Classifications

• • 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

• This invention is directed to the discovery that certain apolipoprotein (ALP) peptide fragments (or domains or moieties) are immunogenically active and can be used to produce type-specific antibodies that recognize ALP's.
• ALP apolipoprotein
• the resulting fragments and ALP type-specific antibodies are useful in another aspect of the invention, assay systems for quantitating ALP levels.
• Lipoproteins are aggregates of lipids and protein which circulate in the blood and are the means by which lipids are transported within the body.
• the lipid portions of these aggregates consist essentially of cholesterol and triglyceride.
• Serum lipoproteins are classified according to their density. These classes include very low density lipoproteins (VLDL) , also known as pre-beta lipoproteins; low density lipoproteins (LDL) , also known as beta-lipoproteins; and high density lipoproteins (HDL) , also known as alpha-lipoproteins.
• VLDL very low density lipoproteins
• LDL low density lipoproteins
• HDL high density lipoproteins
• a fourth class of lipoproteins is chylomicron (CHYLO) , stable droplets containing 86% triglyceride fat, 3% cholesterol, 9% phospholipids, and 2% protein. Chylomicrons are found in the intes ⁇ tinal lymphatics and blood during and after meals, and are the form in which absorbed long-chain fats and cholesterol are transported from the intestine.
• CHYLO chylomicron
• lipoproteins One of the functions of lipoproteins is to carry water insoluble substances, such as. cholesterol and cholesterol esters, for eventual cellular utilization. While all cells require cholesterol for growth, excess accumulation of cholesterol by cells is known to lead to certain diseases, including atherosclerosis. It is now known that the amount of total serum cholesterol can be correlated with the incidence of atherosclero ⁇ sis. However, since all lipoprotein classes contain varying amounts of cholesterol, total serum choles ⁇ terol determination is a complex average of the amount that -each lipoprotein class contributes to the total lipoprotein population of the serum.
• U.S. Patent 4,167,467 to Golias describes an elec- trophoresis method for determining the concentration of HDL free cholesterols in body fluids and simultane ⁇ ously determining the concentration of VLDL and LDL free cholesterols in the fluid sample.
• the method includes applying a direct current across the fluid medium, applying a developing substrate to the electrophoresed lipoproteins, and quantitatively determining the concentration of each lipoprotein free cholesterol.
• the method of Golias purports to be an improvement over the prior art in that direct and simultaneous measurement of each lipoprotein free cholesterol fraction is achieved without precipitation of each fraction.
• U.S. Patent 4,185,963 to Heuck describes a method for determining lipids in blood serum wherein the VLDL, CHYLO, and HDL are extracted from the serum with a polycation, followed by measuring the lipid content of the LDL in the serum.
• U.S. Patent 4,215,993 to Sanders describes a method for isolating HDL from LDL in human serum, followed by quantitative determination of HDL choles ⁇ terol. LDLs are precipitated from the serum without the addition of metal ions to the sample. The pre ⁇ cipitating reagent lowers the pH of the human serum approximately to the isoelectric point of the LDL through the use of an organic buffer.
• U.S. Patent 4,309,188 to Bentzen describes a separation method wherein LDL and HDL are separated on a microcolumn containing a support which has a sulphated polysaccharide covalently bound thereto. Elution with a first pH buffered solution collects the LDL; elution with a second pH buffered solution col ⁇ lects the HDL. Subsequently, LDL/HDL ratios can be determined.
• U.S. Patent 4,039,285 to Teipel discloses a single-sample method for determining concentrations of individual lipoprotein classes and lipids in blood by turbidimetric measurement.
• the ionic strength of the- mixture is raised in steps to cause progressive dis ⁇ solution of each class of complex from that of the highest density lipoprotein to the lowest density lipoprotein.
• Measurement of the turbidity due to the insoluble complexes present at each step allows the concentration of each lipoprotein class and lipid in the blood sample to be calculated.
• Apolipoprotein is the protein moiety which binds the lipid moiety to form the holoprotein. At present, a number of types and subtypes of ALP have been identified.
• Apolipoprotein A includes subtypes A, and A 2 • Apo-A-, is the major apolipoprotein of HDL and is thought to occupy a surface position on HDL particles, surrounding a neutral lipid core. It is also known that Apo-A, activates lecithin:cholesterol acyl trans- ferase, the cholesterol-esterifying enzyme of plasma involved in the production of mature circulating HDL. As mentioned above, there is an inverse correlation between plasma HDL levels and development of coronary artery heart disease. See also, Heiss, G. e_t al. , Circulation, 62:Suppl. IV, 116 (1980).
• the second most abundant apolipoprotein of HDL is Apo-A 2 . It has been reported that Apo-A, binds less total HDL lipid than does Apo-A ⁇ ; however, in an interaction between these apolipoproteins, Apo-A- increases the binding capacity of Apo-A-L. Morrisett et al. , "Lipoproteins: Structure and Function," Annual Review of Biochemistry, 44_: 183, 196-198 (1975). Highly purified LDL has been shown to contain a single molecule of a very large protein, apolipopro ⁇ tein B (Apo-B) , having a molecular weight estimated to be 250,000 to 500,000 daltons.
• Apo-B apolipopro ⁇ tein B
• LDL plays a key role in the -transport of cholesterol to the peripheral tissues where it is bound to cellular receptors and ingested by an endocytosis process. LDL is also known to play an important role in the patho ⁇ logical uptake and deposition of cholesterol, with very high concentrations of LDL implicated as the causative agent of some forms of human atherosclero ⁇ sis. Additionally, moderate elevations of LDL over long periods of time may be an important factor in the development of most human atherosclerosis.
• Apo-B is known to play a number of important roles in triglyceride and cholesterol transport and is required for the forma ⁇ tion and secretion of triglyceride-rich lipoproteins from human liver. It is the only protein always found on LDL and contains a site complementary ' to, and recognized by, the LDL receptor. There is also evi ⁇ dence demonstrating that the presence of a certain allele of pig Apo-B correlates strongly with lipid deposition and plaque formation in pig artery. See Rapacz et al., Exp. and Mol. Path., 27: 429 (1977).
• Apolipoprotein C includes subtypes Apo-C,, Apo-C 2 , and Apo-C.
• Apo-C has been shown to be part of the protein moiety of plasma lipoproteins (Eisenberg, S. et al., J. Biol. Chem., 254: 12603 (1979)).
• Apo-C which makes up 40-80% of the total protein of CHYLO and VLDL, is present in plasma HDL, and plays an important role in the regulation of the activity of the enzyme system lipoprotein lipase.
• apolipoprotein E particularly central to the removal or uptake process for circulating cholesterol- laden lipoproteins.
• Apo-E apolipoprotein E
• An important function of Apo-E is its medi ⁇ ation of cellular uptake of lipoproteins through specific surface receptors. See Mahley, R. W., Klin. Klischer. , 61; 225 (1983).
• Apo-E is known to bind to the low density lipoprotein receptor of fibroblast and various peripheral cells, thereby affecting intra- cellular cholesterol metabolism. It also binds speci ⁇ fically to a hepatic plasma membrane receptor, the Apo-E receptor, and functions as a prime determinant in chylomicron remnant clearance.
• Apolipoprotein E includes three major iso forms, Apo-E 2 , Apo-E.,, and Apo-E.. Amino acid se ⁇ quence analysis has demonstrated that the three iso forms differ in their primary structure. Variant forms of Apo-E 2 have been described, with all forms of Apo-E 2 demonstrating reduced LDL receptor binding activity and reduced Apo-E receptor binding activity. Further, these abnormal forms of Apo-E 2 are associated with the genetic abnormality type III hyperlipopro- teinemia, which appears to be partly due to the defective clearance of cholesterol-rich remnant lipo ⁇ proteins (Weisgraber, H. K. et al. , J. Biol. Chem., 258: 12341 (1983)). This evidence suggests that Apo-E performs a critical role in cholesterol and lipid metabolism as well.
• U.S. Patent 4,399,217 to Holmquist et al. describes a process for the determination of serum lipoproteins by an immuno- enzymatic method.
• Apolipoprotein antibodies are fixed on a support.
• Serum sample' is added, in combination with enzyme-labeled specific apolipoprotein.
• Elimina ⁇ tion of all reagent not fixed on the support, followed by measurement of the enzymatic activity bound to the support produces an indirect determination of the amount of specific apolipoproteins present in the sample being analyzed in a competitive assay.
• the assay requires "type-specific” antibody and specific labeled antigen (apolipoprotein) and a com ⁇ petitive assay system.
• the "type-specific” antibody is produced by immunizing rabbits with purified apo ⁇ lipoprotein obtained by serum lipoprotein fractions separated by ultracentrifugation on a density gradi ⁇ ent.
• ultracentrifugation is somewhat deficient with regard to obtaining highly pure apolipoprotein fractions.
• the "type- specificity" of the resulting antibodies produced by rabbit immunization is deficient as well.
• a need has continued to exist for a highly accurate, truly type-specific assay for apolipoproteins and high specificity antibodies for the same.
• the inventors then successfully- synthesized the polypeptide fragments, conjugated the fragments with carrier proteins, and produced truly type-specific, non-cross-reactive antibodies by immunization.
• Figure 1 shows three peptide sequences (A, B, and C) which are each specific for Apo-A-. as well as three synthetic peptides used to raise Apo-A, specific anti ⁇ bodies (A 1 , B' , and C) .
• Figure 2 shows a peptide sequence which is spe ⁇ cific for Apo-E, as well as a synthetic peptide used
• Figure 3 shows three peptide sequences (A, B, and C) which are specific for Apo-C 3 as well as three synthetic peptides used to raise Apo-C specific antibodies (A 1 , B' , and C ) .
• Figure 4 shows two peptide sequences (A and B) which are each specific for Apo-B as well as two synthetic peptides used to raise Apo-B specific antibodies (A' and B').
• Figure 5 shows two peptide sequences (A and B) which are specific for Apo-A 2 as well as two synthetic peptides used to raise Apo-A 2 specific antibodies (A* and B' ) .
• Figure 6 are diagrams of Western Immuno- blots obtained from SDS-PAGE gels, utilizing the Specific Protocol IV below.
• Figure 7 is a diagram representing immunodot blots obtained from varying amounts of different apolipopro ⁇ teins spotted onto nitrocellulose filters.
• Peptide moieties (fragments) chosen from the determined amino acid sequences of various apolipopro ⁇ teins constitute the starting point in the development comprising the present invention.
• the amino acid sequence for apolipoprotein A has been reported in the literature by Brewer, H. B., Jr. et al., Biochem. Biophys. Res. Commun., 80: 623-630 (1978).
• the amino acid sequence of human Apo-A 2 is published in Morrisett et al. , "Lipoproteins: Structure and Func ⁇ tion," Annual Review Biochemistry, 44: 183-207 (1975).
• the complete amino acid sequence for apo ⁇ lipoprotein E 2 has been reported by Rail, S.
• apolipoprotein C The complete amino acid sequence for apolipoprotein C is also known. Brewer, et al. , J. Biol. Chem. , 249: 4975-4984 (1974) . Amino-terminal sequences for cer ⁇ tain proteolytic fragments derived from apolipoprotein B are known as well, reported by LeBoeuf, R.C. et al. , FEBS Letters, 170: 105-108 (1984).
• Peptide domains corresponding to various segments within the naturally occurring amino acid sequence are obtained.
• the peptide fragments are synthesized by the well known solid phase peptide synthesis described by Merrifield, J. Am. Chem. Soc, 85: 2149 (1962) and Stewart and Young, in Underlying Solid Phase Peptide Synthesis (Freeman, San Francisco, 1969), pp. 27-62, incorporated by reference herein.
• peptide fragment is meant to include both synthetic and naturally-occurring amino acid sequences representing portions of the natural protein, typi ⁇ cally containing 8-20 amino acids in the sequence, more preferably 10-16 amino acids, with 12-15 member oligopeptides representing the preferred chain length.
• derivativeable from a naturally-occurring amino acid sequence are meant to include both synthetic sequences and sequences obtained by fragmenting naturally-occurring sequences to obtain isolated sequences which do not exist in nature as such.
• oligopeptides that, in addition to the chosen sequence, may contain one or more amino acids that may not be present in the naturally-occurring sequence.
• This invention also relates to novel polypetides generated by covalent coupling of two or more distinct peptide moieties (fragments) resulting in an immunospecific peptide comprising two or more domains which are non-contiguous in the natural ALP proteins.
• immunogenicity the property that endows a substance with the capacity to provoke a humoral immune response and the degree to which the substance possesses this property
• immunospecificity the ability of the antibodies provoked by the immune response to bind to specific lipoprotein or apolipo ⁇ protein
• apolipoprotein type-specific is meant to include highly specific immunoreactivity limited to a particular ALP.
• A, B, and C represent three naturally-occurring amino acid sequences found in Apo-A,.
• a 1 , B 1 and C* represent the three peptides which were synthesized and evaluated for their ability to elicit antibody production and represent the actual synthetic peptides which were utilized as the anti ⁇ genic material. Note that each of the synthesized sequences contains an additional cysteine residue at the carboxy terminus to allow for coupling of carrier protein.
• A represents the amino acid sequence for Apo-E 2 which was evaluated for immunogenicity and immunospecificity for Apo-E 2 .
• the synthesized peptide fragment A' includes a cysteine residue at the carboxy terminus to allow for coupling to a carrier protein.
• peptide sequences corres ⁇ ponding to A, B, and C were evaluated for immunogeni ⁇ city and immunospecificity to apolipoprotein C->.
• Each synthetic peptide includes a cysteine residue at the carboxy terminus to allow for coupling to a carrier protein (A 1 , B' and C ) .
• a and B represented therein were evaluated for immunogenicity and immunospecificity to apolipoprotein B.
• the synthe ⁇ tic peptides further corresponding thereto.
• A' and B* include a cysteine added to each carboxy terminus for coupling to a carrier protein.
• a and B represent two naturally-QCCurring amino acid sequences found in Apo-A 2 , which were evaluated for immunogenicity and immunospecificity for Apo-A 2 «
• the synthesized peptide fragments A* and B' include a cysteine residue at the carboxy terminus to allow for coupling to a carrier protein.
• the peptide fragments include one or more epi- topes, i.e. immunogenic domains (determinants), capa ⁇ ble of producing the desired ALP-type specific antibo ⁇ dies and may be the peptide fragment corresponding exactly to the natural sequence or varying to a degree which does not impact on immunogenicity and immuno- specificity relevant to their use according to this invention.
• labelable resi ⁇ due is meant to include a residue such as tyrosine which is present in or has been introduced into the desired sequence in order to make possible the affix ⁇ ing of a detectable label, for example, a radioisotope such as 125I, an enzyme, or a fluorescent tag.
• M is a pharmaceutically acceptable cation or a lower (C,-C..) branched or unbranched alkyl group
• R 2, R3 and R4 are the same or different and selected from the group consisting of hydrogen and a lower ( -Cg) branched or unbranched alkyl group;
• X is the amino acid sequence or peptide fragment as described above;
• amino acid residues may be in their protected or unprotected form, using appropriate amino or carboxyl protecting groups.
• Useful cations M are alkali or alkaline earth metallic cations (i.e., Na, K, Li, 1/2 Ca, 1/2 Ba, etc.) or amine cations (i.e., tetraalkylammonium, trialkylammoniu , where alkyl can be C,- ? ).
• variable length peptides may be in the form of the free amines (on the N-terminus) , or acid-addition salts thereof.
• Common acid addition salts are hydrohalic acid salts, i.e., HBr, HI, or more preferably, HC1.
• ELISA titers for all of the synthetic peptide antisera prepared according to the present invention (using 5 ng peptide per well) is greater than 1:1600 and generally greater than 1:12000.
• ELISA titers for the Apo-A ⁇ antisera using 100 ng per well of native Apo-A was greater than 1:12000.
• the antigenic material (the peptide fragment) may be coupled to a carrier protein such as albumin or key ⁇ hole limpet hemocyanin (KLH) , utilizing techniques well known and commonly used in the art.
• a carrier protein such as albumin or key ⁇ hole limpet hemocyanin (KLH)
• KLH key ⁇ hole limpet hemocyanin
• the carrier protein is KLH, linked to the antigenic material through a cysteine residue.
• the antigenic material can be admix ⁇ ed with an immunologically inert or active carrier.
• Carriers which promote or induce immune responses such as Freund's complete adjuvant, can be utilized.
• the antigenic material the peptide fragment hapten-carrier protein conjugate
• the detection of appropriate antibodies may be carried out by testing the antisera with appropriately labeled tracer-containing molecules. Fractions that bind tracer-containing molecules are then isolated and fur ⁇ ther purified if necessary. These antibodies may then be utilized in various immunoassays to identify and quantitate specific ALP's.
• the immunoassays within the scope of the present invention include both ⁇ com ⁇ petitive assays and immunometric assays.
• the antibody need not necessarily be (although is preferably) generated with the antigenic peptide fragments of the invention.
• the same synthetic peptide used to generate the antibody and containing additionally a tyrosine residue is radioiodine labeled through the tyrosine residue and comprises the tracer-containing molecule.
• Radioactive isotopes which are particularly useful are 3 H, 125 I, 131 I, 32 P, 35 S, 14 C, 51 Cr, 36 C1, 57 Co,
• the peptide sequence may also be labeled using fluorescent labels, enzyme labels, free radical labels, avidin-biotin labels, or bacteriophage labels, using techniques known to the art (Chard, supra) .
• Typical fluorescent labels include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine.
• Typical chemiluminescent compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and the oxalate esters.
• Typical bioluminescent compounds include luciferin, luciferase, and aequorin.
• Typical enzymes include alkaline phosphatase, beta-galactosidase, glucose-6-phosphate dehydrogenase, maleate dehydrogenase, glucose oxidase, and peroxi- dase.
• ELISA enzyme- linked immunosorbent assay
• EMIT enzyme-multiplied immunoassay
• the ALP type-specific antibodies of the present invention are also useful for use in an immunometric assay, also known as sandwich immunoassay.
• immunometric assays include simultaneous sandwich, forward sandwich, and reverse sandwich immunoassays. Each of these terms is well understood by those skill ⁇ ed in the art.
• a sample is first incubated with a solid phase immunoabsorbent containing antibody against the ALP. Incubation is continued for a period of time sufficient to allow the ALP in the sample to bind to the immobilized antibody in the solid phase. After the first incubation, the solid phase immunoabsorbent is separated from the in ⁇ cubation mixture and washed to remove excess ALP and other interfering substances which also may be present in the sample. Solid phase immunoabsorbent-containing ALP bound to the immobilized antibodies is subsequent ⁇ ly incubated for a second time with soluble labeled antibody cross-reactive with a different domain on the ALP.
• a reverse sandwich assay the sample is ini ⁇ tially incubated with labeled antibody, after which the solid phase immunoabsorbent containing immobilized antibody cross-reactive with a different domain on the ALP is added thereto, and a second incubation is- carried out.
• the initial washing step required by a forward sandwich assay is not required, although a wash is performed after the second incubation.
• Reverse sandwich assays have been described, for example, in U.S. Patents 4,098,876 and 4,376,110.
• a simultaneous sandwich assay In a simultaneous sandwich assay, the sample, the immunoabsorbent having immobilized antibody thereon and labeled soluble antibody specific to a different domain are incubated simultaneously in one incubation step.
• the simultaneous assay requires only a single incubation and does not require washing steps.
• the use of a simultaneous assay is a very useful tech ⁇ nique, providing ease of handling, homogeneity, reproducibility, linearity of the assays, and high precision. See U.S. Patent 4,376,110 to David et al. , incorporated by reference herein.
• the sample-containing antigen, solid phase immunoabsorbent with immobilized antibody and labeled soluble antibody are incubated under conditions and for a period of time sufficient to allow antigen to bind to the immobilized antibodies and to the soluble antibodies.
• the s ' pecific concentrations of labeled and immobilized antibodies, the temperature and time of incubation, as well as other such assay conditions, can be varied, depending upon various factors including the concen ⁇ tration of antigen in the sample, the nature of the sample, and the like. Those skilled in the art will be able to determine operative and optimal assay con ⁇ ditions for each determination by employing routine experimentation.
• solid phase immunoabsorbents which have been employed and which can be used in the pres ⁇ ent invention.
• Well known immunoabsorbents include beads formed from glass, polystyrene, polypropylene, dextran, nylon, and other material; tubes formed from or coated with such materials, and the like.
• the immobilized antibodies may be covalently or physically bound to the solid phase immunoabsorbent, by techni ⁇ ques such as covalent bonding via an amide or ester linkage or by absorption.
• the soluble antibody may be labeled with any detectable label, such as a radiolabel, a fluorescent label, an enzyme label, a free radical label, or a bacteriophage label.
• a detectable label such as a radiolabel, a fluorescent label, an enzyme label, a free radical label, or a bacteriophage label.
• the label is a radiolabel or an enzyme label.
• the immunometric assays require two separate and distinct antibodies which are type-specific as regards apolipoprotein.
• One of these antibodies is bound to the solid phase support while the other is detectably labeled.
• the two different antibodies although ALP type-specific . , are cross-reactive with different domains within the anti ⁇ genic protein.
• the two different antibodies may be prepared by using two different synthetic sequences which correspond to different immunogenic and immunospecific segments within the amino acid sequence of the ALP. For example, referring to Figure 1, both the synthetic peptide A 1 and synthe ⁇ tic peptide B'. have been found to be both immunogenic and immunospecific as regards Apo-A,.
• the use of antibodies to each synthetic sequence, one bound to a substrate and the other detectably labeled, is useful in the various sandwich assays.
• antibodies which are type-specific to the same apo ⁇ lipoprotein, but cross-reactive with different domains by producing the antisera in two different species. for example, in rabbit and in mouse, utilizing the same synthetic peptide sequence.
• delayed immunometric assays can also be utilized, as are, for example, described in Chu, U.S. Patent 4,289,747, an Wolters, U.S. Patent 4,343,896.
• kits may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, test tubes, and the like.
• container means such as vials, test tubes, and the like.
• Each of said container means comprises one of the separate elements to be used in the method.
• one of said container means may comprise im unoabsorbent-bound antibody. These antibodies may be bound to a separate solid phase immunoabsorbent or directly to the inner walls of a container.
• a second container may comprise detectably labeled peptide fragment in lyophilized form or in solution.
• the carrier may also contain, in addition, a plurality of containers each of which comprises dif ⁇ ferent, predetermined known amounts of antigen or peptide fragments. These latter containers can then be used to prepare a standard curve from which can be interpolated the results obtained from the sample containing the unknown amount of antigen.
• Peptides conjugated to KLH have been used to immunize laboratory animals, such as rabbits, in order to generate monospecific antibodies capable of binding to the respective apolipoproteins.
• approximately 100 icrograms (ug) of the particular protein to which antibodies are to be raised is admixed with Freund's complete adjuvant and inoculated into the footpads and at subcutaneous sites in rabbits, ten to fourteen days later a comparable amount of protein admixed with incomplete Freund's adjuvant is inoculated subcutaneously; after an addi ⁇ tional ten to fourteen days, the animal is inoculated with an additional 50-100 ug of protein mixed in a ten percent solution of aluminum hydroxide. The animal is subsequently immunized at four week intervals with 50-100 ug of protein.
• the specific antibody titers developed against the respective proteins have been measured by (a) an enzyme-linked immunosorbent assay (ELISA) and by (b) the ability of the antibody to immunoprecipitate radiolabeled protein molecules.
• ELISA enzyme-linked immunosorbent assay
• 10-100 nanograms of the test antigen that is, the particular protein being tested
• is bound to a plastic surface by air-drying of a protein solution on the bottoms of wells in microtiter dishes (Falcon Products or Bellco Products) .
• test antisera Serial dilutions of test antisera are incubated within the wells, unbound antibodies are removed by washing and the bound anti ⁇ bodies are then incubated with an enzyme-conjugated second antibody preparation directed against the immunoglobulins of the species in which the test anti- serum was generated. The amount of enzyme bound in each well is then quantitated by an appropriate color assay. In such testing, sera with high titers of antibody against the test antigen can be diluted several thousand fold and will still show significant color development. It has been found that the storage temperature (room temperature, 4°C, -20°C, and -80°C) of the serum sample to be tested will result in detec ⁇ tion of the same level of apolipoprotein, regardless of temperature.
• room temperature room temperature, 4°C, -20°C, and -80°C
• the test antigen is labeled with a radioisotope such as 125I.
• a radioisotope such as 125I.
• a fixed quantity of the radiolabeled antigen is then incubated with serial dilutions of the test antisera.
• the immunocomplexed antigen is precipitated, either using a second anti ⁇ body directed against the immunoglobulins of the. species in which the test serum was generated, or using a fixed Staphylococcus aureus bacterial sus ⁇ pension, or using Staphylococcus aureus protein A immobilized onto beads.
• serum with high titers of specific antibody can be diluted several thousand fold and still precipitate significant amounts of the radio ⁇ labeled antigen.
• the specific titers of antibodies in test sera samples are determined by subtracting the values (color or precipitated radioactivity) obtained with serum from nonimmunized animals or from animals immunized with a protein which is unrelated to the test protein.
• the specificity of the antisera as well as the immunological relatedness of different proteins can be estimated in either assay by examining the relative effects of serum dilutions on the extent of binding of the antigen.
• proteins are first subjected to a polyacrylamide gel electrophore- sis (PAGE) in the presence of a denaturing and a reducing agent.
• PAGE polyacrylamide gel electrophore- sis
• the proteins are then transferred onto a nitrocellulose membrane electrophoretically in a conducting solution containing Tris-HCl, glycine and methanol (or isopropanol) .
• the nonspecific binding sites on the nitrocellulose membrane are blocked using a Tris-NaCl buffer containing bovine serum albumin (3%-5% wt/vol).
• An appropriate dilution of the specific antiserum in the above buffer is then con ⁇ tacted with the nitrocellulose membrane in order to allow the binding of the antibody molecules to the specific protein.
• Unbound antibody is washed using a Tris-buffered salt solution supplemented with a non- ionic detergent such as Tween 20.
• the protein band(s) that has (have) bound antibody can then be visualized by (1) colorimetric means using an enzyme-conjugated second antibody directed against the immunoglobulins of the species in which the specific antibody was ⁇ generated or (2) autoradiography using radiolabeled protein A from Staphylococcus aureus to bind to the immunoglobulin molecules that had bound to the specif ⁇ ic antigen on the nitrocellulose membrane.
• This technique of immunoblotting allows one to identify the molecular species being recognized by a specific antibody.
• this technique can be applied to establish the type-specificity of an anti-peptide antibody serum by using a mixture of apolipoproteins (such as in plasma or serum) as the test sample.
• immuno dot blot involves spotting a small volume of an aqueous solution on a nitrocellulose filter, fixing the protein on the nitrocellulose using an' acid alcohol mixture such as acetic acid:isopropanol:water (10:20:70 v/v) and then performing the antibody binding technique described above.
• the serum samples to be tested are first treated by boiling and reduction by adding 0.1% SDS solution with 10 mM dithiothrietol to the serum. This solution is then placed in boiling water for 10 minutes. Upon cooling at room tempera ⁇ ture, the sample is then treated with an equal volume of commercially available pansorbin. The sample can then be assayed for APL.
• PBS phosphate buffered saline
• rabbits are bled 7 and 14 days after the third injection (or days 28 and 35 from the beginning) .
• Boosts are the same as the third injection, in A1(0H) 3 , and rabbits are bled 7 and 14 days later.
• the amount of peptide/rabbits which will induce a good response in the boosts can be cut down to 50 ug KLH (coupled to peptide) per rabbit.
• O-phenylen- ediamine dihydrochloride (OPD) solution in phosphate-citrate buffer containing 0.01%
• Block the nonspecific binding sites by soaking in a blocking buffer, i.e. Buffer B containing 3% BSA and 0.1% NP40.
• Buffer B is 10 mM Tris base, 150 mM NaCl, pH 7.4
• Electrophoretically-purified human apolipoproteins A-l, B and C-III were purchased from Calbiochem- Behring, Inc. (LaJolla, California).
• Solutions containing 100 ug per ml of these proteins and a solution containing 100 ug/ml of purified human serum VLDL (as source of apolipoprotein E) were incubated in 1% sodium dodecyl sul ⁇ ate (SDS) at 100°C for 10 min.
• SDS sodium dodecyl sul ⁇ ate
• Synthetic peptide antisera derived from peptide A* from the Apo-C ⁇ sequence listed in Figure 3 yielded the result shown in lane 2 and a similar result was obtained with anti ⁇ sera derived with peptide B' listed in Figure 3.
• Synthetic peptide antisera from the Apo-B peptide A' shown in Figure 4 yielded the result shown in lane 4.
• the positions for the bands indentified in each lane correspond to the known molecular weights for Apo-A, (24,000), Apo-B (200,000 to 500,000), Apo-E (33,000), and Apo-C 3 (12,000) .
• Immunodot blot analyses were performed using puri ⁇ fied Apo-A 1 , Apo-B and Apo-C III (C,) to corroborate the specificity of the synthetic peptide antisera. This procedure was performed as described in "General Protocol III" and the autoradiographic results are schematically represented in Figure 7. Strips of nitrocellulose filter paper were spotted with differ ⁇ ent quantities of Apo-B, Apo-A, or Apo-C, as indicated by the numbers, in micrograms, adjacent to each spot.
• a mixture of unrelated protein (1 ug per spot) was included on each filter strip and at the bottom of each strip, a mixture of the standards (1 ug) plus the indicated ug quantities of Apo-B, Apo-A, and Apo-C .
• Filter strips were subsequently incubated with synthe ⁇ tic peptide antisera (1:1000 dilution) and processed as described in- "Protocol III.”
• Strips are labeled from left to right Apo-B (with Apo-B spots and tested with antisera derived from peptide A 1 of Figure 4; the same result was obtained, although not shown here, using antisera derived with peptide B' of Figure 4) , Apo-A, (with Apo-A, spots and tested with antisera derived from peptide A 1 of Figure 1; the same result was obtained although not shown here, using antisera derived from peptide B* of Figure 1), Apo-C 3 (with Apo-C, spots and tested with antisera derived
• Both a sandwich ELISA and a competition ELISA were developed using antibodies raised against certain synthetic apolipoprotein peptide fragments.
• step (b) In the competition ELISA, wells were coated with antibodies raised against synthetic peptides specific for Apo-A, .
• the serum was diluted as in step (a) above.
• Serial dilutions of the serum were incubated in the wells with 5 to 15 nanogra s of biotinylat.ed Apo-A, protein (tracer) and the extent of competition for binding to the immobilized antibody was monitored after rinsing the wells using streptavidin-horseradish peroxidase conjugate binding followed by color formation.
• Standard curves using sera with known concentra ⁇ tions of Apo-A were generated for both assays (a) and (b) in order to quantitate the amounts of Apo-A, in test serums samples.
• Antibodies raised against synthetic peptides specific for apolipoprotein A were affinity purified and used to develop a single, antibody (biotinylated) ELISA for serum testing.
• the affinity column was prepared with the synthetic peptide and Sepharose.
• the specific antibody was eluted with 50 mM glycine at a pH of 2.5.
• the peak fraction of eluted antibodies was shown by SDS-PAGE to consist predominantly of heavy and light chains from IgG, with a small amount of IgM heavy chains.

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