COMPLEX-SPECIFIC STANDARDIZATION OF IMMUNOLOGICAL METHODS FOR THE QUANTIFICATION OF S100A12

Abstract

The present invention relates to mutants of S100A12 having at least one mutation in the high-affinity calcium binding hand or the low-affinity calcium binding hand or the zinc binding region. The present invention also relates to methods of detecting S100A12 dimers in a sample as well as methods of diagnosis using the S100A12 mutant of the invention, as well as to diagnostic compositions and kits comprising such an S100A12 mutant. The present invention further relates to a method of generating an antibody that specifically binds to an S100A12 dimer using the S100A12 mutant of the invention, as well as to an antibody that specifically binds to an S100A12 dimer.

Claims

1. A mutant of S100A12 comprising at least one mutation in one of the following regions: a) the high-affinity calcium binding hand of S100A12; and/or b) the low-affinity calcium binding hand of S100A12; and/or c) the zinc binding region of S100A12.

2. The mutant of claim 1, wherein the mutant is capable of forming S100A12 dimers.

3. The mutant of claim 1 or 2, wherein the mutant does not significantly form S100A12 tetramers or S100A12 hexamers.

4. The mutant of any one of the preceding claims, wherein the mutant has a lower binding affinity to calcium as compared to the corresponding wild type S100A12.

5. The mutant of any one of the preceding claims, wherein the mutant has a lower binding affinity to zinc as compared to the corresponding wild type S100A12.

6. The mutant of any one of the preceding claims, wherein the S100A12 is a mammalian S100A12, preferably a human, canine, equine, or bovine S100A12, preferably a human S100A12.

7. The mutant of any one of the preceding claims, wherein the mutant comprises at least one mutation at a) an amino acid position corresponding to a position ranging from amino acid position 58 to amino acid position 74 of the human S100A12 as set forth in SEQ ID NO: 1; or b) an amino acid position corresponding to a position ranging from amino acid position 16 to amino acid position 34 of the human S100A12 as set forth in SEQ ID NO: 1; or c) an amino acid position corresponding to a position ranging from amino acid position 83 to amino acid position 92 of the human S100A12 as set forth in SEQ ID NO: 1

8. The mutant of any one of the preceding claims, wherein the mutant comprises at least one mutation at an amino acid corresponding to amino acid His16, Ser19, Lys22, His24, Asp26, Thr27, Glu32, Asp62, Asn64, Asp66, Glu73, His86, or His90 of human S100A12.

9. The mutant of any one of the preceding claims, wherein the mutant comprises at least one mutation at an amino acid corresponding to amino acid Glu32, Asp62, Asn64, Asp66, or Glu73 of human S100A12.

10. The mutant of any one of the preceding claims, wherein the mutant comprises a glycine or an alanine at an amino acid position corresponding to position His16, Ser19, Lys22, His24, Asp26, Thr29, Ser31, Glu32, Asp62, Asn64, Asp66, Glu73, His86, or His90 of human S100A12.

11. The mutant of any one of the preceding claims, wherein the mutant comprises an alanine at an amino acid position corresponding to position His16, Ser19, Lys22, His24, Asp26, Thr29, Ser31, Glu32, Asp62, Asn64, Asp66, Glu73, His86, or His90 of human S100A12.

12. The mutant of any one of the preceding claims, wherein the mutant comprises a glycine or an alanine at an amino acid position corresponding to position Glu32, Asp62, Asn64, Asp66, or Glu73 of human S100A12.

13. The mutant of any one of the preceding claims, wherein the mutant comprises an alanine at an amino acid position corresponding to position Glu32, Asp62, Asn64, Asp66, or Glu73 of human S100A12.

14. The mutant of any one of the preceding claims, wherein the mutant comprises an alanine at an amino acid position corresponding to position Asn64 or Glu73 of human S100A12.

15. The mutant of any one of the preceding claims, wherein the mutant comprises a set of amino acid mutations selected from a) Glu73.fwdarw.Ala; b) Asn64.fwdarw.Ala; c) Asn64.fwdarw.Ala, Glu73.fwdarw.Ala;

16. The mutant of any one of the preceding claims, wherein the mutant has a sequence identity of at least about 75%, 80%, 85%, 88%, 90%, 95%, 98%, or 99% as compared to the sequence of SEQ ID NO: 01.

17. The mutant of any one of the preceding claims, wherein the mutant has a sequence identity of at least about 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% as compared to any one of the sequences of SEQ ID NO: 05 (=E73A), SEQ ID NO: 06 (N64A), or SEQ ID NO: 07 (N64A E73A).

18. The mutant of any one of the preceding claims further comprising a peptide tag.

19. The mutant of claim 18, wherein the peptide tag is selected from the group consisting of an oligohistidine tag, a strep-tag, a strep-tag II, a myc-tag, a FLAG tag, and an HA tag.

20. A method for detecting an S100A12 dimer in a sample, the method comprising detecting an S100A12 mutant according to any one of claims 1 to 18.

21. The method of claim 20, wherein the S100A12 mutant or fusion protein is used as a standard.

22. The method of claim 20 or 21, wherein the method is a quantitative enzyme-linked immunosorbent assay (ELISA).

23. The method of claim 22, wherein the method comprises (i) pre-coating a microplate with a capture antibody capable of binding S100A12, (ii) optionally contacting the pre-coated capture antibody with the sample to be analyzed and the standard as defined in any one of claims 1 to 17, (iii) optionally washing away unbound sample and standard, (iv) optionally contacting bound sample and standard with an enzyme-conjugated detecting antibody, (v) optionally washing away free amounts of the detecting antibody, (vi) optionally contacting the bound detecting antibody with the substrate of the conjugated enzyme, (vii) optionally finishing the enzymatic reaction, (viii) optionally photometrically determining the absorbance of the sample and the standard, and (ix) optionally determining the amount of S100A12 dimer in the sample by comparing the absorbance of the sample with the absorbance of the standard.

24. The method of claim 23, wherein the capture antibody of step (i) is capable of binding an S100A12 dimer but not essentially an S100A12 tetramer or an S100A12 hexamer.

25. The method of claim 24, wherein the capture antibody is an antibody having a binding specificity to an epitope of a vertebrate S100A12, wherein the epitope corresponds to an epitope in the range from amino acid position 58 to amino acid position 74 of the human S100A12.

26. The method of any one of claims 20 to 25, further comprising comparing the amount of S100A12 dimer with the total amount of S100A12 in the sample.

27. The method of any one of claims 20 to 26, wherein the sample is selected from the group consisting of a stool sample, a blood sample, a serum sample, a plasma sample, an urine sample, a tissue extract sample or a cell culture sample, wherein the sample is preferably a serum sample, a plasma sample or a stool sample.

28. The method of any one of claims 20 to 27, wherein the sample is obtained from a subject suspected of having an acute or chronic inflammatory disease.

29. The method of claim 28, wherein the disease is selected from group consisting of rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, immune reconstituation inflammatory syndrome (IRIS), sepsis, systemic inflammatory response syndrome (SIRS), pneumonia, osteomyelitis, autoinflammatory syndromes, hyperzincemia, systemic inflammation, atherosclerosis, acute coronary syndrome, myocardial infarction, Crohn's disease, colitis ulcerosa, glomerulonephritis (SLE), diabetes, an inflammatory skin disease, psoriasis, inflammatory bowel disease, vasculitis, allograft rejection, glomerulonephritis, systemic lupus erythematosus, pancreatitis, a cancer, dermatomyositis and polymyositis, multiple sclerosis, allergies, autoimmune diseases, cardiovascular diseases, infections, pulmonary inflammation, systemic onset juvenile idiopathic arthritis (SOJIA), acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS).

30. A kit comprising a S100A12 mutant of any one of claims 1 to 19.

31. The kit of claim 30, wherein the S100A12 mutant is a standard.

32. The kit of claim 30 or 31 further comprising a capture antibody as defined in claim 23 or 24.

33. A method for the diagnosis of a disease comprising detecting a S100A12 mutant as defined in any one of claims 1 to 19 and detecting an S100A12 in a sample obtained from a subject.

34. The method of claim 33, wherein the disease is an acute or chronic inflammatory disease.

35. The method of claim 34, wherein the disease is selected from group consisting of rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, immune reconstituation inflammatory syndrome (IRIS), sepsis, systemic inflammatory response syndrome (SIRS), pneumonia, osteomyelitis, autoinflammatory syndromes, hyperzincemia, systemic inflammation, atherosclerosis, acute coronary syndrome, myocardial infarction, Crohn's disease, colitis ulcerosa, glomerulonephritis (SLE), diabetes, an inflammatory skin disease, psoriasis, inflammatory bowel disease, vasculitis, allograft rejection, glomerulonephritis, systemic lupus erythematosus, pancreatitis, a cancer, dermatomyositis and polymyositis, multiple sclerosis, allergies, autoimmune diseases, cardiovascular diseases, infections, pulmonary inflammation, systemic onset juvenile idiopathic arthritis (SOJIA), acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS).

36. The method of any one of claims 33 to 35, wherein the subject is a human subject.

37. The method of any one of claims 33 to 36 comprising detecting S100A12 dimer in a sample obtained from the subject.

38. The method of any one of claims 33 to 37, wherein the method comprises: a) quantifying the amount of S100A12 dimer in a sample obtained from the subject using the S100A12 mutant, and b) comparing the amount of S100A12 dimer as determined in a) to reference data from a subject known to not suffer from an acute or chronic inflammatory disease.

39. The method of claim 38, wherein an increased amount of S100A12 dimer as compared to the reference data indicates that the subject suffers from an acute or chronic inflammatory disease.

40. A method of monitoring the progression or regression of an acute or chronic inflammatory disease associated with an increased amount of S100A12 dimer in a subject, the method comprising: a) quantifying the amount of S100A12 dimer in a sample obtained from the subject using an S100A12 mutant as defined in any one of claims 1 to 18, and b) comparing the amount of S100A12 dimer determined in a) with the amount of S100A12 dimer in a sample that was obtained from the subject at an earlier date, wherein the result of the comparison of b) provides an evaluation of the progression or regression of the inflammatory disease in the subject.

41. The method of claim 40, wherein an increased amount of S100A12 heterodimer as compared to the reference data indicates a progression of the inflammatory disease in the subject.

42. The method of claim 40, wherein no change or a decreased amount of S100A12 dimer as compared to the reference data indicates no progression or a regression of the inflammatory disease in the subject.

43. The method of any one of claims 33 to 41, wherein the method is an in vitro method.

44. A diagnostic composition comprising an S100A12 mutant of any one of claims 1 to 19.

45. Use of an S100A12 mutant of any one of claims 1 to 19 in a method of detecting an S100A12 dimer in a sample.

46. A method of generating an antibody that specifically binds to dimeric S100A12, wherein said antibody binds to an epitope of S100A12 that is accessible in the dimeric S100A12 and that is not accessible in the tetrameric and hexameric S100A12.

47. An antibody that is obtainable by the method of claim 46.

48. An antibody that specifically binds to dimeric S100A12.

49. The antibody of claim 47 or 48, wherein the antibody does not essentially bind to tetrameric S100A12.

50. The antibody of any one of claims 47 to 49, wherein the antibody does not essentially bind to hexameric S100A12.

51. The antibody of any one of claims 47 to 50, wherein the antibody specifically binds to an epitope of a mammalian S100A12 protein, wherein the epitope is within the range of the amino acid positions that correspond to positions 58 to position 74 of the human S100A12 set forth in SEQ ID NO: 01.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0080] FIG. 1: Sequence alignment between human S100A12 (SEQ ID NO: 01) and human S100A9 (SEQ ID NO: 08). Positions involved in the complexation of Ca.sup.2+ are highlighted.

[0081] FIG. 2: Multiple sequence alignment between human S100A12 (SEQ ID NO: 01), canine S100A12 (SEQ ID NO: 02), equine S100A12 (SEQ ID NO: 03), and bovine S100A12 (SEQ ID NO: 04). Positions involved in the complexation of Ca.sup.2+ are highlighted in grey, positions involved in the complexation of Zn.sup.2+ are depicted in bold italic letters.

[0082] FIG. 3: Mutants of human S100A12. FIG. 3A shows the amino acid sequence of a Glu73.fwdarw.Ala mutant of human S100A12 (SEQ ID NO: 05). FIG. 3B shows the amino acid sequence of an Asn64.fwdarw.Ala mutant of human S100A12 (SEQ ID NO: 06). FIG. 3C shows the amino acid sequence of an Asn64.fwdarw.Ala, Glu73.fwdarw.Ala mutant of human S100A12 (SEQ ID NO: 07).

[0083] FIG. 4: Molecular masses determined by ESI-MS. The molecular masses of the recombinant S100A8 and S100A9 proteins are determined by ESI-MS under denaturing conditions and compared with their theoretical calculated masses. S100A8 refers to human S100A8 protein of Uniprot accession no. P05109 (version 1 as of 1 Jan. 1988, SEQ ID NO: 09). S100A9 refers to human S100A9 protein of Uniprot accession no. P06702 (version 1 as of 1 Jan. 1988, SEQ ID NO: 08). S100A9 (N69A) refers to a mutant of human S100A9 having a single Asn69.fwdarw.Ala substitution. S100A9 (E78A) refers to a mutant of human S100A9 having a single Glu78.fwdarw.Ala substitution. S100A9 (N69A+E78A) refers to a mutant of human S100A9 having a double Asn69.fwdarw.Ala and Glu78.fwdarw.Ala substitution.

[0084] FIG. 5: MALDI mass spectra in the absence and presence of calcium under native solvent conditions. (a) MALDI mass spectra of recS100A8/S100A9 wt complexes in the absence (left) and presence (right) of calcium using 2,6-dihydroxy-acetophenone as matrix. The mass spectra between m/z 20,000 and 100,000 together with an inset between m/z 46,000 and 50,000 are shown. T.sup.+, singly charged; T.sup.2+, doubly charged; T.sup.3+, triply charged tetramer. (b) MALDI mass spectra of recS100A8/S100A9(N69A) mutant complexes in the absence (left) and presence (right) of calcium. RecS100A8/S100A9(E78A) and recS100A8/S100A9(N69A+E78A) showed almost identical results (data not shown).

[0085] FIG. 6: ESI mass spectra in the absence and presence of calcium under native solvent conditions. (a) ESI mass spectra of recS100A8/S100A9 wild-type complexes. The mass spectra between m/z 1000 and 4000 together with an inset between m/z 2800 and 4000 are shown. In the absence of calcium (left) the main signals observed correspond to heterodimers with charge states 10+ and 9+. In the presence of calcium (right) signals corresponding to tetramers occurred at charge states 16+, 15+, 14+ and 13+ in the range between m/z 3000-3800. (b) ESI mass spectra of recS100A8/S100A9(N69A) mutant complexes. In the absence and presence of calcium the main signals correspond to heterodimers with charge states 10+ and 9+, no tetramers were found. recS100A8/S100A9(E78A) and recS100A8/S100A9(N69A+E78A) showed almost identical results (data not shown).

[0086] FIG. 7: Density gradient centrifugation of S100A8/S100A9 proteins. wt and mutant recS100A8/S100A9 complexes were loaded on a glycerol gradient in the presence of either 1 mM EGTA or 100 M Ca2+. After centrifugation, successive fractions of the gradients were analyzed by SDS-PAGE. In the presence of EGTA wt and mutant complexes showed an almost identical distribution centered in the low density fractions of the gradient. Addition of calcium induced a marked shift for recS100A8/S100A9 wt to higher glycerol densities, whereas for the mutant complexes recS100A8/S100A9(N69A) and recS100A8/S100A9(E78A) no shift was observed.

[0087] FIG. 8: Glycerol centrifugation of S100A12 proteins. Wild type (wt) and mutant recS100A12 complexes were loaded on a 15% glycerol solution in the presence of either EGTA or Ca.sup.2+ or Zn.sup.2+ or Ca.sup.2+/Zn.sup.2+. Buffer conditions were 20 mM HEPES, 140 mM NaCl, pH 7.4. After centrifugation, successive fractions of the gradients were analyzed by the colorimetric assay Bradford. In the presence of EGTA wt and mutant complexes showed an almost identical distribution centered in the low density fractions of the gradient. Addition of calcium or zinc alone induced no shift for wtS100A12 to higher glycerol fractions. Addition of calcium plus zinc induced a marked shift for wtS100A12 to higher glycerol fractions, whereas for the mutant complex recS100A12(E73A) no shift was observed. FIG. 8A: Protein concentrations of collected fractions determined by Bradford assay. Sample A: S100A12 wild type+2 mM EGTA, sample B S100A12 wild type+5 mM CaCl.sub.2, sample C: S100A12 wild type+1 mM Zn, sample D S100A12 wild type+200 M Zn, sample E: S100A12 wild type+5 mM CaCl.sub.2+200 M Zn, sample F: S100A12 wild type+2.5 mM CaCl.sub.2+100 M Zn FIG. 8B: Summary of collected and analysed fractions of wtS100A12 (see FIG. 8A) in the presence or absence of bivalent cations as indicated in the figure. FIG. 8C Summary of collected and analysed fractions of wtS100A12 and recS100A12E73A mutant in the presence (5 mM CaCl.sub.2+200 M Zn, 2.5 mM CaCl.sub.2+100 M Zn) or absence (2 mM EGTA) of bivalent cations as indicated in the figure.

EXAMPLES

[0088] The following examples illustrate the invention. These examples should not be construed as to limit the scope of this invention. The examples are included for purposes of illustration and the present invention is limited only by the claims.

Example 1: Differentiation Between S100A8/S100A9 Heterodimers and Teteramers Using MALDI-MS and ESI-MS

[0089] Electrospray ionization mass spectrometry (ESI-MS) confirms the theoretically calculated masses without the N-terminal methionine (SwissProt) for mutated and non-mutated recombinant S100A8/S1009 proteins (FIG. 4).

[0090] S100A8 and S100A9 exist as heterodimers in the absence of calcium, and these heterodimeric complexes associate to (S100A8/S100A9).sub.2 tetramers upon calcium-binding. FIG. 5(a) shows the matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) spectra of the recS100A8/S100A9 wt proteins. In the absence of calcium, samples show intense signals of singly charged heterodimers. In contrast, in the presence of Ca2+ wt S100A8/S100A9 show a base peak in first shot spectra that corresponds to a singly charged heterotetramer (T+: 48 kDa) composed of two molecules recS100A8 and two molecules of recS100A9, respectively. Other prominent signals are detected at molecular masses of around 24 kDa and 16 kDa, representing doubly charged (T2+) or triply charged (T3+) tetramers in accordance with results reported earlier for the native proteins purified from human granulocytes. The number of Ca2+ bound to the tetramers was calculated from the difference between the observed masses of the tetramers and the sum of the calculated theoretical molecular masses of the monomeric components.

[0091] The oligomerization properties of the recS100A8/S100A9 mutant complexes can be determined by MALDI-MS. As shown exemplarily for the N69A mutant in FIG. 5(b), all mutant S100A9 proteins display signals for singly charged heterodimers in the absence of calcium. In contrast to the results obtained with the recS100A8/S100A9 wt proteins no heterotetramers can be observed for the mutant complexes in the presence of calcium (FIG. 5). The base peaks under these conditions exclusively represent S100A8/S100A9(N69A), S100A8/S100A9(E78A) and S100A8/S100A9(N69A+E78A) heterodimers. All MALDI-MS experiments presented here were also confirmed by ESI-MS measurements (see FIG. 6).

[0092] Density gradient centrifugation can be employed in order to confirm the different complex formation patterns obtained in the mass spectrometric studies (FIG. 7). In EGTA-containing samples the recS100A8/S100A9 wt and mutant complexes are found in the same range of fractions of the glycerol gradient (19(2)%), indicating that in the absence of calcium the formation of heterodimers is preferred in wt proteins and all S100A9 mutants. In the presence of calcium, wt complexes shifted to fractions of significantly higher glycerol concentrations (23(2)%), as observed earlier for S100A8/S100A9 purified from granulocytes. This shift reflects the calcium-induced formation of high-molecular (S100A8/S100A9).sub.2 tetramers. In contrast, after addition of calcium, the mutant complexes recS100A8/S100A9(N69A) and recS100A8/S100A9(E78A) show no shift to higher glycerol concentrations, confirming that heterotetramer formation is disturbed.

Example 2: Differences in Oligomerization States of S100A12 Wild Type and S100A12 Mutants

[0093] Glycerol centrifugation can be employed in order to confirm the different complex formation patterns as previously observed for S100A8/S100A9 complexes. In EGTA-containing samples the recS100A12 wt and mutant rec S100A12E73A complexes are found in the same range of glycerol fractions 4-10 indicating that in the absence of calcium/zinc the formation of homodimers is preferred in wt proteins and the tested S100A12 mutant. In the presence of calcium or zinc alone wtS100A12 was found in the same fractions as observed before under EGTA-conditions, indicating that for S100A12 in contrast to S100A8/S100A9 calcium alone is not sufficient to induce oligomerization. However, in the presence of calcium and zinc, wild type S100A12 complexes shifted to fractions of significantly higher numbers (9-15). This shift reflects the calcium/zinc-induced formation of high-molecular S100A12 tetramers and hexamers. In contrast, after addition of calcium and zinc, the mutant complex recS100A12(E73A) shows no shift to higher glycerol fractions, confirming that tetramer/hexamer formation is disturbed (FIG. 8).