Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/775—Apolipopeptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

• the present invention relates to antibodies and polypeptides useful for immunologically determining the amount of Apo Al in a vascular fluid sample.
• 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) .
• VLDL very low-density lipoproteins
• HDL high-density lipoproteins
• Apo Al is now believed to mediate the removal of cholesterol from tissues and to activate LCAT.
• 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.
• 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.
• 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.
• Such 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.
• epitope A 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
• the present invention c ⁇ ntemplates 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
• 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:
• 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
• Still further contemplated is a method of assaying the amount of Apo Al in a vascular fluid sample comprising the steps of:
• 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.
• 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:
• 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.
• Tl through T4 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 ( ⁇ ) , 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.
• 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.
• Al polypeptide refers to a polypeptide whose amino acid residue sequence is homologous (similar in structure) to a portion of the Apo Al molecule.
• 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-,
• 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.
• Each polypeptide designation used herein indicates the corresponnding residues in the Apo Al protein from which the polypeptide amino acid residue sequence was derived.
• 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
• 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.
• a suitable, selectively removable protecting group is utilized for amino acids containing a reactive side group such as lysine.
• 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.
• any remaining terminal and side group protecting groups are removed sequentially or concurrently, to afford the final polypeptide.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• an Apo Al polypeptide of the present invention 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) .
• 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.
• 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 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.
• 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).
• 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.
• a preferred method for determining monoclonal antibody affinity is by equilibrium - competitive inhibition analysis.
• 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). -
• step (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.
• 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.
• 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.
• 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. —
• DMEM Dulbecco's minimal essential medium
• 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.
• hybridoma useful in producing a subject monoclonal antibody i.e., MAB AI-16
• 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 .
• ATCC American Type Culture Collection
• 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.
• 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.
• 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.
• 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.
• a diagnostic system of the present invention typically includes a label or indicating means capable of signaling the formation of a complex-containing a polypeptide or antibody molecule of the present invention.
• complex refers to the product of a specific binding reaction such as an antibody-antigen or receptor-ligand reaction. Exemplary complexes are immunoreaction products.
• 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.
• fluorochromes such as fluorescein isocyanate (FIC) , fluorescein isothiocyante (FITC) , 5-dimethylamine-l- naphthalenesulfonyl chloride (DANSC) , t
• the indicating group is an enzyme, such as horseradish peroxidase (HRP) , glucose oxidase, or the like.
• HRP horseradish peroxidase
• glucose oxidase or the like.
• additional reagents are required to visualize the fact that a receptor-ligand complex (immunoreactant) has formed.
• 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 ⁇ 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.
• a beta emitter such ⁇ :L1 indium of 3 H.
• labeling of, polypeptides and proteins is well known in the art.
• 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.
• 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.
• 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.
• the specific binding agent binds the reagent species when that species is present as part of a complex.
• the specific binding agent is labeled.
• the agent is typically used as an amplifying means or reagent.
• 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.
• 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.
• 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.
• 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. ⁇
• 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.
• 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.
• 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.
• exemplary assay methods are described herein, the invention is not so limited.
• 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 monoclonal antibody of the present invention preferably AI-16
• an Apo Al polypeptide of the present invention preferably AI1-15 or AI1-21.
• 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.
• 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.
• 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.
• 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.
• 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.
• Determining the amount of the Apo AI- containing immunoreaction product can be accomplished by assay techniques well known in the art, and typically depend on the type of indicating means used.
• 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.
• the present invention contemplates a double antibody or "sandwich" immunoassay comprising the steps of:
• 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.
• a subject monoclonal antibody preferably MAB AI-16.
• the first antibody is operatively linked to a solid matrix.
• step (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.
• 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.
• the subject monoclonal antibody of step (c) (i) is labeled, preferably with an enzyme, and the second immunoreaction product formed is a labeled product.
• 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.
• 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
• 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) .
• amino acid residue sequences of polypeptides AI1-15 and AI1-21 were previously shown in Table 1.
• sequences of polypeptides AI1-10 and AI5-15 are shown in Table 2 below.
• 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) .
• 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 apr ⁇ tinin.
• the HDL was then isolated from this adjusted plasma by sequential ultracentrifugation using solid potassium bromide (KBr) for density adjustment.
• 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.
• LLB lipoprotein buffer
• 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.
• delipidated Apo Al contains not only Apo Al, but also other proteins associated with the HDL, such as Apo Al.
• 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,
• 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.
• TEMED N,N,N',N'-tetramethyl- ethylemediamine
• 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
• 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.
• CFA complete Freund's adjuvant
• IFA incomplete Freund's adjuvant
• 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.
• DMEM Dulbecco's Modified Eagle Medium
• HEPES [4-(2-hydroxyethyl)-l-piperidineethanesulfonic acid] buffer
• 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.
• PEG polyethylene glycol 4000 molecular weight
• 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.
• HAT hypoxanthine/aminopterin/thymidine
• 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
• 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.
• 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.
• 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 ⁇ - labeled solid-phase immunoreaction products.
• 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.
• 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.
• PBS phosphate-buffered saline, pH 7.2
• 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.
• 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.
• 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:
• the immunoreaction admixtures were maintained about 16 hours at 4 degrees C on a rotating platform to permit formation of solid-phase immunoreaction products.
• 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
• the labeling immunoreaction admixtures so formed were maintained for 4 hours at 4 degrees C on a rotating platform.
• the wells were washed with SPRIA as previously described and the amount of 125 I-labeled solid-phase immunoreaction product formed was determined.
• MAB AI-16 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.

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