Compilation of Detection Reagents, In-Vitro Method for Detecting Mutated Leptin, and Use of a Detection Reagent

Abstract

The invention relates to a compilation of detection reagents, wherein the compilation comprises a first and a second detection reagent, wherein the first detection reagent binds non-mutated leptin with a first binding value, but does not bind mutated leptin or binds it with a maximum of 50% of the binding value of non-mutated leptin, and wherein the second detection reagent binds both mutated and non-mutated leptin with a second binding value. The invention furthermore relates to an in-vitro method for detecting mutated leptin and the use of a detection reagent.

Claims

1.-23. (canceled)

24. An in-vitro method for detecting mutated leptin, the method comprises the following steps: determining the binding of non-mutated leptin from serum or plasma to human leptin receptor which binds non-mutated leptin, but does not bind mutated leptin or binds it with a maximum of 50% of the binding value of non-mutated leptin, giving a first binding value, wherein the mutated leptin in the amino acid sequence comprises at least one of the following amino acid changes D100Y, N103K, L72S, R105W, G133V, S141C or L161G; and determining the binding of both mutated leptin and non-mutated leptin from serum or plasma to a polyclonal or monoclonal antibody, wherein the polyclonal or monoclonal antibody binds both mutated leptin and non-mutated leptin, giving a second binding value, wherein mutated leptin is present when the first binding value is smaller than the second binding value, and wherein the binding values are determined by means of enzyme-linked immunosorbent assay (ELISA), radio immunoassay (RIA), fluorescence immunoassay (FIA), luminescence immunossay (LIA).

25. The method according to claim 1, wherein the human leptin receptor comprises at least one of isoform A, isoform B, isoform C, isoform D or isoform E.

26. The method according to claim 1, wherein the leptin receptor is a soluble isoform.

27. The method according to claim 1, wherein the determination of the first binding value and the second binding value takes place in one test sample.

28. The method according to claim 1, wherein the determination of the first binding value and the second binding value takes place in different test samples and at different points in time.

29. The method according to claim 1, wherein the determination of the first binding value and the second binding value takes place using different determination methods.

30. The method according to claim 1, wherein a quotient Q formed from the first and the second binding value:
Q=(first binding value)/(second binding value) is between 0.8 and 1.2.

31. The method according to claim 1, wherein a quotient Q formed from the first and the second binding value:
Q=(first binding value)/(second binding value) is 0.8 or less.

32. The method according to claim 1, wherein, for the in-vitro diagnosis of obesity, a quotient Q formed from the first and the second binding value:
Q=(first binding value)/(second binding value) between 0.8 and 1.2 indicates homozygosity in the obesity gene with respect to non-mutated leptin; between 0.3 and 0.7 indicates heterozygosity in the obesity gene with respect to mutated and non-mutated leptin; and less than 0.2 indicates homozygosity with respect to mutated leptin.

33. An in-vitro method comprising using a test system for detecting mutated leptin in an isolated sample of an individual, wherein the test system comprises: a human leptin receptor which binds non-mutated leptin with a first binding value, but does not bind mutated leptin or binds it with a maximum of 50% of the binding value of non-mutated leptin, in the in-vitro diagnosis of obesity, wherein the mutated leptin in the amino acid sequence has at least one of the following amino acid changes D100Y, N103K, L72S, R105W, G133V, S141C or L161G.

34. The method according to claim 33, wherein the test system determines homozygosity or heterozygosity with respect to the obesity gene, which encodes mutated leptin and/or non-mutated leptin, of a mammal.

35. The method according to claim 33, wherein the human leptin receptor comprises at least one of isoform A, isoform B, isoform C, isoform D or isoform E.

36. The method according to claim 33, wherein the leptin receptor is a soluble isoform.

37. The method according to claim 1, wherein the mutated leptin in the amino acid sequence has at least one of the following amino acid changes: D100Y, N103K, or L72S.

38. The method according to claim 1, wherein the leptin receptor is SEQ ID NO: 10.

39. The method according to claim 30, wherein the quotient Q is between 0.9 and 1.1.

40. The method according to claim 30, wherein the quotient Q is between 0.3 and 0.7.

41. The method according to claim 30, wherein the quotient Q is between 0.4 and 0.6.

42. The method according to claim 30, wherein the quotient Q is less than 0.2.

43. The method according to claim 30, wherein the quotient Q is less than 0.1

44. The method according to claim 32, wherein, for the in-vitro diagnosis of obesity, the quotient Q between 0.9 and 1.1 indicates homozygosity in the obesity gene with respect to non-mutated leptin.

45. The method according to claim 32, wherein, for the in-vitro diagnosis of obesity, the quotient Q between 0.4 and 0.6 indicates heterozygosity in the obesity gene with respect to mutated and non-mutated leptin.

46. The method according to claim 32, wherein, for the in-vitro diagnosis of obesity, the quotient Q less than 0.1 indicates homozygosity with respect to mutated leptin.

47. The method according to claim 1, wherein the non-mutated leptin amino acid sequence is SEQ ID NO: 1, and wherein the mutated leptin sequence is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and combinations thereof.

48. The method according to claim 33, wherein said test system is an immunoassay selected from at least one of enzyme-linked immunosorbent assay (ELISA), radio immunoassay (RIA), fluorescence immunoassay (FIA), luminescence immunossay (LIA), Western blot, chromatography, cell assay, particle-based assay immuno precipitation, flow cytometry or biochip.

49. The method according to claim 33, wherein the mutated leptin in the amino acid sequence has at least one of the following amino acid changes: D100Y, N103K or L72S.

50. A method of treating a patient suffering from obesity, comprising: a) providing a first sample isolated from the patient at a first time, b) determining binding of non-mutated leptin to a first detection reagent in said first sample and obtaining a first value, c) providing at least a second sample isolated from said patient at a second time, wherein said second time is later than said first time, d) determining binding of both mutated leptin and non-mutated leptin from the second sample to a second detection reagent and obtaining a second value, e) calculating a quotient Q, wherein
quotient Q=(first binding value)/(second binding value), f) determining the genetic status of the patient based upon quotient Q, wherein a quotient Q of 0.1 or less is an indication that the patient is homozygous, or wherein a quotient Q between 0.8 and 0.1 is an indication that the patient is heterozygous, and g) developing a suitable treatment regimen based upon the patient's genetic status.

Description

FIGURES

[0161] FIG. 1a shows the human leptin precursor sequence, which comprises the mature leptin (amino acids 22 to 187) as well as the N-terminal signal peptide (amino acids 1 to 21).

[0162] FIG. 1b shows the human leptin precursor sequence of a mutated leptin, which contains a change from aspartic acid (D) to tyrosine (Y) at the amino acid position 100.

[0163] FIG. 1c shows the human leptin precursor sequence of a mutated leptin, which contains a change from asparagine (N) to lysine (K) at the amino acid position 103.

[0164] FIG. 1d shows the human leptin precursor sequence of a mutated leptin, which contains a change from leucine (L) to serine (S) at the amino acid position 72.

[0165] FIG. 2a shows the human leptin receptor sequence of isoform A.

[0166] FIG. 2b shows the human leptin receptor sequence of isoform B.

[0167] FIG. 2c shows the human leptin receptor sequence of isoform C.

[0168] FIG. 2d shows the human leptin receptor sequence of isoform D.

[0169] FIG. 2e shows the human leptin receptor sequence of isoform E.

[0170] FIG. 2f shows the fragment Thr20 to Asp839 of the human leptin receptor sequence of isoform B.

EXAMPLES

Example 1a: In-Vitro Detection of Total Leptin

[0171] Polystyrene microtiter plates customary in the trade (Greiner Bio-One GmbH, Frickenhausen, DEC8 type, Flat Bottom, No. 705071) were used for the production of a total leptin ELISA.

[0172] A commercially available monoclonal mouse anti-human leptin IgG1 primary antibody in a concentration of 2 g/ml in phosphate-buffered saline (PBS) at pH 7.4 was adsorptively bound to these polystyrene microtiter plates as solid phase.

[0173] For this, the plates were incubated, cooled (2 C. to 8 C.), overnight in a wet chamber. The antibody solution was then extracted by suction, and unsaturated binding sites on the polystyrene surface were saturated with bovine serum albumin (BSA) in a 1% solution in PBS over 4 hours at pH 7.4. The solution was extracted by suction and the plates were used directly or stored, dried, until use.

[0174] The measurements were carried out in duplicate. All incubations were carried out at room temperature (20 C. to 25 C.).

1. 100 l dilution buffer (50 mM PBS buffer pH 7.4 with 0.5% BSA and 0.05% Tween-20 detergent) was placed in all required wells of the microtiter plates.
2. 20 l dilution buffer was pipetted into the first 2 wells (blank value). Following this, in each case 20 l leptin-standard or 20 l of the sample to be measured was added.
3. The wells of the plate were covered with adhesive film and shaken at 200 to 350 rpm for 1 hour.
4. At the end of the incubation time, the solutions were extracted by suction and the plate was washed five times with 300 l wash buffer WB (50 mM PBS buffer pH 7.4 with 0.5% BSA)/well. The wash buffer remained in the wells for approximately 15 seconds before the extraction by suction.
5. Following the last washing step, 100 l of a horseradish peroxidase (POD) conjugate of an anti-mouse IgG1 antibody was pipetted into each well and the batch was shaken at 200 to 350 rpm for 30 minutes.
6. After completion of this incubation, the plate was washed 5, as described in step 4).
7. 100 l of the substrate solution, stabilized H.sub.2O.sub.2 tetramethylbenzidne, was pipetted into each well.
8. The plate was incubated in the dark for 15 minutes as the substrate is light-sensitive.
9. At the end of the Incubation time, 100 l 0.1 M sulfuric acid was pipetted into each well.
10. The measurement of the color reaction took place within 30 minutes in a photometer for microtiter plates (ELISA Reader) at 450 nm (reference filter 590 nm).

[0175] Samples which achieved higher absorbances than the standard with the highest concentration lay outside the standard curve. For reliable determination, these samples were measured again in a second performance of the test at higher dilution.

[0176] As ELISA standards, recombinant leptin (Mediagnost, Reutlingen, DE) was used in concentrations of 1, 10, 25, 50 and 100 ng/ml.

[0177] The average blank value OD was subtracted from the average values of the optical density (OD) of the standard concentrations and of the samples.

[0178] The standard concentrations (x-axis) were plotted against the measured optical density (y-axis) and a standard curve was generated.

[0179] The leptin concentration of the controls and of the samples respectively is obtained from the standard curve.

Example 1b: In-Vitro Detection of Functional Leptin

[0180] For the detection of functional leptin, the test setup was varied such that a leptin receptor which binds non-mutated leptin, but does not bind mutated leptin or binds it with a maximum of 50% of the binding value of non-mutated leptin, was bound to polystyrene microtiter plates as solid phase.

[0181] A commercially available recombinantly produced molecule with the amino acid sequence Thr20-Asp839 of the human leptin receptor (Recombinant Human Leptin R Fc Chimera, Catalog Number 389-LR/CF, R & D Systems, Minneapolis, Minn., USA) was used as leptin receptor.

[0182] The leptin receptor was adsorptively bound to polystyrene microtiter plates in a concentration of 1.5 g/ml in 10 mM sodium carbonate buffer at pH 9.6 overnight in a wet chamber in a cooled environment (2 C. to 8 C.).

[0183] The coating solution was then extracted by suction, and unsaturated binding sites on the polystyrene surface were saturated with bovine serum albumin in a 1% solution in PBS over 4 hours at pH 7.4.

[0184] The solution was extracted by suction and the plates were used directly or stored dried until use. The measurements were carried out in duplicate. All Incubations were carried out at room temperature (25 C.).

[0185] The samples or controls to be measured were diluted with dilution buffer (50 mM PBS buffer pH 7.4 with 0.5% BSA and 0.05% Tween-20 detergent) in a ratio of 1:10.

1. 100 l standard and 100 l pre-diluted control or sample solution respectively were pipetted into all required wells of the microtiter plates.
2. The wells of the plate were covered with adhesive film and shaken at 350 rpm for 2 hours.
3. At the end of the incubation time, the solutions were extracted by suction and the plate was washed three times with 300 l wash buffer WB/well. The wash buffer remained in the wells for approximately 15 seconds before the extraction by suction.
4. Following the last washing step, 100 l of a biotin conjugate of a commercially available polyclonal rabbit anti-human leptin antibody was pipetted into each well and the batch was shaken at 350 rpm for 30 minutes.
5. After completion of this Incubation, the plate was washed three times, as described in step 3.
6. Following the last washing step, e.g. 100 l of a streptavin horseradish peroxidase conjugate Is pipetted into each well and the batch is shaken at 350 rpm for 30 minutes.
After completion of this incubation, the plate was washed 3, as described in step 3).
7. 100 l of the substrate solution, stabilized H.sub.2O.sub.2 tetramethylbenzidine, was pipetted into each well.
8. The plate was incubated in the dark at 20 C. to 25 C. for 15 minutes as the substrate is light-sensitive.
9. At the end of the incubation time, 100 l 0.1M sulfuric acid was pipetted into each well.
10. The measurement of the color reaction took place within 30 minutes in a photometer for microtiter plates (ELISA Reader) at 450 nm (reference filter 590 nm).

[0186] Samples which achieved higher absorbances than the standard with the highest concentration lay outside the standard curve. For reliable determination, these samples were measured again in a second performance of the test at higher dilution.

[0187] As ELISA standards, recombinant leptin (Mediagnost, Reutlingen. DE) was used in concentrations of 0, 0.2, 1, 2.5, 5, 7.5 and 10 ng/ml.

[0188] The average blank value OD was subtracted from the average values of the optical density (OD) of the standard concentrations and of the samples.

[0189] The standard concentrations (x-axis) were plotted against the measured optical density (y-axis) and a standard curve was generated.

[0190] The leptin concentration of the diluted controls and of the samples respectively is obtained from the standard curve and, after multiplication by the dilution factor, the leptin concentration of the undiluted controls and samples respectively is then obtained.

[0191] Non-mutated functional leptin was measured in this test in a concentration which corresponds to the concentration measured for total leptin in the leptin ELISA.

Example 1c: Evaluation

[0192] Human leptin cDNA (NCBI Reference Sequence: NM_000230.2) was used to generate point mutations by site-directed mutagenesis.

[0193] For this, the Q5 Site-Directed Mutagenesis Kit from New England Biolabs GmbH (Frankfurt am Main, DE) was used, according to the manufacturer's specifications.

[0194] HEK293 cells were transiently transfected with human leptin (pcDNA3.1+leptin_wt) or mutated leptin (pcDNA3.1+leptin_D100Y or pcDNA3.1+leptin_N103K), as described in Sambrook et al. (Molecular cloning: a laboratory handbook, 2.sup.nd edition, 1998, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA).

[0195] 48 h after the transfection, the respective cells were lysed and 100 l of the supernatants were used as samples 1 to 3 in the ELISA tests described in Example 1a and Example 1b.

[0196] Serum samples from 5 blood donors and purified recombinant human leptin and purified recombinant human leptin mutation D100Y were also used.

[0197] Measurement results from various samples using the tests described in Example 1a and Example 1b are shown in Table 1:

TABLE-US-00001 TABLE 1 Comparison of measured values of total against functional leptin Measured Measured values values functional total Sample no.: leptin leptin HEK293 transiently expressed cell culture supernatants 1 Leptin non-mutated 490.2 ng/ml 577.7 ng/ml 2 Leptin mutation D100Y <0.5 ng/ml 193.1 ng/ml 3 Leptin mutation N103K <0.35 ng/ml 37.1 ng/ml recombinant leptin, purified 4 Leptin non-mutated 277.7 g/ml 335.2 g/ml 5 Leptin mutation D100Y 108 ng/ml 8.03 g/ml International WHO standard for human leptin WHO IS NIBSC 97/594 6 nominal 2 1.83 ng/ml 1.84 ng/ml ng/ml: 7 nominal 6 6.05 ng/ml 6.1 ng/ml ng/ml: Serum/plasma samples blood donor 8 CS-2 2.81 ng/ml 2.46 ng/ml 9 DO-2226 4.61 ng/ml 5.05 ng/ml 10 IDB-883- 7.25 ng/ml 8.31 ng/ml 11 IDB-8960 23.96 ng/ml 25.05 ng/ml 12 BB-3935 34.12 ng/ml 34.84 ng/ml

[0198] It can be seen from Table 1 that a distinction can be made between mutated leptin and non-mutated leptin with the compilation according to the invention or the method according to the invention.

[0199] Sample no. 1 contains exclusively non-mutated leptin. The quotient of the concentration of non-mutated leptin (functional leptin) and total leptin is approximately 0.85 (490.2/577.7).

[0200] Sample no. 2 contains exclusively mutated leptin with the mutation D100Y. The quotient of the concentration of non-mutated leptin (functional leptin) and total leptin is <0.01 (0.5/193.1).

[0201] Sample no. 3 contains exclusively mutated leptin with the mutation N103K. The quotient of the concentration of non-mutated leptin (functional leptin) and total leptin is <0.01 (0.35/37.1).

[0202] Sample nos. 4 and 5 contain human leptin in a concentration of 2 ng/ml and 6 ng/ml respectively in accordance with the WHO standard. The quotient of the concentration of non-mutated leptin (functional leptin) and total leptin is 0.99 (1.83/1.84) and 0.99 (6.05/6.1) respectively.

[0203] Sample nos. 8 to 12 are serum/plasma samples from healthy blood donors. The quotient of the concentration of non-mutated leptin (functional leptin) and total leptin is 1.14 (2.81/2.46), 0.91 (4.61/5.05), 0.87 (7.25/8.31), 0.96 (23.96/25.05) and 0.98 (34.12/34.84) respectively.

Example 2

[0204] In the case of an obese infant (not shown in Table 1), a total leptin concentration of 30.1 ng/ml and a concentration of non-mutated leptin (functional leptin) of 0.3 ng/ml were determined in serum/plasma samples. The quotient of the concentration of non-mutated leptin (functional leptin) and total leptin is therefore 0.01 (0.3/30.1).

[0205] In the case of the father and mother, the concentration of non-mutated leptin (functional leptin) and total leptin respectively were then determined in the blood plasma/serum:

[0206] In the case of the father, a total leptin concentration of 2.3 ng/ml and a concentration of non-mutated leptin (functional leptin) of 1.2 ng/ml were determined in serum/plasma samples. The quotient of the concentration of non-mutated leptin (functional leptin) and total leptin is therefore 0.52 (1.2/2.3).

[0207] In the case of the mother, a total leptin concentration of 11.4 ng/ml and a concentration of non-mutated leptin (functional leptin) of 4.8 ng/ml were determined in serum/plasma samples. The quotient of the concentration of non-mutated leptin (functional leptin) and total leptin is therefore 0.42 (4.8/11.4).

[0208] From the measured concentration values of non-mutated leptin and total leptin and, respectively, the quotients calculated therefrom, it is immediately apparent that the father and mother are heterozygous with respect to mutated leptin and non-mutated leptin respectively.

[0209] The child of the father and mother is, however, homozygous with respect to mutated leptin.

[0210] The mutated leptin from father, mother and child was leptin with a D100Y mutation.

[0211] The compilation according to the invention and/or the method according to the invention therefore surprisingly allow an extremely reliable differential diagnosis in the case of obesity, in particular with respect to homozygosity and heterozygosity in the obesity gene which encodes leptin and mutated leptin respectively, at the protein level, in particular using an immunological test system or immunological test method. Immunological test methods can be designed as rapid tests. In particular, the method according to the invention is substantially quicker to carry out than genetic analyses, even within the framework of mass screenings.