METHODS FOR THE DETECTION OF ANTIBODIES AGAINST MEMBERS OF THE CARDIAC RECEPTOR FAMILY

Abstract

The present invention relates to methods for the detection of antibodies against members of the cardiac receptor family; kits for performing the methods of the invention; the use of the methods of the invention for the diagnosis, therapy and/or prophylaxis of one or more diseases, which are related to one or more members of the cardiac receptor family, and the use of the methods of the invention for a) the identification of modulators of the binding properties of antibodies against members of the cardiac receptor family orb) the identification of therapeutic agents for the treatment of one or more of the said diseases.

Claims

1. A method of detecting in a sample to be investigated the presence and/or the binding properties of analyte antibodies reactive with one or more antigenic molecules, said method comprising the steps of: (a) providing one or more first antigenic molecules selected from the cardiac receptor family (CRF); and (b) providing one or more second antigenic molecules selected from the CRF; and (c) contacting said first antigenic molecules as provided by step (a) and said second antigenic molecules as provided by step (b) simultaneously or successively with the sample to be investigated, wherein analyte antibodies when present in said sample can interact with said antigenic molecules so as to form complexes comprising: [first antigenic molecule]-[analyte antibody]-[second antigenic molecule]; and (d1) prior to, or concurrent with, or subsequent to, step (c), immobilizing the one or more first antigenic molecules using a first immobilizing means to a solid support such that the complexes as formed in step (c) are immobilized; and/or (d2) prior to, or concurrent with, or subsequent to, step (c), labeling said one or more first antigenic molecules with a second labeling means such that the complexes as formed in step (c) are labeled with the second labeling means; and (e) prior to, or concurrent with, or subsequent to, step (c), labeling said one or more second antigenic molecules with a first labeling means such that the complexes as formed in step (c) are labeled with the first labeling means; and (g) detecting or quantifying the presence of complexes [first antigenic molecule]-[analyte antibody]-[second antigenic molecule], formed in or subsequent to step (c), so as to provide indication of analyte antibodies present in said sample.

2. The method of claim 1, further comprising: (f) prior to, or concurrent with, or subsequent to, step (c), providing a reference sample comprising at least one compound; (h) contacting said reference sample with said sample, said one or more first antigenic molecules, said one or more second antigenic molecules or said complexes; and (I1) determining that at least one of the at least one compound is a modulator that is configured to decrease or increase the affinity of said one or more first antigenic molecules or said one or more second antigenic molecules with said analyte antibodies, or (I2) determining that at least one of the at least one compound is not a modulator that is configured to decrease or increase the affinity of said one or more first antigenic molecules or said one or more second antigenic molecules with said analyte antibodies.

3. The method of claim 1, wherein said first antigenic molecules and said second antigenic molecules are identical.

4. The method according to claim 1, wherein said first antigenic molecules and/or said second antigenic molecules are embedded in a membrane environment.

5. The method according to claim 1, wherein the said analyte antibody to be detected in said sample is an endogenous autoantibody or a monoclonal antibody.

6. The method according to claim 1, wherein one or more of the said means selected from the group consisting of said first labeling means, said second labeling means, said immobilization means, and said at least one compound are provided prior to the contacting said antigenic molecules and said analyte antibodies.

7. A kit useful for the performance of the method according to claim 1 comprising: (a) one or more first antigenic molecules selected from the cardiac receptor family as defined in claim 1; (b) one or more second antigenic molecules selected from the cardiac receptor family as defined in claim 1; (c1) immobilization means as defined in claim 1 and/or (c2) second labeling means as defined in claim 1; and (d) first labeling means as defined in claim 1.

8. The kit of claim 7, wherein (a) the one or more said first antigenic molecules are labeled with a the second labeling means; and (b) the said one or more second antigenic molecules are labeled with a the first labeling means.

9. The kit of claim 7, wherein (a) the said one or more first antigenic molecules are immobilized to a solid support; and (b) the one or more said second antigenic molecules are labeled with a the first labeling means.

10. The method according to claim 1, further comprising: diagnosing a presence or onset of a disease related to the cardiac receptor family based upon the detection of step (g).

11. The use of the method according to claim 1, further comprising: identifying a pharmaceutically effective compound for treatment and/or prophylaxis of a disease related to the cardiac receptor family based upon the detection of step (g).

12. A method of using the kit according to claim 7, comprising: diagnosing a presence or onset of a disease related to the cardiac receptor family.

13. A method of using the kit according to claim 7, comprising: identifying a pharmaceutically effective compound for the treatment and/or prophylaxis of a disease related to the cardiac receptor family.

Description

EXAMPLE 1

Construction of Fusion Proteins

EXAMPLE 1A

Construction of Beta1-Adrenergic Receptor-Luciferase-Fusion Protein

[0106] The DNA (Seq. ID No. 1) encoding amino acids 2-551 of the firefly luciferase (Seq. ID No. 2 on plasmid pSP-luc+NF) was amplified by PCR (polymerase chain reaction') using primers P1 (Seq. ID No. 3) and P2 (Seq. ID No. 4) containing EcoRI and BamHI restriction sites, respectively. Plasmid pIRESneo was digested with EcoRI and BamHI restriction endonucleases; the obtained fragment was replaced by the DNA encoding firefly luciferase obtained from the aforementioned PCR, thus resulting in plasmid pIRESneo-Luc. The cDNA (Seq. ID No. 5) encoding amino acids 1-469 of the human beta 1 adrenergic receptor (Seq. ID No. 6) was amplified by PCR using primers P3 (Seq. ID No. 7) and P4 (Seq. ID No. 8) containing EcoRV and EcoRI restriction sites, respectively. pIRESneo-Luc was digested with EcoRV and EcoRI restriction endonucleases and the obtained fragment was replaced by the DNA sequence encoding human beta 1 adrenergic receptor obtained from the previous PCR resulting in vector pIRESneo-B1-Luc containing Seq. ID No. 9 encoding the labelled fusion protein Seq. ID No. 10.

EXAMPLE 1B

Construction of Beta2-Adrenergic Receptor-Luciferase Fusion Protein

[0107] The cDNA (Seq. ID No. 11) encoding amino acids 1-413 of the human beta 2 adrenergic receptors (Seq. ID No. 12) was amplified by PCR using primers P5 (Seq. ID No. 13) and P6 (Seq. ID No. 14) containing Not1 and EcoR1 restriction sites, resp.. pIRESneo-Luc was digested with NotI and EcoRI restriction endonucleases and the obtained fragment was replaced with the DNA sequence encoding beta 2 adrenergic receptor obtained from the previous PCR resulting in vector pIRESneo-B2-Luc containing Seq. ID No. 15 encoding the labelled fusion protein Seq. ID No. 16.

EXAMPLE 1C

Construction of M2 Muscarinic Receptor-Luciferase Fusion Protein

[0108] The cDNA (Seq. ID No. 17) encoding amino acids 1-466 of the M2 muscarinic receptor (Seq. ID No. 18) was amplified by PCR using primers P7 (Seq. ID No. 19) and P8 (Seq. ID No. 20) containing EcoRV and EcoR1 restriction sites, respectively. Plasmid pIRESneo-Luc was digested with EcoRV and EcoR1 restriction endonucleases and the obtained fragment was replaced with the DNA sequence encoding the M2 muscarinic receptor obtained from the previous PCR resulting in vector pIRESneo-M2-Luc containing Seq. ID No. 21 encoding the labelled fusion protein Seq. ID No. 22.

EXAMPLE 1D

Construction of a Fusion Protein of the Beta2-Adrenergic Receptor and the Epitope Recognized by PGA14 Antibodies

[0109] The cDNA (Seq. ID No. 11) encoding amino acids 1-413 of the beta2-adrenergic receptor (Seq. ID No. 12) was amplified by PCR using primers P9 (Seq. ID No. 23) and P10 (Seq. ID No. 24) containing BamHI and HindIII restriction sites, respectively (P10 containing the coding sequence for the 16 amino acid epitope recognized by PGA14 antibodies). pFastBac1 vector was digested with BamHI and HindIII restriction endonucleases and the obtained fragment was replaced by the DNA sequence encoding the fusion protein of beta2 adrenergic receptor and the PGA14 epitope obtained from the aforementioned PCR resulting in vector pFastBac1-B2-PGA14tag.

EXAMPLE 2

Manufacturing of B1-Luc, B2-Luc and M2-Luc Producing Cells

EXAMPLE 2A

Manufacturing of B1-Luc, B2-Luc and M2-Luc Producing HEK 293

[0110] HEK 293 cells were grown in DMEM-F12 supplemented with 10% fetal bovine serum at 5% CO.sub.2 and 37 C. HEK 293 cells were transfected with one of the plasmids pIRESneo-B1-Luc, pIRESneo-B2-Luc or pIRESneo-M2-Luc using FuGENE6 transfection reagent (obtained from Roche Deutschland Holding GmbH, Grenzach-Wyhlen, Germany) according to the manufacturer's instruction. 48 hours after transfection, the selection was started with 0.8 mg/ml G418 (Gibco BRL, Invitrogen). Stable clones expressing high levels of fusion protein were selected.

EXAMPLE 2B

Manufacturing of B1-Luc, B2-Luc and M2-Luc Cell Extracts

[0111] Confluent HEK293 cells (producing either B1-LUC, B2-LUC or M2-LUC) grown in a 75 cm.sup.2 plate were harvested and resuspended in PBS. Cells were washed by centrifugation with PBS. The obtained cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.5; 100 mM NaCl; 10% glycerol; 1% triton X-100). The suspension was centrifuged, the supernatant was removed from debris and stored at 80 C.

EXAMPLE 2C

Manufacturing of Recombinant Baculovirus Expressing the Fusion Protein B2-PGA14Tag

[0112] The B2-PGA14tag sequence obtained from example 1D was transferred to bacmid DNA by site-specific recombination in bacteria. The bacmid was then used to generate a fully recombinant baculovirus in SD insect cells according to the protocols of the manufacturer (Bac-to-Bac expression system manual, Invitrogen).

EXAMPLE 2D

Manufacturing of B2-PGA14Tag Fusion Protein

[0113] Suspensions of High Five insect cells were grown in Express Five serum-free medium to density of 210.sup.6 cells/ml. Cells were then infected (transduced) with recombinant B2-PGA14tag-baculovirus at a multiplicity of infection (MOI) of 1. 72 hours post-infection the cells were harvested and the extract was collected and stored at 80C. as described in Example 2B.

COMPARISON EXAMPLE 3

Immunoprecipitation Assay for B2 Autoantibodies (B2-aAb's)

[0114] The B2-Luc cell extract obtained from example 2B was diluted 20-fold with buffer containing 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1% Triton X-100, 10% glycerol, 5 mg/ml BSA. 100 l of the diluted extract (about 10.sup.7 RLU) was mixed with 10 l of a sample (serum probe) and incubated overnight at 4 C. Immune complexes were subsequently precipitated by addition of 10 l of 10% protein A-sepharose (POROS, Life Technologies) suspension in the same buffer for 1 h at room temperature with shaking. The Protein A-sepharose was precipitated and washed 3 times with 1 ml washing buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Triton X-100). Finally, luciferase activity of the precipitated immune complexes was measured in a Berthold luminometer (AutoLumat Plus LB 953) for 10 sec. Results were expressed as RLU bound. Table 1 shows the activities of B2-aAb positive sera and B2-aAb negative sera.

EXAMPLE 4

Bridge Assays

EXAMPLE 4A

Bridge Assay for the Detection of B2-aAb

[0115] Polystyrene tubes coated with PGA14 antibody (ICI immunochemical) were incubated overnight at 4 C. with 200 l of SF6 insect cell medium containing B2-PGA14tag. After B2-PGA14tag immobilization the tubes were washed twice with 1 ml of buffer 20 mM Tris-HCl pH 7.5, 50 mM NaCl, 10% glycerol. Then each tube was incubated overnight at 4C. with a mixture of 100 l of the same buffer containing 10 mg/ml BSA and 100 l of a sample (serum probe). Tubes were washed twice with 1 ml of the same buffer and incubated overnight at 4C. with 200 l of B2-Luc obtained as described above in example 2B, diluted in the same buffer with BSA (about 4010.sup.6 RLU of luciferase activity). After incubation tubes were washed four times and luciferase activity was measured in a Berthold luminometer (AutoLumat Plus LB 953) for 10 sec. The results shown in Table 1 are expressed as RLU (relative light units) bound and are compared with the results obtained from the conventional assay of Example 3.

TABLE-US-00001 TABLE 1 Signals obtained from positive and negative sera in a conventional immunoprecipitation (Example 3) and the assay of the invention (Example 4). Bridge- Precipitate Assay Precipitate Bridge-Assay Table 1 Example 3 Example 5 Example 3 Example 5 [RLU] Positive Sera Positive Sera Negative Sera Negative Sera 1 82348 33734 3358 553 2 60722 45749 4835 340 3 87350 49528 3289 464 4 69774 38763 5210 533 5 124829 96762 4427 748 6 174171 69349 4008 643 7 105881 58823 4159 462 8 133034 95768 2776 668 9 172382 73908 4858 587 10 375973 187864 4324 523 11 3787 573 12 3389 640 13 3104 707 14 4349 747 15 2898 621 16 4341 602 17 2790 883 18 3464 751 19 2916 511 20 2720 704 21 2200 843 Mean 138646 75025 3676 624 Average (arithm.) Control 128 624

EXAMPLE 4B

Detection Limit of the Assay for B2-aAb from Human Serum

[0116] Each 3 B2-aAb positive and negative sera were diluted in 20 mM Tris-HCl, pH7.5, 50 mM NaCl, 10% glycerol, 10 mg/ml BSA. The assay was performed as described in Example 4A. Background signal was obtained by buffer only.

TABLE-US-00002 TABLE 2 Dilution of B2-aAb positive and negative sera Table 2 [RLU] undiluted 1:3 1:10 1:40 Positive Sera 86342 31807 7324 1751 71164 21127 8632 1097 34843 15402 2584 956 Negative Sera 937 678 478 328 848 652 392 442 1021 748 507 382 Background 368

EXAMPLE 4C

Retrieval Rate of B2-aAb in Human Sera

[0117] Three human B2-aAb positive sera (P1-P3) were mixed 1:1 (K1-K3) with negative sera and analyzed as described in Example 4A (Table 3).

TABLE-US-00003 TABLE 3 Retrieval rate of B2-aAb Table 3 [RLU] P1 86342 K1 937 P1 + K1 51486 P2 71164 K2 848 P2 + K2 32659 P3 34843 K3 1021 P3 + K3 13286

EXAMPLE 4D

Direct Coating of the Antigen to Plastic Surface

[0118] Three human B2-aAb positive sera and three B2-aAb negative sera were analyzed as described in Example 4A, though with the difference, that the B2-PGA14tag fusion complex from Example 2D was incubated prior to detection with direct uncoated polystyrene tubes (ICI immunochemical intelligence GmbH, Berlin). Controls were analyzed exactly as described in 4A (Table 4).

TABLE-US-00004 TABLE 4 Effect of tube coating Table 4 [RLU] P1 427 P2 365 P3 429 K1 508 K2 337 K3 488 Control P1 69754 Control K1 953 Control without serum 392

EXAMPLE 4E

Specific Detection

[0119] Three human B2-aAb positive sera and three B2-aAb negative sera were analyzed as described in Example 4A, with the difference, that the assay was performed with 1:500 diluted HRP-coupled rabbit anti-human IgG (DIANOVA, Hamburg Germany) instead of the B2-Luc extract from Example 2B (Table 5).

TABLE-US-00005 TABLE 5 Effect of the method of detection RLU P1 2632746 P2 2357224 P3 2468791 K1 2586937 K2 2703475 K3 2638210 Control P1 69754 Control K1 953 Control without serum 392

EXAMPLE 4F

Detection Limit of anti-B2 Antibodies

[0120] Known amounts of Antibodies against B2 (sc-569 Santa Cruz, Calif., USA) were diluted in buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10% glycerin, 10 mg/ml BSA), as shown in Table and analyzed as described in Example 4A.

TABLE-US-00006 TABLE 6 Detection Limit of Anti-B2 Antibodies Table 6 Antikrper [ng/ml] RLU 0 24973 1 24396 2 25762 5 28067 10 33372 20 45107 50 76020 100 93872 200 186542 500 391738 1000 557850 2000 948346 5000 1707022 10000 2389831 20000 2867797

EXAMPLE 4G

Hetero-bridge

[0121] Three B2-aAb positive sera were analyzed as described in Example 4A. Detection of the B2-aAb-crossreactivity was performed using incubation with either 200 L of the diluted B2-Luc extract, the diluted B1-Luc extract or the diluted M2-Luc extract from Example 2B (4010.sup.6 RLU luciferase activity of B1 and B2, 0,510.sup.6 RLU luciferase activity of M2) in buffer with BSA at 4 C. over night (Table 7).

TABLE-US-00007 TABLE 7 Crossreactivity of B2-aAb and B1 or M2 receptors B2-receptor- B2-receptor- B2-receptor- 14PGA + 14PGA + 14PGA + B2-receptor Luc B1-receptor Luc M2-receptor Luc Positive Sera 79856 12043 401 83198 3756 998 61461 8237 247 Negative Sera 537 923 269 601 754 393 723 968 344

EXAMPLE 5

Clinical Relevance of B2-aAb Level in Humans

[0122] Table 8 shows the mean average values (and standard deviation) of the weight (adiposity and diabetes-risk) and age of patient sera exhibiting the presence of B2-aAb in humans.

TABLE-US-00008 TABLE 8 B2-aAb positive sera B2-aAb negative (n = 13) sera (n = 80) Weight [kg] Mean average 77.2 +/ 7.4 70.7 +/ 11.4 (arithm.) Age (years) Mean average 77.3 +/ 5.5 85.0 +/ 5.5 (arithm.)