METHOD TO IDENTIFY ATHEROGENIC APO(A) ISOFORMS

Abstract: The following description refers to a method and a diagnostic kit for the identification of atherogenic apo(a) isoforms. The method consists in an electrophoretic run of samples to be tested and of a mixture of standards, the transfer (by blotting) of migrated proteins onto an appropriate membrane, and the immunologic identification of apo(a)isoforms. The presence of atherogenic apo(a) isoforms can be easily identified on the basis of their rate of migration (MW 640 kilodalton (kD) max.).

Description: METHOD TO IDENTIFY ATHEROGENIC APO(A) ISOFORMS Field of the invention The present invention refers to a method for the identification of the gene product responsible for the atherosclerotic disease based on the detection of atherogenic apo(a) isoforms. Prior art Lipoprotein(a) [Lp(a)] represents a plasma lipoprotein, consisting of a lipidic moiety and a proteic moiety. The lipidic moiety is similar in structure to low-density lipoprotein (LDL); the proteic moiety consists of two glycoproteins, linked by one or more disulphide bridge bond/s: apo B-100 and apoprotein(a) [apo(a)], typical of Lp(a) and responsible for its immunologic specificity. McLean et al. (Nature, 1987, 300: 132-137) cloned and sequenced the apo(a) gene. They found that the apo(a) structure consists of rings, the so-called kringles, amino acidic sequences containing six cysteines that form three intrachain disulphide bridges, responsible for the characteristic bending. They also found that, in apo(a), the repetition of a variable number of copies (from 3 to 40) of the kringle IV-2 sequence is responsible for the length polymorphism of this gene. The different corresponding apoproteins are defined as isoforms in that they differ in the molecular weight (MW) only. A technique for apo(a) genotype determination from DNA, based on pulsed field gel electrophoresis (PFGE) (Lackner et al., Hum. Mol. Genet, 1993, 2:933-940) proved the existence of at least 34 allels, differing exclusively in the apo(a) gene size, which varies due to the constant units (kringle IV-2 ) repeat. However, tests based on PFGE are too complex and expensive to be applied to clinical practice. For example, the blood for lymphocytes isolation must be freshly drawn, lymphocytes are to be included in agarose and digested with different enzymes, the entire operation is very complex and does not give reproducible results. Various techniques for the identification of apo(a) isoforms based on their different elecrophoretic mobility are now available. A different number of isoforms can be identified depending on the experimental conditions. It follows that the techniques based on the eiectrophoretic mobility of apo(a) differ in resolution power resulting in a different nomenclature assigned to the proteins identified. There is some controversy in the scientific literature regarding the importance of apo(a) polymorphism as a genetic marker for the prediction of coronary heart disease (CHD), rising in part from the non-uniform nomenclature of isoforms, which does not allow a comparison between clinical investigations. In order to investigate the genetic predisposition to the onset of cardiovascular diseases Geroldi et al. (CCA, 221 , 1993, 159-169) proposed a capillary immunoblotting technique useful for the characterisation of apo(a) polymorphism. To develop this method, the authors used an eiectrophoretic run on agarose gel and various standards for apo(a) phenotyping, e.g. isoforms F1 = 400 kD, S1 = 520 kD, S2 = 580 kD, S3 = 640 kD, as described in early kits commercialised by Immuno AG, Vienna, Austria. In this test, standards were run in more lanes of the same electrophoresis. For example, as shown in Fig. 1 (corresponding to Fig. 1 reported in Geroldi D. et al., CCA, 221 , 1993, 159-169) these standards were located in the 1st, 8th and 15th eiectrophoretic lanes. A standard curve to correlate distance with molecular weight had to be plotted (as shown in Fig. 2). Then the migration distance of the serum samples to be examined was measured and, on the basis of the standard curve, the molecular weight of the subject's apo(a) isoform/s was determined. Gazzaruso C. et al., Journal of Cardiovascular Risk, 1998, Vol. 5, No.1 , hypothesized the existence of a cut-off, conventionally chosen between 640 and 655 kD to statistically interpret the polymorphisms observed. Due to the great importance of the detection of atherogenic isoforms, a less laborious and expensive test allowing a rapid determination of the genetic predisposition to the onset of CHD is highly desirable in clinical practice. Summary The Applicant has solved the aforesaid technical problems and has developed a simple method allowing to establish immediately whether the separation of standards is optimal or not and whether the plasma sample under examination contains atherogenic (pathogenic) apo(a) isoforms. Therefore, the present invention refers to a diagnostic method consisting in an eiectrophoretic run on agarose gel of the samples to be tested together with a mixture containing at least the standard having MW of 640 kD, preferably at least two standards having MW of 640 and 655 kD, respectively, and in the transfer onto an appropriate membrane of the isoforms that are later to be immunologically revealed. Said method allows the clinical analyser to identify the presence of the standard having MW of 640 kD or of the two standards having MW of 640 and 655 kD, easily and immediately recognize the presence of apo(a) isoforms below 640 kD, and establish the presence of a pathological sample. The mixture containing at least the two standards having MW of 640 and 655 kD, which is preferred according to the invention, offers the advantage that, when the two standards are distinctly separated, the separation has been optimal and a sharp separation due to the presence of a kringle has taken place. Various clinical investigations proved that the presence of isoforms having MW of 640 kD as a maximum, is a good discriminating index between the group of patients with overt CHD or extracoronary atherosclerosis and the general population. Therefore, the clinical relevance of genetic polymorphism is evidenced only by two genes: the high- molecular-weight normal apo(a) gene and the low-molecular-weight gene, responsible for atherosclerosis. It follows that there is no need for the laborious calculations and procedures required by the methods known in the art. Consequently, it is possible to distinguish normal apo(a) isoforms above 655 kD and pathological apo(a) isoforms below 640 kD with the naked eye and conclude whether the sample is pathological or not. According to a further feature of the present invention, here is provided a diagnostic kit for the determination of atherogenic apo(a) isoforms in one or more blood samples. The kit includes a mixture containing at least the standard having MW of 640 kD, preferably at least the two standards having MW of 640 and 655 kD; an anti Lp(a) antibody either directly conjugated with an enzyme (preferably peroxidase) or revealed with another anti-antibody (antigammaglobulin antibody) conjugated with an enzyme; the substrates necessary for the enzymatic reaction. Description of the figures Figure 1 : This Figure corresponds to Figure 1 reported in Geroldi D. et al., CCA, 221 , 1993, 159-169 and shows the final result of an immunoblotting corresponding to an eiectrophoretic run of various apo(a) isoforms. Lanes 1 , 8, 15, 21 and 27 refer to standard isoforms; the samples to be tested are caused run in the other lanes. As standards, commercial isoforms F1 , S1 , S2, S3 (Immuno AG's kit) are used. Once the eiectrophoretic run has been performed, atherogenicity values and predictions are obtained by plotting a standard curve, as shown in Figure 2. Figure 2: Once the eiectrophoretic pattern of Figure 1 has been obtained, a standard curve, shown in the Figure, is to be plotted. The ordinate refers to the migration values of the standards and the abscissa the molecular weights. Once the curve has been plotted (passing through the four points F1 , S1 , S2 and S3) isoforms migration is measured and their molecular weight determined. Figura 3: This Figure shows the eiectrophoretic run in agarose according to the method of the present invention. Figura 4: This figure refers to the eiectrophoretic run of Figure 3. A horizontal line is drawn at the level of each of the two cut-off standards. The two standards having MW of 640 and 655 kD, where the 640 KD represents the lower cut-off, are visible; they are loaded onto the outermost lanes (lanes A and C). Commercial standards known to the art (loaded onto lane B) are hardly capable of discriminating the pathological isoforms. By simply drawing at least one line between the two extreme 640 kD standards, it immediately appears that the samples in lanes 4, 6, 7, 9, 10, and 12 show an atherogenic apo(a) isoform, whereas sample 8 shows two atherogenic apo(a) isoforms. All the aforesaid samples refer to subjects at genetic risk with ischemic pathology. Detailed description of the invention A salient advantage of the method according to the present invention is that it allows a clear and rapid readout of the eiectrophoretic pattern and enables the clinical analyser to understand at a glance whether the separation of the standards has been good, and predict whether the subject is prone to CHD or any other form of extracoronary atherosclerosis or not. In the method according to the present invention, the mixture used contains at least the standard having MW of 640 kD, preferably at least the two standards having MW of 640 and 655 kD, and more preferably said two standards only. Standards may be prepared from serum samples characterized by the presence of the 640 kD or 655 Kd apo(a) isoform or both. The serum standards containing one or the other isoform separately may also be mixed up to contain both isoforms. Once the eiectrophoretic run has been performed, a good separation of the two standards means that they differ by a kringle, that the separation has been optimal, and that the two bands are exactly referred to molecular weights of 640 and 655 kD, which represent the cut-off range. At that point, the clinical analyser shall do nothing more than to check whether the bands resulting from the eiectrophoretic runs of the samples to be studied or examined are above (higher molecular weight) or below (lower molecular weight) the cut-off. If bands relating to a molecular weight below the cut-off (i.e. lower than 640 kD) are present, they will represent one or more atherogenic isoforms, thus indicating the patient's inherited predisposition to the onset of CHD (Figure 4). If only the standard of 640 kD is used, it will be enough to check whether the samples to be tested contain one or more apo(a) isoforms below the value of 640 kD. In the tests of the prior art, such as for example the test shown in Figure 1 , a mixture of various standards (generally 4 or 5) was used and an eiectrophoretic run was performed. After that, a standard curve had to be plotted (such as for example the one shown in Fig. 2) to identify a correlation between distance and molecular weight. Said standard curve had to be plotted immediately, since the colour starts fading away within few hours. Therefore, the samples could not be stored, which evidently caused technical and organisational difficulties to the clinical analyser. Finally, the migration distance of the serum samples to be examined was measured; on the basis of the standard curve, the molecular weight of apo(a) isoform/s of the subject was determined. Consequently, the number of operations to be performed by the clinical analyser as well as the use of a great number of rather expensive standards made the test performence extremely laborious and complex. To conclude, the test according to the invention offers clear advantages, as it involves a reduced number of operations (curve plotting is no longer necessary) and of the standards to be used. Although the method according to the invention does not require the determination of the standard curve, this may be anyhow determined if extremely precise MW values have to be known i.e. for analytic purposes. The method according to the invention consists essentially in the following steps: a) eiectrophoretic run on agarose gel of a mixture containing at least the standard with MW of 640 kD, preferably at least the two standards with MW of 640 and 655 kD at least in one lane, loaded preferably in the outermost lanes, and of the blood sample/s to be tested in one or more lane/s; b) once the eiectrophoretic run has been performed, transfer of proteins onto a support (blotting) for their identification by specific antibodies; addition of specific antibodies allowing the identification of standards and isoforms; the enzyme-specific substrates used may be as described by Geroldi et al. in CCA, 221 , 1993, 159-169; d) examination of the pattern of apo(a) isoforms obtained; if apo(a) isoforms having MW below 640 kD are present, apo(a) isoforms will be atherogenic. The support used in step b) may be a cellulose membrane or another commercially available support for blotting. The specific antibody used in step c) may be either directly marked or revealed by a marked anti-antibody. Marking may be enzymatic, preferably by peroxidase or phosphatase. A preferred diagnostic method according to the invention is described in more detail below. I) Blood samples Serum or plasma samples are treated with a chelating agent, preferably EDTA. The treated samples may be used either immediately or stored a few months, preferably at -20°C and more preferably at -80°C. Patient's fasting is not required. II) Gel preparation for the eiectrophoretic run The gel may be prepared according to any method known in the art, and preferably as follows. A 0.8 to 2.8%, preferably 1.5%, agarose in a buffer solution is chosen. The buffer used is preferably a buffer for gel, which includes equimolar Tris with boric acid (from 50 to 250 mM, preferably 90 mM); EDTA (from 0.5 to 5 mM, preferably 2 mM); SDS (sodium dodecyl sulphate) (from 0.05 to 2%, preferably 0.1%). Agarose is suspended in the buffer (preferably 100 ml), allowed to boil until complete dissolution, e.g. for a few minutes, poured into an appropriate vessel for immersion electrophoresis (preferably 15 x 20 cm in size) and allowed to cool, e.g. for 1 hr approx. Ill) Samples pretreatment If stored samples are used, only just thawed out serum or plasma (from 5 to 30 μL, preferably 10 μL) will be selected, and diluted with a gel loading buffer (from 20 to 50 μL, preferably 30 μL) containing β-mercaptoethanol (1 part), 0.5% bromophenol blue (2 parts) in a 5% glycerol solution, and a 5% SDS solution (10 parts). The diluted sample solutions are mixed thoroughly and maintained at room temperature for a few minutes, preferably for 20 minutes. IV) Eiectrophoretic run The apparatus used is immersion electrophoresis type, filled up with a buffer solution for electrophoresis containing equimolar Tris with boric acid (from 50 to 250 mM, preferably 45 mM); EDTA (from 0.5 to 5 mM, preferably 2 mM); SDS (sodium dodecyl sulphate) (from 0.05 to 2%, preferably 0.1%). Before the eiectrophoretic run starts, the gel is soaked in the aforesaid solution to equilibrate. A quantity (e.g. 20 μL) of pretreated samples and standards are seeded in wells located in the gel at some distance, preferably 3 cm, from the cathode. Once samples and standards seeding has been completed, the eiectrophoretic run is carried out, generally for 6 to 18 hrs approx., and preferably for 13 to 14 hrs. V) Transfer of proteins (Blotting) Blotting can be performed according to one of the following methods, i.e. Capillary or Diffusion blotting or Electroblotting (also called Western blotting). The solid support may consist of nitrocellulose membranes (0.2 μm), such as for example Hoefer code No. TM-NC2 Roll used in Immuno AG kit or polyvinylidene difluoride (PVDF) membranes with 0.45 micrometer pores available from Millipore (USA) or extra Hybond-C membranes (Amersham, USA). Prior to use, the PVDF membrane must be kept immersed in pure methanol for 3 to 5 sec and then in the buffer used for electrophoresis for 10 min approx. Capillary blotting Capillary blotting is carried out according to usual procedures, by applying a weight of 2 kg approx. on blotting paper layers for 10 hrs approx. Diffusion Blotting Proteins are transferred to the membrane by diffusion. According to this method, the membrane, i.e. nitrocellulose membrane, is soaked in eiectrophoretic buffer and placed in contact with the agarose gel in a sealed box, in order to avoid drying, at a temperature of 45-50°C, for a time ranging from 30' to 2 hours. The membrane is then treated as usually with dry powder milk, as described. Electroblotting Electrotransfer is performed by a Trans-blot cell apparatus (Biorad, USA). The membrane is kept immersed in the transfer buffer (5 to 30 mM, preferably 10 mM, Tris-base; 5 to 50 mM, preferably 40 mM, glycine; 5% methanol; and water, q.s.) for 10 min. approx. Transfer starts by applying a 50 V current for 5 to 10 hrs, preferably for 7 hrs. VI) Identification of apo(a) isoforms Two variants are possible for the reactions with the antibody: A) polyclonal anti Lp(a) antibody from sheep (Immuno AG test) subsequently revealed with peroxidase-conjugated rabbit anti-sheep immunoglobulins (Dako, Denmark); or B) monoclonal anti Lp(a) peroxidase-conjugated antibody available from Boehringer (Germany). The membrane is kept immersed for a few hours in the same buffer solution as used for the eiectrophoretic run, added with detergent (e.g. 0.2% Tween-20) and powdered skim milk (Difco). Then, it is incubated under gentle stirring for 2 to 3 hrs with dilute antibodies solutions (from 1 :100 to 1 :5,000) in the buffer solution containing powdered skim milk and detergent. The membrane is washed at least twice for 10 min with the buffer added with 1% NP-40, 0.1 % SDS e 0.5% desoxycholate. If variant a) is chosen, incubation with the secondary antibody will be necessary, followed by two washings. The identification is performed by immunoperoxidasic reaction of precipitating chromogenes (diaminobenzidine) or preferably by chemiluminescence, according to ECL detection system (Amersham). According to a further embodiment, samples and standard detection is performed by immunoprecipitation, according to one of the methods known in the art. According to a preferred embodiment, immunoprecipitation is performed by samples and standard electrophoresis on 0.8% agarose placed on pre-cut supports such as Gel-Bond films (22X11 cm, SPA S.p.A., Milano Italy). Agarose is dissolved in electrophoresis buffer (Tris equimolar with 90 mM boric acid, 2 mM EDTA, 0,1 % SDS). The serum samples (10 μl) and the standards are loaded at the catode of the gel. Two eiectrophoretic buffer strips are then placed on the gel, aligned with the electrodes and the eiectrophoretic separation is then performed at 250 Volts, for the time needed for optimal separation of the samples (usually 2-6 hours). After electrophoresis, the gel is placed in contact with adsorbing paper soaked with an anti Lp(a) antibody (i.e. Dako anti-Lp(a) antibody diluted 1 :3 in electrophoresis buffer) in a humid box, for a time comprised between 30' and 6 hours, preferably 2 hours. The adsorbing paper is then removed and the gel is washed with electrophoresis buffer few times (at least 3 times). The agarose gel is then dried and proteins are stained with dyes such as Coomassie Blue 0.2% in a solution comprising methanol and acetic acid in H20 (450 ml Methanol, 450 ml H 2 0 and 100 ml CH 3 COOH) and destained, according to methods known in the art. The interpretation of the data is performed as described before and similarly to what is shown in figure 4, placed that at least the 640 or 655 standards or both, are eletrophoretically run together with the samples. According to a further embodiment, the eiectrophoretic run of serum samples and standards is performed under non-reducing conditions (i.e. without β- mercaptoethanol); lipoproteins are then stained with a lipid-specific dye, such as Sudan Black. In this case serum samples are chosen among those with a Lp(a) content > 5-10 mg/100 ml (> 5-10%) as determined by commercial kits. By this method, all plasma lipoproteins are stained, and the eiectrophoretic pattern shows HDL, as well as Lp(a), VLDL and LDL. The presence of atherogenic Lp(a) isoforms is visualized as described before, and similarly to what is shown in figure 4, placed that at least the 640 or 655 standards or both, are eletrophoretically run together with the samples. The present invention also provides a diagnostic kit for the determination of atherogenic apo(a) isoforms and, therefore, for the identification of subjects genetically prone to coronary or extracoronary disease. The kit according to the invention preferably consists of: i) two standards having MW of 640 and 655 kD; ii) monoclonal antibody marked with anti-lipoprotein(a)-POD peroxidase, Boehringer Mannheim Biochemicals, Cat. No.1399322; iii) reagents for Western blotting identification (ECL, Amersham); iv) and, optionally, all concentrated solutions for buffers preparation. Particular embodiments of the present invention are described in the following examples. Example 1 I) Blood samples Serum or plasma samples were treated with EDTA. The treated samples were frozen at -80°C and used after a few months' storage. II) Gel preparation for the eiectrophoretic run 1.5% Agarose (Sigma A-9539) (100 ml) was prepared in a buffer for gel containing equimolar Tris with boric acid (90 mM); EDTA (2 mM); and 0.1 % SDS. Agarose was suspended in 100 ml buffer and allowed to boil for a few minutes until complete dissolution, poured into an appropriate vessel for immersion electrophoresis (preferably 15 x 20 cm in size) and allowed to cool for 1 hr approx. III) Samples pretreatment Only just thawed out serum or plasma (10 μL) was diluted with a gel loading buffer (30 μL) containing β-mercaptoethanol (1 part), 0.5% bromophenol blue (2 parts) in a 5% glycerol solution, and a 5% SDS solution (10 parts). The diluted sample solutions were mixed thoroughly and maintained at room temperature for 20 min. IV) Eiectrophoretic run The apparatus used was an immersion electrophoresis type, filled up with a buffer solution for electrophoresis containing equimolar Tris with boric acid (45 mM); EDTA (2 mM); SDS (0.1%). Before the run started, the gel had been kept immersed in the aforesaid solution to equilibrate for 1 hr approx. Pretreated samples and standards (20 μL) were seeded in wells, located in the gel at a 3 cm distance from the cathode. Once samples and standards seeding had been completed, the eiectrophoretic run was carried out at 60 V, 0.04 A, for 13 hr. V) Transfer of proteins (Blotting) Blotting can be performed by electroblotting (Western blotting), using, as solid support, extra Hybond-C membranes (Amersham), as follows: the membrane was maintained in the transfer buffer (10 mM Tris-base, 40 mM glycine, 5% methanol; and water, q.s.) for 10 min. After assembling for transfer with Trans-blot cell apparatus (Biorad), transfer was started by applying 50 V current for 7 hrs approx., then 90 V current, for 1 hr or more. VI) Identification of apo(a) isoforms The membrane was incubated overnight in TBS (Tris buffer at pH 7.6: 20 mM Tris- base, 137 mM NaCI, 3.8 ml 1 M HCI made up to one litre) added with 0.2% Tweed-20 and 5% skim milk (Difco) in a refrigerator (30 ml/100 cm2 in a vessel possibly similar in size to the filter). The buffer where the membrane had been incubated was replaced with fresh TBS added with 1 % detergent Nonidet P40 (NP-40), 5% skim milk and 1 :500 anti- Lp(a)-POD antibody from Boehringer (30U/ml solution), in an extremaly small volume, e.g. 2 ml approx./100 cm 2 filter. The membrane was then incubated at room temperature under gentle stirring for 2 to 3 hrs; the vessel was covered with parafilm in order to avoid drying of the membrane. Periodical controls were done to check whether the buffer was homogeneously dispersed on the filter. The filter was washed twice for 10 min at room temperature in TBS added with 1% NP-40, 0.1 % SDS, and 0.5% deoxycholate. Finally the identification is performed. Chemiluminescence is conduced according to ECL or ECL Plus detection system (Amersham). Clinical interpretation The clinical interpretation is extremely simplified since the Mendelian inheritance of the multiallelic monogenic genetic character (apo[a]) (until today, the possible isoforms are at least 34) becomes biallelic monogenic (normal isoform and atherogenic isoform) of easy applicability to clinical genetic studies. From the technical or methodological point of view, in fact, the method and relevant kit refer to the identification of apo(a) isoforms. Data interpretation (isoforms position) will allow the physician to decide whether the subject suffers from or is prone to the onset of coronary or extracoronary pathology. It is the physician's evaluation to make the most correct interpretation of the test result on the basis of the patient's symptoms, and psychological aspects. Clinical applications Investigations on the population The test allows the identification of asymptomatic subjects genetically prone to the onset of coronary or extracoronary diseases, due to their inherited atherogenic isoform. Major efforts are just concentrated on said subjects to eliminate modifiable risk factors and attain an early determination of coronary alterations before irreversible damages develop (new diagnostic protocols starting from the atherogenic isoform biological entity). Diseases commonly regarded as predisposing the coronary disease Common diseases, such as diabetes and arterial hypertension seem to be a factor that accelerates the development of coronary diseases in genetically prone subjects due to their inherited atherogenic isoforms. As far as said patients are concerned, it will be of vital importance to apply the aforesaid preventive measures and to correctly treat the original diseases. Atherosclerotic disease of extracoronary districts and other applications The method and the kit according to the invention allow the identification of subjects at risk for extracoronary atherosclerosis: cerebrovascular diseases (ictus), supra-aortal trunks atherosclerosis, peripheral obliterative artheropathy. Preliminary studies evidenced the prognostic role of atherogenic isoforms also for diabetic microangiopathy, very likely due to the thrombophilic effect of lipoprotein Lp(a). Therefore, the performance of this test is of vital importance for patients with diabetes mellitus expecially in view of the development of chronic complications. Information relating to legal and occupational medicine A clinical manifestation of coronary or extracoronary disease is sudden death; the atherogenic isoform might be predictive of said event and/or allow post-mortem evaluation with all its medico-legal consequences. In occupational medicine, some manufacturing processes have been found to favour ischemic cardiopathy. The identification of genetically prone subjects might be a reason for unfitness. Etiological therapy of subjects with atherogenic isoforms The goal of possible etiological therapies of coronary disease will consist in inhibiting the expression of the atherogenic isoform at the hepatic level (by means of a new drug or a genie therapy).

Claims: Claims 1. Diagnostic method for the identification of atherogenic apo(a) isoforms comprising the following steps: eiectrophoretic run of the samples to be tested and of a mixture containing at least the apo(a) standard with MW of 640 kD; once the eiectrophoretic run has been performed, transfer of the proteins (blotting) onto a support allowing their identification by specific antibodies; addition of specific antibodies allowing the apo(a) isoforms identification; detection of the presence of apo(a) jsoforms having MW below 640 kD. 2. The method as claimed in claim 1 , wherein said mixture of standards contains at least the two standards having MW of 640 and 655 kD, respectively. 3. The method as claimed in claim 1 , wherein said mixture contains only the two apo(a) standards having MW of 640 and 655 kD. 4. The method as claimed in claim 1 consisting of the following steps: I) serum or plasma samples treatment with a chelating agent and storage until use; II) preparation of the gel for the eiectrophoretic run with 0.8% to 2.8% agarose in a buffer for gel containing equimolar Tris with boric acid (from 50 to 250 mM), EDTA (from 0.5 to 5 mM); SDS (from 0.05 to 2%) by resuspending agarose in said buffer, and allowing it to boil until complete dissolution, and pouring it into an appropriate vessel for immersion electrophoresis; III) dilution of freshly thawed serum or plasma (from 5 to 30 μL) sample with a buffer solution (from 20 to 50 μL) containing β-mercaptoethanol (1 part), 0.5% bromophenol blue (2 parts) in a 5% glycerol solution, and a 5% SDS solution (10 parts); IV) carrying out an eiectrophoretic run using an apparatus for immersion electrophoresis, filled up with a buffer solution for electrophoresis containing equimolar Tris with boric acid (from 50 to 250 mM); EDTA (from 0.5 to 5 mM); SDS (from 0.05 to 2%); pretreated samples and standards are seeded in wells located in the gel at some distance from the cathode; once seeding has been completed, the eiectrophoretic run starts; V) transfer of proteins onto a solid support is performed by capillary blotting or electroblotting; VI) identification of apo(a) isoforms by using A) polyclonal anti Lp(a) antibody from sheep subsequently revealed with peroxidase-conjugated rabbit anti-sheep immunoglobulins or B) peroxidase-conjugated monoclonal anti Lp(a) antibody; the identification is completed by immunoperoxidasic reactions of precipitant chromogens or by chemiluminescence; VII) establishing the presence of atherogenic apo(a) isoforms in the samples visually. 5. Diagnostic kit for the determination of the presence of atherogenic apo(a) isoforms in a blood samples, comprising a mixture containing at least the apo(a) standard having MW of 640 kD; an anti Lp(a) antibody either directly conjugated with an enzyme or revealed with another anti-antibody conjugated with an enzyme; the substrates necessary for the enzymatic reaction. 6. The diagnostic kit as claimed in claim 5, wherein said mixture of standards contains at least the two apo(a) standards having MW of 640 and 655 kD. 7. The diagnostic kit as claimed in any of claims 5 and 6, wherein said anti Lp(a) antibody is directly conjugated with peroxidase or phosphatase. 8. The diagnostic kit as claimed in any of claims 5 - 7, wherein standards and isoforms are identified by chemiluminescence.

Inventors: GEROLDI DIEGO, GEROLDI, DIEGO
Applicants: GEROLDI DIEGO, UNIV PAVIA, GEROLDI, DIEGO, UNIVERSITA' DEGLI STUDI DI PAVIA
Application year: 2000
Main classification: G01N33
IPC codes: G01N33