epitopes, i.e., epitopes whose expression was significantly increased or reduced upon storage at a similar range of temperatures. Brief Summary of the Invention The present invention cφntemplates an Apo Al polypeptide consisting essentially of no more than about 40 amino acid residues and having as a part of its amino acid residue sequence a sequence represented by the formula: -DEPPQSPWDRVKDLA-. Also contemplated is a monoclonal antibody containing anti-Apo Al antibody molecules that i munoreact with: (a) Apo AI/HDL (b) isolated Apo Al (c) Apo Al CNBrl, and (d) the polypeptide DEPPQSPWDRVKDLA, but do not immunoreact with: (e) Apo Al CNBr2, (f) Apo Al CNBr3, ' (g) Apo Al CNBr4, (h) the polypeptide DEPPQSPWDR, and (i) the polypeptide QSPWDRVKDLA. in another embodiment, the present invention contemplates a diagnostic system, in kit form, that includes, in an amount sufficient to perform at least one assay, an Apo Al polypeptide consisting essentially of no more than about 40 amino acid residues and having as a part of itss ammino acid residue sequence a sequence represented by the . formula: -DEPPQSPWDRVKDLA-. A diagnostic system, in kit form, that includes, in an amount sufficient to perform at least one assay, a monoclonal antibody containing anti-Apo Al antibody molecules that immunoreact with: (a) Apo AI/HDL (b) isolated Apo Al (c) Apo Al CNBrl, and (d) the polypeptide DEPPQSPWDRVKDLA, but do not immunoreact with: (e) Apo Al CNBr2, (f) Apo Al CNBr3, (g) Apo Al CNBr4, (h) the polypeptide DEPPQSPWDR, and (i) the polypeptide QSPWDRVKDLA, is also contemplated. Still further contemplated is a method of assaying the amount of Apo Al in a vascular fluid sample comprising the steps of: (1) forming an immunoreaction admixture by admixing a vascular fluid sample with: (a) an anti-Apo Al monoclonal antibody having antibody molecules that immunoreact with: (i) Apo AI/HDL (ii) isolated Apo Al (iii) Apo Al CNBrl, and (iv) the polypeptide DEPPQSPWDRVKDLA, but do not immunoreact with: (V) Apo Al CNB42 (vi) Apo Al CNB43 (vii) Apo Al CNBr4 (viii) the polypeptide DEPPQSPWDR, and (ix) the polypeptide QSPWDRVKDLA, and (b) an Apo Al polypeptide consistinng essentially of no more than about 40 amino acid residues and having as a part of its amino acid residue sequence a sequence represented by the formula: -DEPPQSPWDRVKDLA-, (2) maintaining said immunoreaction- admixture for a time period sufficient to form an Apo Al-containing immunoreaction product, and (3) determining the amount of product formed in step (2) , and thereby the amount of Apo Al in the vascular sample. In preferred assay methods, the anti-Apo Al MAB of Step (1) (a) is MAB AI-16 and the polypeptide of step (1) (b) is a polypeptide selected from the group consisting of: (i) DEPPQSPWDRVKDLA, and (ϋ) DEPPQSPWDRVKDLATVYVDV. Brief Description of the Drawings In the drawings forming a portion of this disclosure: Figure 1 illustrates the amino acid residue sequence of Apo Al CNBrl,- as reported by Brewer et al., Biochem. Biophvs. Res. Comm.. 80:623-630 (1978), from residue positions 1 through 86. Apo Al CNBrl, which is formed by cleavage at the methionine (M) residue located at position 86, corresponds in sequence to positions 1 through 86 with the carboxyterminal methionine being converted to homoserine lactone. The positions of the 4 fragments produced upon trypsin cleavage of CNBrl, designated Tl through T4, and the position of the one fragment produced upon cleavage of fragment T3 with BNPS- skatole, designated SI, are also indicated. Figure 2 illustrates the ability of Apo AI/HDL, HDL present in fresh plasma and polypeptide AI1-15 to competitively inhibit MAB AI-16 binding to Apo AI/HDL. Protein concentration were determined according to the method of Markwell et al. , Anal. Biochem.. 87:206-120 (1978). The logit transformed apo Al (Δ) , plasma (O) and polypeptide AI1-15 (•) data displayed a slopes of -1.96, and -2.47 and -2.60, respectively. Figure 3 illustrates the immunoreactivity [molar (M) amount of peptide required for 50% inhibition (ID 50 ) of antibody binding in the solid- phase competitive RIA described in Example 6] of each of the peptides AI1-10, AI1-15, AI1-21 and AI5-15 for MAB AI-16. The ID 50 's were obtained following linear regression analysis of the logit transformed competition curves and are shown in the uppoer portion of the figure. The affinity of MAB AI-16 for peptides AI1-15 and AI1-21 were determined to be essentially identical because the slpes' of the logit transformed lines produced by those peptides was -2.60 and -2.57, respectively. The concentration of peptide competitor is shown in moles per liter (MM) determined as described in Figure 2. Detailed Description of the Invention A. Definitions Amino Acid: All amino acid residues identified herein are in the natural L-configuration. In keeping with standard polypeptide nomenclature, _____ Biol. Chem.. 243:3557-59, (1969), abbreviations for amino acid residues are as shown in the following Table of Correspondence: TABLE OF CORRESPONDENCE SYMBOL AMINO ACID 1-Letter 3-Letter Y Tyr L-tyrosine G Gly glycine F Phe L-phenylalanine M Met L-methionine A Ala L-alanine S Ser L-serine I lie L-isoleucine L Leu L-leucine T Thr L-threonine V Val L-valine P Pro L-proline K Lys L-lysine H His L-histidine Q Gin L-glutamine E Glu L-glutamic acid W Trp L-tryptophan R Arg L-arginine D Asp L-aspartic acid N Asn L-asparagine C Cys L-cysteine It should be noted that all amino acid residue sequences are represented herein by formulae whose left to right orientation is in the conventional direction of amino-terminus to carboxy-terminus. Furthermore, it should be noted that a dash at .the beginning or end of an amino acid residue sequence indicates a bond to a further sequence of one or more amino acid residues up to a total of about fifty residues in the polypeptide chain. Apo AI/HDL: Designates Apo Al when it is present on HDL particles. Delipidated APO Al: Refers to Apo Al that is substantially free of associated lipids. . Isolated APO Al: Designates Apo Al that is substantially free of both associated lipids and other proteins, such as those, like Apo All, that are typically found on HDL in addition to Apo Al. Polypeptide and Peptide: Polypeptide and peptide are terms used interchangeably herein to designate a linear series of no more than about 40 amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. Protein: Protein is a term used herein to designate a linear series of greater than about 50 amino acid, residues connected one to the other as in a polypeptide. B. Polypeptides As used herein, the phrase "Apo Al polypeptide" refers to a polypeptide whose amino acid residue sequence is homologous (similar in structure) to a portion of the Apo Al molecule. In one embodiment, an Apo Al polypeptide of the present invention consists essentially of at least about 15 and no more than about 40, and preferably no more than about 25, amino acid residues and has as a portion of its sequence a sequence represented by the formula: -DEPPSQPWDRVKDLA-, In another embodiment, an Apo Al polypeptide of this invention consists essentially of at least about 15 and no more than 25, preferably no more than about 21 amino acid residues and has as a portion its sequence a sequence represented by the formula: -DEPPQSPWDRVKDLATVYVDV-. Preferred Apo Al polypeptides are shown in Table 1. Table 1 Designation 1 Amino Acid Residue Seouence AI1-15 DEPPQSPWDRVKDLA AI1-21 DEPPQSPWDRVKDLATVYVDV 1 Each polypeptide designation used herein indicates the corresponnding residues in the Apo Al protein from which the polypeptide amino acid residue sequence was derived. Preferably, an Apo Al polypeptide of this invention is further characterized by its ability to immunologically mimic an epitope (antigenic determinant) expressed by Apo Al on substantially all HDL. An Apo Al polypeptide of the present invention also referred to herein as a subject polypeptide, can be synthesized by any of the techniques that are known to those skilled in the polypeptide art, including recombinant DNA techniques. Synthetic chemistry techniques, such as a splid-phase Merrifield-type synthesis, are preferred for reasons of purity, antigenic specificity, freedom from undesired side products, ease of production 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, Second Edition, .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. l, Academic Press (New York) , 1965 for classical solution synthesis, each of which is incorporated herein by reference. — Appropriate protective groups usable in such synthesis are described in the above texts and in J.F.W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press, New York, 1973, which is incorporated herein by reference. In general, the solid-phase synthesis methods contemplated comprise the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group. A different, selectively removable protecting group is utilized for amino acids containing a reactive side group such as lysine. Using a solid phase synthesis as exemplary, the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group. The protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected is admixed and reacted under conditions suitable for forming the amide linkage with the residue already attached to the solid support. The protecting group of 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, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and solid support) are removed sequentially or concurrently, to afford the final polypeptide. It should be understood that a subject polypeptide need not be identical to the amino-acid residue sequence of Apo Al, so long as it includes the required sequence and is able to immunoreact with antibodies that immunoreact with Apo Al. Thus, substitutions of one amino acid for another, either conservative or non-conservative, where such changes provide for certain advantages in their use are contemplated. 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, lexicine or methionine for another, or the substitution of one polar residue for another such as between arginine and lysine, between glutamic and aspartic acids or between glutamine and asparagine and the like. The term "conservative substitution" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that such a polypeptide also displays the requisite binding activity. When a polypeptide of the present invention has a sequence that is not identical to the sequence of a Apo Al because one or more conservative or non- conservative substitutions have been made, usually no more than about 30 number percent, more usually no more than 20 number percent, and preferably no more than 10 number percent of the amino acid residues are substituted, except that the proline residue at position 99 cannot be substituted or deleted where additional residues have been added at either terminus for the purpose of providing a "linker" by which the polypeptides of this invention can be conveniently affixed to a label or solid matrix, or carrier, the linker residues do not form Apo Al epitopes, i.e., are not similar is structure to the Apo Al. Labels, solid matrices and carriers that can be used with the polypeptides of this invention are described hereinbelow. Amino acid residue linkers are usually at least one residue and can be 40 or more residues, more often 1 to 10 residues, but do not form Apo Al epitopes. Typical amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like. In addition, a subject polypeptide can differ, unless otherwise specified, from the natural sequence of Apo Al by the sequence being modified by terminal-NH 2 acylation, e.g., acetylation, or thioglycolic acid amidation, by terminal-carboxlyamidation, e.g. , with ammonia, methylamine, and the like. When coupled to a carrier to form what is known in the art as a carrier-hapten conjugate, an Apo Al polypeptide of the present invention is capable of inducing antibodies that immunoreact with Apo Al, preferably Apo Al when it is part of an HDL particle (Apo AI/HDL) . In view of the well established principle of immunologic cross-reactivity, the present invention therefore contemplates antigenically related variants of the polypeptides shown in Table 1. An "antigenically related variant" is a subject polypeptide that contains at least about 15 and no more than about 40 amino acid residues, includes the amino acid residue sequence DEPPQSPWDRVKDLA and is capable of inducing antibody molecules that immunoreact with a polypeptide from Table 1 and Apo Al. C. Antibodies and Monoclonal Antibodies The term "antibody" in its various grammatical forms is used herein as a collective noun that refers to a population of im unoglobulin — molecules and/or immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope. An "antibody combining site" is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen. The phrase "antibody molecule" in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contain the paratope, including those portions known in the art as Fab, Fab', F(ab') 2 and F(v) . Fab and F(ab') 2 portions of antibodies are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibodies by methods that are well known. See for example, U.S. Patent No. 4,342,566 to Theofilopolous and Dixon. Fab' antibody portions are also well known and are produced from F(ab') 2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide. An antibody _ containing intact antibody molecules are preferred, and are utilized as illustrative herein. A polyclonal antibody of the present invention is characterized as being capable of immunoreacting with 1) a subject polypeptide containing no more than 25 amino acid residues " ; and 2) Apo AI/HDL. A polyclonal antibody of the present invention is further characterized as being substantially free of antibody molecules that immunoreact with Apo Al CNBr2, CNBr3 and CNBr4. The phrase "monoclonal antibody" in its various grammatical forms refers to a population of antibody molecules that contains only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen, e.g., a bispecific monoclonal antibody. A monoclonal antibody (MAB) of the present invention (subject MAB) is characterized as immunoreacting with: (a) Apo AI/HDL, (b) isolated Apo Al, (c) Apo Al CNBrl, and (d) polypeptide AI1-15, but as not immunoreacting with: (e) Apo Al CNBr2, (f) Apo Al CNBr3, (g) Apo Al CNBr4, (h) polypeptide AI1-10, and (i) polypeptide AI5-15. A preferred MAB of the present invention immunoreacts with Apo AI/HDL, and thus displays a ratio of immunoreactivities for Apo AI/HDL and polypeptide AI1-15 in the range of about 1:5 to about 5:1, preferably about 1:2.5 to about 2.5:1, and more preferably about 1.5:1 to about 1:1.5. • — As used herein, the term "immunoreactivity: in its various grammatical forms refers to the concentration of antigen required to achieve a 50% inhibition of the immunoreaction between a given amount of the antibody and a given amount of Apo AI/HDL. That is, immunoreactivity is the concentration of antigen required to achieve a B/B 0 value of 0.5, where B 0 is the maximum amount of antibody bound in the absence of competing antigen and B is the amount of antibody bound in the presence of competing antigen, and both Bo and B have been adjusted for background. See, Rodbard, Clin. Chem.. 20:1255-1270 (1974). More preferably, a monoclonal antibody of the present invention has identical (indistinguishable) affinities for native Apo AI/HDL and polypeptide AI1-15. That is, a preferred monoclonal antibody has an affinity for polypeptide AI1-15 that, when separately determined, are indistinguishable (equivalent) by statistical analysis to within a confidence limit of p<0.1, preferably p<0.05, more preferably p<0.01. Methods for determining the affinity of a monoclonal antibody for an antigen and comparing those affinities for equivalence are well known in the art. See, for example, Muller, J. Immunol. Meth.. 34:345- 352 (1980) and Sokal et al_. Biometry. W. H. Freeman & Co., (1981). A preferred method for determining monoclonal antibody affinity is by equilibrium - competitive inhibition analysis. In that method, the ability of Apo AI/HDL and polypeptide AI1-15 to compete with Apo AI/HDL for binding to the monoclonal antibody being characterized are separately determined and compared for equivalence. See Tsao et al., J. Biol. Chem.. 257:15222-15228 (1982). - For example, determining whether or not the affinities displayed by a monoclonal antibody for Apo AI/HDL and polypeptide AI1-15 are identical (indistinguishable) can be performed in the following manner: (a) The percent of a known amount of the monoclonal antibody bound to solid-phase Apo AI/HDL in the presence of polypeptide AI1-15 present as liquid- phase competitor is determined at various known competitor concentrations. The logit transformation of each percent bound determination is then plotted against competitor (liquid-phase polypeptide) concentration. [Logit (Y)=log e (Y/l-Y) where Y is the percent binding of monoclonal antibody in the presence of a given amount of competitor. ] (b) Using the same amount of monoclonal antibody as in step (a) , the percent of antibody bound to solid-phase Apo AI/HDL in the presence of Apo AI/HDL present as liquid-phase competitor is determined at the same concentrations as the competitor in step (a) . The logit transformation of each percent bound is then plotted against competitor (liquid-phase Apo AI/HDL) concentration. (c) Linear regression analysis is preformed on each of the plots obtained in steps (a) and (b) to obtain their respective slopes. (d) The slopes obtained for Apo AI/HDL and the slope obtained for polypeptide AI1-15 are then compared using a test for equality of slopes, such as that described by Sokal et al., supra. p.485, Box 14.5. A subject monoclonal antibody, typically containing whole antibody molecules can be prepared using the polypeptide-induced hybridoma technology described by Niman et al., Proc. Natl. Sci.. UTS.A.. 80:4949-4953 (1983), which description is incorporated herein by reference. Briefly, to form the hybridoma from which the monoclonal antibody composition is produced, a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with a polypeptide of this invention. It is preferred that the myeloma cell line be from the same species as the lymphocytes. Typically, a mouse of the strain 129 G1X + 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 that are available from the American Type Culture Collection, Rockville, MD, under the designations CRL 1580 and CRL 1581, respectively. Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 1500. Fused hybrids are selected by their sensitivity to HAT. Hybridomas producing a monoclonal antibody of this invention are identified using the radioimmunoassay (RIA) described in Example 6. A monoclonal antibody of the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate polypeptide specificity. The culture is maintained under conditions and for a time period 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 by well known techniques. — Media useful for the preparation of these compositions are both well known in the art and commercially available and include- synthetic culture media, inbred mice and the like. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol. 8:396 (1959)) supplemented with 4.5 gm/1 glucose, 20 mm glutamine, and 20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c. The monoclonal antibody produced by the above method can be used, for example, in diagnostic and therapeutic modalities wherein formation of an Apo Al-containing immunoreaction product is desired. A hybridoma useful in producing a subject monoclonal antibody, i.e., MAB AI-16, is hybridoma H131E4, said hybridoma being deposited pursuant to Budapest Treaty Requirements with the American Type Culture Collection (ATCC) , Rockville, MD 20852 U.S.A. on 29 March 1988 and given the ATCC designation . It should be noted that hybridoma ATCC 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 culturing hybridoma by ATCC per se. D. Diagnostic Systems A diagnostic system in kit form of the present invention includes, in an amount sufficient for at least one assay, a subject Apo Al polypeptide and/or a subject monoclonal antibody, as separately packaged immunochemical reagents. Instructions for use of a packaged immunochemical reagent are also typically included. As used herein, the term "package" refers to a solid matrix or material such as glass, plastic, paper r foil and the like capable of holding within fixed limits a polypeptide, polyclonal antibody or monoclonal antibody of the present invention. Thus, for example, a package can be a glass vial used to contain milligram quantities of a contemplated polypeptide or it can be a microtiter plate well to which microgram quantities of a contemplated polypeptide have been operatively affixed, i.e., linked so as to be capable of being immunologically bound by an antibody. "Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like. In preferred embodiments, a diagnostic system of the present invention further includes a label or indicating means capable of signaling the formation of a complex-containing a polypeptide or antibody molecule of 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. Exemplary complexes are immunoreaction products. As used herein, the terms "label" and "indicating means" in their various grammatical forms refer to single 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 label or indicating means can be linked to or incorporated in 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 those atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well- known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins methods and/or systems. The labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC) , fluorescein isothiocyante (FITC) , 5-dimethylamine-l- naphthalenesulfonyl chloride (DANSC) , tetramethylrhodamine isothiocyanate (TRITC) , lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like. A description of immunofluorescence analysis techniques is found in DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool. Marchalonis, et al., eds., John Wiley & Sons, Ltd., pp. 189-231 (1982) , which is incorporated herein by reference. In preferred embodiments, the indicating group is an enzyme, such as horseradish peroxidase (HRP) , glucose oxidase, or the like. In such cases where the principal indicating group is an enzyme such as HRP or glucose oxidase, additional reagents are required to visualize the fact that a receptor-ligand complex (immunoreactant) has formed. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine. An additional reagent useful with glucose oxidase is 2,2'-azino-di-(3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS) . - Radioactive elements are also useful labeling agents and are used illustratively herein. An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as 124 I, 125 I, 128 I, 132 ι and 51 Cr represent one class of gamma ray emission-producing radioactive element indicating groups. Particularly preferred is 12 ^I. Another group of useful labeling means are those elements such as 11 C, 18 F, 15 0 and 13 N which themselves emit positrons. The positrons so emitted produce gamma rays upon encounters with electrons*present in the animal's body. Also useful is a beta emitter, such ι:L1 indium of 3 H. The linking of labels, i.e., labeling of, polypeptides and proteins is well known in the art. For instance, antibody molecules produced by a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium. See, for example, Galfre et al., Meth. Enzvmol.. 73:3-46 (1981). The techniques of protein conjugation or coupling through activated functional groups are particularly applicable. See, for example, Aurameas, et al., Scand. J. Immunol.. Vol. 8 Suppl. 7:7-23 (1978), Rodwell et al., Biotech.. 3:889-894 (1984), and U.S. Pat. No. 4,493,795. The diagnostic systems can also include, preferably as a separate package, a specific binding agent. A "specific binding agent" is a molecular entity capable of selectively binding a reagent species of the present invention or a complex containing such a species, but is not itself a polypeptide or antibody molecule composition of the present invention. Exemplary specific binding-agents are second antibody molecules, complement proteins or fragments thereof, S. aureus protein A, and the like. Preferably the specific binding agent binds the reagent species when that 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 typically used as an amplifying means or reagent. In these embodiments, the labeled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a reagent species- containing complex. The diagnostic kits of the present invention can be used in an "ELISA" format to detect the quantity of Apo Al in a vascular fluid sample such as blood, serum, or plasma. "ELISA" refers to an enzyme- linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen present in a sample. A description of the ELISA technique is 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 U.S. Patents No. 3,654,090; No. 3,850,752; and No. 4,016,043, which are all incorporated herein by reference. Thus, in preferred embodiments, an Apo Al polypeptide, or monoclonal antibody of the present invention can be affixed to a solid matrix to form a solid support that comprises a package in the subject diagnostic systems. ~ A reagent is typically affixed to a solid matrix by adsorption from an aqueous medium although other modes of affixation applicable to proteins and polypeptides well known to those skilled in the art, can be used. Useful solid matrices are also well known in the art. Such materials are water insoluble and include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, NJ) ; agarose; beads of polystyrene beads about 1 micron to about 5 millimeters in diameter available from Abbott Laboratories of North Chicago, IL; polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-based webs such as sheets, strips or paddles; or tubes, plates or the wells of a microtiter plate such as those made from polystyrene or polyvinylchloride. The reagent species, labeled specific binding agent or amplifying reagent of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form. Where the indicating means is an enzyme, the enzyme's substrate can also be provided in a separate package of a system. A solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system. The packaging materials discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems. Such materials include glass and plastic (e.g., polyethylene, polypropylene and polycarbonate) bottles, vials. plastic and plastic-foil laminated envelopes and the like. ~ E. Assay Methods The present invention contemplates various immunoassay methods for determining the amount of Apo Al in a biological fluid sample using a polypeptide, polyclonal antibody or monoclonal antibody of this invention as an immunochemical reagent to form an immunoreaction product whose amount relates, either directly or indirectly, to the amount of Apo Al in the sample. Those skilled in the art will understand that there are numerous well known clinical diagnostic chemistry procedures in which an immunochemical reagent of this invention can be used to form an immunoreaction product whose amount relates to the amount of Apo Al present in a body sample. Thus, while exemplary assay methods are described herein, the invention is not so limited. Various heterogenous and homogeneous protocols, either competitive or noncompetitive, can be employed in performing an assay method of this invention. For example, the present invention contemplates a competitive method for assaying the amount of Apo Al in a vascular fluid sample which comprises the steps of: (a) Forming an immunoreaction admixture by admixing a vascular fluid sample with: (i) a monoclonal antibody of the present invention, preferably AI-16, and (ii) an Apo Al polypeptide of the present invention, preferably AI1-15 or AI1-21. Preferably, the vascular fluid sample is 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 obtained from a person who has fasted at least about 12 hours as is known in the art. Such a sample is referred to as a "fasting" sample. It is also noted that where serum or plasma is used as the sample, that sample need not be subjected treatment with a denaturing or chaotropic agent for purposes of altering the expression of the Apo Al epitope being assayed. Preferably, the amount of monoclonal antibody that is admixed is known. Further preferred are embodiments where the monoclonal antibody is labeled, i.e., operatively linked to an indicating means such as an enzyme, radionuclide and the like. Preferably, the Apo Al polypeptide is present as part of a solid support, i.e. , operatively linked to a solid matrix, so that the immunoreaction admixture formed has a solid and a liquid phase. Further preferred are embodiments wherein the amount of polypeptide present in the immunoreaction admixture is an amount sufficient to form an excess of epitopes relative to the number of antibody combining sites present in the immunoreaction admixture capable of immunoreacting with those epitopes. (b) The immunoreaction admixture is maintained under biological assay conditions for a predetermined time period such as about 10 minutes to about 16-20 hours at a temperature of about 4 degrees C to about 45 degrees C that, such time being sufficient for the Apo Al present in the sample to immunoreact with (immunologically bind) a portion of the anti-Apo Al antibody combining sites present in the monoclonal antibody to form an Apo Al-containing immunoreaction product. Biological assay conditions are those that maintain the biological activity of the immunochemical reagents of this invention and the Apo AI sought to be assayed. Those conditions include a temperature range of about 4 degrees C to about 45 degrees C, a pH value range of about 5 to about 9 and an ionic strength varying from that of distilled water to that of about one molar sodium chloride. Methods for optimizing such conditions are well known in the art. (c) The amount of Apo Al-containing immunoreaction product that formed is determined, and thereby the amount of Apo Al in the sample. Determining the amount of the Apo AI- containing immunoreaction product, either directly or indirectly, can be accomplished by assay techniques well known in the art, and typically depend on the type of indicating means used. In preferred competitive assay methods, the amount of product determined in step (c) is related to the amount of immunoreaction product similarly formed and determined using a control sample in place of the vascular fluid sample, wherein the control sample contains a known amount of a subject polypeptide, preferably AI1-15 or AI1-21. In another embodiment, the present invention contemplates a double antibody or "sandwich" immunoassay comprising the steps of: (a) Forming a first immunoreaction admixture by admixing a vascular fluid sample with a first antibody, preferably a monoclonal antibody, wherein the antibody and Apo AI/HDL present in the sample are capable of forming a first immunoreaction product that can immunoreact with a subject monoclonal antibody, preferably MAB AI-16. Preferably the first antibody is operatively linked to a solid matrix. (b) Maintaining the first immunoreaction admixture so formed under biological assay conditions for a time period sufficient to form the first immunoreaction product. Preferably, the first- immunoreaction product is then separated from the sample. (c) Forming a second immunoreaction admixture by admixing the first immunoreaction product with: (i) a monoclonal antibody of the present invention, preferably MAB AI-16, and (ii) an Apo Al polypeptide of the present invention, preferably AI1-15 or AI1-21. Preferably, step (ii) is performed prior to step (i) or substantially simultaneously therewith, i.e., within about 5-10 minutes, preferably within about 1-2 minutes. (d) Maintaining the second immunoreaction admixture so formed under biological assay conditions for a true period sufficient to form the second or "sandwich" immunoreaction product. (e) Determining the amount of second immunoreaction product that formed, and thereby the amount of Apo Al in the sample. Preferably, the subject monoclonal antibody of step (c) (i) is labeled, preferably with an enzyme, and the second immunoreaction product formed is a labeled product. In preferred double antibody assay methods, the amount of immunoreaction product determined in step (e) of the double antibody method is related to the amount of immunoreaction product similarly formed and determined using a control sample in place of the vascular fluid sample, wherein the control sample contains a known amount of a subject polypeptide, preferably AI1-15 or AI1-21. Examples The following Examples illustrate, but do not limit, the present invention. 1. Preparation of Antigens A. Polypeptides Polypeptides AI1-10, AI1-15, AI1-21, and AI5-15, were synthesized using the classical solid-phase technique described by Merrifield, Adv. Enzvmol.. 32:221-96 (1969) as adapted for use with a Model 430A automated peptide synthesizer (Applied Biosystems, Foster City, CA) . Polypeptide resins were cleaved by hydrogen fluoride, extracted and analyzed for purity by high-performance liquid chromatograph using a reverse-phase C18 column. (Waters Associates, Mildord, MA) . The amino acid residue sequences of polypeptides AI1-15 and AI1-21 were previously shown in Table 1. The sequences of polypeptides AI1-10 and AI5-15 are shown in Table 2 below. Table 2 Peptide Designation Amino Acid Residue Sequence AI1-10 DEPPQSPWDR AI5-15 QSPWDRVKDLA B. Preparation of Apo AI/HDL HDL was isolated from plasma obtained by plasmaphoresis of normal fasting-donor blood at the local blood bank (San Diego Plasma Center, San Diego, CA) . For that purpose, plasma so obtained was adjusted to contain a final concentration of 5 millimolar (mM) benzamidine, l mM diisopropyl fluorophosphate, 10 mM ethylenediaminetetraacetic acid (EDTA) , 10 milligrams per milliliter (mg/ml) soybean trypsin inhibitor and 10,000 units per ml aprόtinin. The HDL was then isolated from this adjusted plasma by sequential ultracentrifugation using solid potassium bromide (KBr) for density adjustment. First, the adjusted plasma was centrifuged at about 2000,000 x g for 18 to 24 hours and the bottom layer of the resulting supernatant was recovered. Solid KBr was admixed to the bottom layer until the density was greater than 1.063 grams per milliliter (g/ml) . The resulting admixture was then layered under a 0.1% EDTA solution containing KBr at density of 1.063 g/ml and centrifuged at 200,000 x g for more than 48 hours. The bottom layer was again recovered and to it was admixed solid KBr until the density was adjusted to greater than 1.21 g/ml. That adjusted layer was layered 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 admixed until the density was greater than 1.063 g/ml. That adjusted top layer was layered under a 0.1% EDTA solution containing KBr at a density of 1.063 g/ml, and still further centrifuged at 200,000 x g for more than 48 hours. The middle layer was recovered and solid KBr was admixed to it until the density was adjusted to greater than 1.21 g/ml. That adjusted middle layer was layered 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, having a density equal to 1.063 to 1.21 g/ml, was recovered. The recovered HDL was dialyzed against lipoprotein buffer (LLB; water containing 0.15 mM NaCl, 0.3 mM EDTA, and 0.005% alpha-tecopherol) and the resulting Apo AI/HDL was stored under sterile conditions-and used within 3 days. C. Preparation of Delipidated Apo Al Delipidated Apo Al was prepared by organically extracting the lipids from Apo AI/HDL. A sample of the Apo AI/HDL prepared in Example IB was first dialyzed against 0.01 percent EDTA having a pH value of 7.5 overnight (approximately 18 hours) , then dialyzed against 0.003 percent EDTA for approximately 12 hours, and subsequently lyophilized at 10 to 20 milligrams of protein per tube. To each tube were admixed 35 ml of absolute ethanol:anhydrous ether (1:1) at 4 degrees C. Following mixture, the solution was maintained for 20 minutes at -20 degrees C. The solution was then centrifuged for 30 minutes at 1000 x g at zero degrees C, the supernatant was poured off and the Apo Al-containing pellet was retained. The ethanol ether extraction as described above was performed twice again for a total of three extractions. Subsequently, 35 ml of anhydrous ether at 4 degrees C were admixed to the sample. The admixture was maintained for 30 minutes at -20 degrees C, spun at 1000 x g for 30 minutes at - 20 degrees C, and the Apo Al-containing pellet was recovered and dried using nitrogen gas to form delipidated Apo Al. It should be noted that delipidated Apo Al contains not only Apo Al, but also other proteins associated with the HDL, such as Apo Al. D. Preparation of Isolated APO Al Apo Al was isolated from delipidated Apo Al by size fractionation using high pressure liquid chromatography (HPLC) following the procedures of Kinoshita et al., J. Biochem.. 94:615-617 (1983). About 300 g of delipidated Apo Al 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 (Beckman Instruments Inc. , Fullerton, CA) . Fractions containing the isolated Apo Al were stored at minus 20 degrees C. Apo Al was also isolated by delipidation and electrophoresis on polyacrylamide as described herein. E. Preparation of Polvacrylamide-HDL HDL was immobilized in polyacrylamide by admixing the following designated amounts of separately prepared solutions to form a cross-linking reaction admixture: (a) 4.3 ml of LLB containing 50 mg HDL, (b) 1.25 ml water containing 28% (w/v) acrylamide (c) 2.5 ml water containing 2% (w/v) N,N'methylene-bisc arcyla ide (d) 1.25 ml of LLB (e) 1.2 ml water containing 1% (w/v) ammonium persulfate. The cross-linking reaction was allowed to proceed for about 16 hours at 37°C. Because cross- linking did not occur, TEMED (N,N,N',N'-tetramethyl- ethylemediamine) was subsequently admixed producing cross-linking within about 90 minutes at 37°C. The resulting polyacrylamide mass was mechanically homogenized in the presence of 20 ml LLB and then washed with LLB by centrifugation filtration to form polyacrylmide-HDL 2. Generation of Monoclonal Antibodies Balb/c ByJ mice (Scripps Clinic and Research Foundation Vivarium, La Jolla, CA) were immunized intraperitoneally (i.p.) with 50 ug of polyacrylamide- HDL as immunogen in complete Freund's adjuvant (CFA) and 500 units of interferon-gamma followed by " a second and third immunization, each about three weeks apart, in incomplete Freund's adjuvant (IFA) without interferon. About nine months after the last adjuvant-containing immunization, the mice received a boost of 50 ug of native HDL intravenously (i.v.) in normal saline 4 days prior to fusion and a second similar perfusion boost one day later. The animals so treated were sacrificed and the spleen of each mouse was harvested. A spleen cell suspension was then prepared. Spleen cells were then extracted from the spleen cell suspension by centrifugation for about 10 minutes at 1000 r.p.m., at 23 degrees C. Following removal of supernatant, the cell pellet was resuspended in 5 ml cold NH 4 CI lysing buffer, and was incubated for about 10 minutes. To the lysed cell suspension were admixed 10 ml Dulbecco's Modified Eagle Medium (DMEM) (GIBCO) and HEPES [4-(2-hydroxyethyl)-l-piperidineethanesulfonic acid] buffer, and that admixture was centrifuged for about 10 minutes at 1000 r.p.m. at 23 degrees C. The supernatant was decanted, the pellet was resuspended in 15 ml of DMEM and HEPES, and was centrifuged for about 10 minutes at 1000 r.p.m. at 23 degrees C. The above procedure was repeated. The pellet was then resuspended in 5 ml DMEM and HEPES. An aliquot of the spleen cell suspension was then removed for counting. Fusions were accomplished in the following manner using the non- secreting mouse myeloma cell line P3X63Ag8.653.1, a subclone of line P3x63Ag 8.653 (ATCC 1580). Using a myeloma to spleen cell ratio of about 1 to 10 or about 1 to 5, a sufficient quantity of myeloma cells were centrifuged into a pellet, washed twice in 15 ml DMEM and HEPES, and centrifuged for 10 minutes at 1000 r.p.m. at 23 degrees C. ~ Spleen cells and myeloma cells were combined in round bottom 15 ml tubes. The cell mixture was centrifuged for 10 minutes at 1000 r.p.m. at 23 degrees C, and the supernatant was removed by aspiration. Thereafter, 200 ul of 50 percent (weight per volume) aqueous polyethylene glycol 4000 molecular weight (PEG; ATCC Baltimore, MD) at about 37 degrees C were admixed using a 1 ml pipette with vigorous stirring to disrupt the pellet, and the cells were gently mixed for between 15 and 30 seconds. The cell mixture was centrifuged 4 minutes at 700 r.p.m. At about 8 minutes for the time of adding the PEG, 5 ml of DMEM plus HEPES buffer were admixed slowly to the pellet, without disturbing the cells. After 1 minute, the resulting admixture was broken up with a 1 ml pipette, and was incubated for an additional 4 minutes. This mixture was centrifuged for 7 minutes at 1000 r.p.m. The supernatant was decanted, 5 ml of HT (hypoxanthine/thymidine) medium were slowly admixed to the pellet, and the admixture was maintained undisturbed for 5 minutes. The pellet was then broken into large chunks, and the final cell suspension was placed into T75 flasks (2.5 ml per flask) into which 7.5 ml HT medium had been placed previously. The resulting cell suspension was incubated at 37 degrees C to grow the fused cells. After 245 hours 10 ml of HT medium were admixed to the flasks, followed 6 hours later by admixture of 0.3 ml of 0.04 mM aminopterin. 48 hours after fusion, 10 ml of HAT (hypoxanthine/aminopterin/thymidine) medium were admixed to the flasks. Three days after fusion, viable cells were plated out in 96-well tissue culture plates at about 2xl0 4 viable cells per well (768 total wells) in HAT buffer medium as described in Kennett et al., -Curr. Top. Microbiol. Immunol.. 81:77 (1978). The cells were fed seven days after fusion with HAT medium and at approximately 4-5 day intervals thereafter as needed with HT medium. Growth was followed microscopically, and culture supernatants were collected about two weeks later and assayed 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 ul of PBS containing 5 ug/ml Apo AI/HDL were admixed into the wells of microtiter plates. The plates were maintained overnight (about 16 hours) at 4 degrees C to permit the Apo AI/HDL to adhere to well walls. After washing the wells four times with SPRIA buffer (2.68 mM KC1, 1.47 mM KH 2 P0 4 , 137 mM NaCl, 8.03 mM Na 2 HP0 4 , 0.05% Tween-20, 0.1 KlU/ml Traysol, 0.1% BSA, 0.015% NaN 3 ) , 200 ul of SPRIA buffer containing 3% normal goat serum (NGS) and 3% bovine serum albumin (BSA) were admixed to each well to block excess protein binding sites. The plates were maintained for 30 minutes at 20 degrees C, the wells emptied by shaking, and blotted dry to form a solid-support, i.e., a solid matrix to which Apo AI/HDL was operatively affixed. To each well was then admixed 50 ul of hybridoma tissue culture supernatant to form a solid- liquid phase immunoreaction admixture. The admixture was maintained for 2 hours at 37 degrees C to permit formation of solid-phase immunoreaction products. After washing the wells as previously described, 50 ul of 125 I-labeled goat anti-mouse IgG at 0.25 ug protein per ml were admixed to each well to form a labeling reaction admixture. That admixture was maintained for 1 hour at 37 degrees C to permit formation of J- 25 ι- labeled solid-phase immunoreaction products. After washing the wells as previously described, the amount of 125 I-labeled product bound to each well was determined by gamma scintillation. Hybridoma AI-16 was selected from about 16 hybridoma cultures that secreted anti-HDL antibodies into their culture media. Hybridoma AI-16 was determined to have a IgG 2a immunoglobuliin heavy chain and was further characterized as described herein. 3. Monoclonal Antibody Preparation Purification Ascites fluids were obtained from 10 week old Balb/c mice, which had been primed with 0.3 ml of mineral oil and injected intraperintoneally with 5xl0 6 hybridoma cells. The average time for development of ascites was 9 days. Following clarification by centrifugation at 15,000 x g for 15 minutes at 23 degrees C, ascites fluids produced by hybridoma H135D3 were pooled and stored frozen at -20 degrees C. Purified AI-16 monoclonal antibody each of the five hybridomas were prepared by fast protein liquid chromatography (FPLC) using a Pharmacia Mono Q HR5/5 anion exchange column (Pharmacia Fine Chemicals, Piscataway, NJ) using a 0-0.5 molar (M) NaCl gradient in 10 mM Tris, pH 8.0 following directions supplied with the column. Purified Mabs were concentrated using an Amicon stirred ultrafiltration cell (Danvers, MA; PM 30 membrane) to a concentration of 1 mg/ml, dialyzed into PBS (phosphate-buffered saline, pH 7.2) and stored at -70 degrees C. 4. Radioiodination Radioiodination of HDL, Apo Al and immunochemically purified goat anti-mouse Ig was performed enzymatically utilizing the Enzymobead iodination procedure and Enzymobeads obtained -from Biorad, (Burlingame, CA) . The Enzymobead iodination was utilized to characterize the antigens and antibodies for the solid phase radioimmunoassay as discussed below. 5. Apo Al-Cvanogen Bromide Fragment Specificity The Apo Al CNBr fragment specificity of MAB AI-16 was determined by Western blot analysis according to the method in Curtiss et al. , Proceeding of the Workshop on Lipoprotein Heterogeneity, Ed. by Lippel, NIH Publication No. 87-2646 p. 363-377 (1987). Briefly CNBr fragmentation was performed on isolated Apo Al dissolved in 90% formic acid. CNBr was added in a 13,000 molar excess and the reaction mixture was maintained about 15 hours at about 20 degrees C. Following lyophilization, the resulting CNBr fragments were solubilized inn 1% SDS, 0.01 M Tris, pH 8.2 and subjected to isoelectric focusing in 6% polyacrylamide slab gels containing 8 M urea and 2% ampholine (pH 4 to pH 6) as described by Curtiss et al., J. Biol. Chem.. 260:2982-93 (1985). Electrophoretically separated proteins were transferred to nitrocellulose for immunoreaction with MAB AI-16. Production of immunoreaction products was detected by radioiodimated goat anti-mouse Ig followed by autoradiography. The results of these studies indicate that MAB AI-16 does not immunoreact with Apo Al CNBr fragments CNBr2, CNBr3 and CNBr4 but does immunoreact with CNBrl. It should also be noted that these results indicate that MAB AI-16 immunoreacts with isolated Apo Al. 6. MAB AI-16 Immunoreactivity The immunoreactivity of MAB AI-16 for native Apo AI/HDL, deamidated Apo AI/HDL and various polypeptides was examined by a competitive RIA- performed as follows: One hundred ul of PBS (0.15 M NaCl, 0.01 M NaP0 4 , pH 7.2) containing 10 ug/ml Apo AI/HDL were admixed to the wells of microtiter plates. The plates were maintained for 1 hour at 20 degrees C on a rotating platform to allow the Apo AI/HDL to adhere to the wells and form solid supports. After aspirating excess liquid from the wells, 200 ul of block solution (3% BSA, 3% NGS in PBS) was admixed to each well, and the wells were maintained for 30 minutes at 20 degrees C on a rotating platform. Subsequently, the blocking solution was removed by aspiration and the " wells were wash 3 times with SPRIA buffer. To each well was then admixed first 50 ul of PBS containing 3% BSA and various concentrations of competitor antigen, i.e., Apo AI/HDL, or peptide, and second, 50 ul of MAB AI-16 in the form of clarified ascites diluted 1:1.25X10 5 in PBS containing 3% BSA to form competitive immunoreaction admixtures. In control wells either competing antigen or antibody was replaced by PBS containing 3% BSA. The immunoreaction admixtures were maintained about 16 hours at 4 degrees C on a rotating platform to permit formation of solid-phase immunoreaction products. After washing the wells as previously described, 100 ul of 125 I-labeled goat anti-mouse Ig ( 1 5 I-goat anti-mouse Ig diluted to 2X10 5 trichloracetic acid precipitable disintegrations per minute per 100 ul in PBS containing 3% BSA) were admixed to each well. The labeling immunoreaction admixtures so formed were maintained for 4 hours at 4 degrees C on a rotating platform. Subsequently, the wells were washed with SPRIA as previously described and the amount of 125 I-labeled solid-phase immunoreaction product formed was determined. - The ability of MAB AI-16 to immunoreact with Apo AI/HDL, HDL in fresh plasma and polypeptides AI1- 15 was compared by using each as a competitor in the above described RIA. The results of this study are shown in Figure 2. The slopes of the Apo AI/HDL, plasma HDL and polypeptide AI1-15 logit transformed data were found to be -1.96, -2.42 and -2.60. MAB AI- 16 demonstrated 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 are similar if not identical. However, the affinity of the MAB AI-16 for delipidated apo Al was considerably less, indicating that the amino terminal portion of apo Al is altered by delipidation. To compare the immunoreactivity of MAB AI-16 for the polypeptides shown in Tables 1 and 2, a stock solution of each was prepared at a concentration of 10 ug/ml. Fifty microliters of the stock or 2 fold serial dilutions of the stock were used as competitor in the RIA described in this Example. The results of this study, graphically illustrated in Figure 3; indicate that peptides AI1-10 and AI5-15 were not capable of inhibiting the binding of MAB AI-16 to solid-phase APO AI/HDL. However, both peptides AI1-15 and AI1-21 were good competitors. Furthermore, slope analysis of the logit transformed curves indicted that the affinity of MAB AI-16 for peptides AI1-15 and AI1- 21 was the same. The foregoing specification, including the specific embodiments and examples, is intended to be illustrative of the present invention and is not to be taken as limiting. Numerous other variations and modifications can be effected without departing from the true spirit and scope of the present invention.

Claims: What Is Claimed Is: 1. An Apo Al polypeptide consisting essentially of no more than 40 amino acid residues and having as a part of its amino acid residue sequence a sequence represented by the formula: -DEPPQSPWDRVKDLA-. 2. The polypeptide of claim 1 wherein said polypeptide has an amino acid residue sequence represented by a formula selected from the group consisting of: a) DEPPQSPWDRVKDLA, and b) DEPPQSPWDRVKDLATVYVDV. 3. A monoclonal antibody containing anti- Apo Al antibody molecules that immunoreact with: (a) Apo AI/HDL (b) isolated Apo Al (c) Apo Al CNBrl, and (d) the polypeptide DEPPQSPWDRVKDLA, but do not immunoreact with: (e) Apo Al CNBr2, (f) Apo Al CNBr3, (g) Apo Al CNBr4, (h) the polypeptide DEPPQSPWDR, and (i) the polypeptide QSPWDRVKDLA. 4. The monoclonal antibody of claim 3 wherein said antibody molecules are those produced by the hybridoma having ATCC designation . 5. A diagnostic system, in kit form, comprising, in an amount sufficient to perform at least one assay, an Apo Al polypeptide represented by a formula selected from the group consisting of: (a) DEPPQSPWDRVKDLA, and (b) DEPPQSPWDRVKDLATVYVDV. 6. The diagnostic system of claim 5 wherein said polypeptide is operatively linked to a solid matrix. 7. The diagnostic system of claim -5 further comprising, in an amount sufficient to perform at least one assay, a monoclonal antibody containing anti-Apo Al antibody molecules that immunoreact with: (a) Apo AI/HDL (b) isolated Apo Al (c) Apo Al CNBrl, and (d) the polypeptide DEPPQSPWDRVKDLA, but do not immunoreact with: (e) Apo Al CNBr2, (f) Apo Al CNBr3, (g) Apo Al CNBr4, (h) the polypeptide DEPPQSPWDR, and (i) the polypeptide QSPWDRVKDLA. 8. The diagnostic system of claim 7 wherein said antibody molecules are those produced by the hybridoma having ATCC designation . 9. The diagnostic system of claim 7 wherein said antibody molecules are operatively linked to an enzyme indicating means. 10. A diagnostic system, in kit form, comprising, in an amount sufficient to perform at least one assay, a monoclonal antibody containing anti-Apo Al antibody molecules that immunoreact with: (a) Apo AI/HDL (b) isolated Apo Al (c) Apo Al CNBrl, and (d) the polypeptide DEPPQSPWDRVKDLA, but do not immunoreact with: (e) Apo Al CNBr2, (f) Apo Al CNBr3, (g) Apo Al CNBr4, (h) the polypeptide DEPPQSPWDR, and (i) the polypeptide QSPWDRVKDLA. 11. The diagnostic system of claim 10 wherein said antibody molecules are those capable of being produced by the hybridoma having ATCC designation . 5 12. The diagnostic system of claim 11 wherein said antibody molecules are operatively linked to an enzyme indicating means. 13. A method of assaying the amount of Apo Al in a vascular fluid sample comprising the steps of: 0 (a) forming an immunoreaction admixture by admixing a vascular fluid sample with: (i) an anti-Apo Al monoclonal antibody produced by the hybridoma having ATCC designation , and 5 (ii) an Apo Al polypeptide selected from the group consisting of: a) DEPPQSPWDRVKDLA, AND b) DEPPQSPWDRVKDLATVYVDV. (b) maintaining said immunoreaction Q admixture for a time period sufficient to form an Apo Al-containing immunoreaction product, and (c) determining the amount of product formed in step (b) , and thereby the amount of Apo Al in the vascular fluid sample. 14. The method of claim 13 wherein said polypeptide is operatively linked to a solid-matrix, said antibody is operatively linked to an enzyme label, and said product formed in step (b) is a labeled immunoreaction product. 15. A hybridoma that produces antibody molecules capable of immunoreacting with Apo AI/HDL and the polypeptide DEPPQSPWDRVKDLA, and has the ATCC designation .

Inventors: CURTISS LINDA K, SMITH RICHARD S, CURTISS, LINDA, K., SMITH, RICHARD, S.
Applicants: SCRIPPS CLINIC RES, SCRIPPS CLINIC AND RESEARCH FOUNDATION
Application year: 1989
Main classification: G01N33
IPC codes: A61K39, C07K7, C07K14, C07K16, C07K19, C12N5, C12N15, C12P21, C12R1, G01N33