NOVEL METHODS AND REAGENTS FOR DIAGNOSING SJÖGREN'S SYNDROME

Abstract

The inventive subject matter relates to novel antibodies to lacritin splice variants, improved methods for detecting lacritin splice variants, and methods for diagnosing Sjgren's Syndrome.

Claims

1. A method for detecting lacritin-c in a subject, comprising detecting whether lacritin-c is present in a tear sample from a human subject by contacting the tear sample with an anti-lacritin-c antibody and detecting binding between lacritin-c and the antibody.

2. The method of claim 1, wherein said anti-lacritin-c antibody is produced in a non-human animal.

3. The method of claim 2, wherein said animal is selected from the group consisting of rabbit, chicken, goat, guinea pig, hamster, horse, mouse, rat, pig, and sheep.

4. The method of claim 3, wherein said animal is a rabbit.

5. The method of claim 3, wherein said animal is a goat.

6. The method of claim 1, wherein said anti-lacritin-c antibody is an antibody against SEQ ID NO. 2.

7. The method of claim 1, wherein said anti-lacritin-c antibody is polyclonal.

8. The method of claim 1, wherein said anti-lacritin-c antibody is monoclonal.

9. A method for detecting lacritin in a subject, comprising detecting whether lacritin is present in a tear sample from a human subject by contacting the tear sample with an anti-lacritin antibody and detecting binding between lacritin and the antibody.

10. The method of claim 9, wherein said anti-lacritin antibody is produced in a non-human animal.

11. The method of claim 10, wherein said animal is selected from the group consisting of rabbit, chicken, goat, guinea pig, hamster, horse, mouse, rat, pig, and sheep.

12. The method of claim 11, wherein said animal is a rabbit.

13. The method of claim 9, wherein said anti-lacritin antibody is an antibody against SEQ ID NO. 1.

14. The method of claim 9, wherein said anti-lacritin antibody is polyclonal.

15. The method of claim 9, wherein said anti-lacritin antibody is monoclonal.

16. A method for diagnosing Sjgren's Syndrome in a subject, comprising: (a) detecting whether lacritin-c monomer is present in a tear sample obtained from a human subject by contacting the tear sample with a non-human animal anti-lacritin-c antibody and detecting binding between lacritin-c monomer and said antibody; and (b) diagnosing the subject with Sjgren's Syndrome when lacritin-c monomer in the tear sample is not detected.

17. A method for diagnosing Sjgren's Syndrome in a subject, comprising: (a) detecting whether lacritin monomer is present in a tear sample obtained from a human subject by contacting the tear sample with antibody to SEQ ID NO. 1, and detecting binding between lacritin monomer and said antibody to SEQ ID NO. 1; (b) detecting whether lacritin-c monomer is present in said tear sample by contacting the tear sample with antibody to SEQ ID NO. 2, and detecting binding between lacritin-c and said antibody to SEQ ID NO. 2; and (c) diagnosing the subject with Sjgren's Syndrome when lacritin monomer and/or lacritin-c monomer in the tear sample is not detected.

18. A method for diagnosing Sjgren's Syndrome in a subject, comprising: (a) in a tear sample of a subject suspected to suffer from Sjgren's Syndrome, determining the amount of lacritin-c present in said tear sample with an immunoassay, wherein said immunoassay comprises: (i) contacting the sample with an anti-lacritin-c antibody; (ii) measuring the amount of bound lacritin-c in the sample; and (b) comparing the amount of lacritin-c monomer with reference amount(s) obtained from one or more sample(s) of subject(s) known not to suffer from Sjgren's Syndrome, wherein Sjgren's Syndrome is diagnosed if the amount of lacritin-c monomer is decreased as compared to said reference amount(s).

19. The method of claim 10, wherein said anti-lacritin-c antibody is an antibody to SEQ ID NO. 2.

20. A method for diagnosing Sjgren's Syndrome in a subject, comprising: (a) in a tear sample of a subject suspected to suffer from Sjgren's Syndrome, determining the amount of lacritin and the amount of lacritin-c present in said tear sample with an immunoassay, wherein said immunoassay comprises: (i) contacting the sample with antibody microarray comprising an anti-lacritin antibody and an anti-lacritin-c antibody; (ii) measuring the amounts of bound lacritin and lacritin-c in the sample; and (b) comparing the amounts of lacritin monomer and lacritin-c monomer with reference amounts obtained from samples from subject(s) known not to suffer from Sjgren's Syndrome, wherein Sjgren's Syndrome is diagnosed if the amounts of either or both of lacritin monomer and lacritin-c monomer are decreased as compared to said reference amounts.

21. The method of claim 20, wherein said anti-lacritin antibody is an antibody to SEQ ID NO. 1, said anti-lacritin-c antibody is an antibody to SEQ ID NO. 2, or both.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 is a photograph which depicts a Western blot analysis of human tear samples for lacritin according to one aspect of the present inventive subject matter.

[0023] FIG. 2 is a series of photographs which depict Western blot analysis of human tear samples for lacritin according to one aspect of the present inventive subject matter.

[0024] FIG. 3 is a drawing which depicts natural and recombinant lacritin splice variants, and pictorially depicts the binding regions for the Lacripep, N-Term, and 13 antibodies according to one aspect of the present inventive subject matter.

[0025] FIG. 4 is a photograph which depicts a Western blot analysis of human tear samples for lacritin-c according to one aspect of the present inventive subject matter.

[0026] FIG. 5 is a series of photographs which depict Western blot analysis of human tears with lacritin antibodies according to one aspect of the present inventive subject matter.

[0027] FIG. 6 is a pair of photographs which depict Western blot quantitative analysis of human tear samples as compared to purified recombinant lacritin of known concentrations according to one aspect of the present inventive subject matter.

[0028] FIG. 7 is a graph depicting a standard curve for determining concentrations of tear lacritin proteins according to one aspect of the present inventive subject matter.

[0029] FIG. 8 is a photograph which depicts a multiplex Western blot analysis according to one aspect of the present inventive subject matter.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

[0030] The term SEQ ID NO. 1 as used herein refers to the sequence

TABLE-US-00001 EDASSDSTGADPAQEAGTS.

[0031] The term N-Term Antibody as used herein refers to polyclonal antibodies produced in rabbit against SEQ ID NO. 1, the first 19 amino acids in the N-terminus of mature lacritin.

[0032] The term Lacripep Antibody as used herein refers to polyclonal antibodies produced in goat against 19 amino acids in the N-94/C-6 C-terminus of lacritin.

[0033] The term SEQ ID NO. 2 as used herein refers to the sequence

TABLE-US-00002 SKSLSLCQINNLEKSLAAGPHHTSTHRDKPG.

[0034] The term I3 Antibody as used herein refers to polyclonal antibodies produced in rabbit against SEQ ID NO. 2, 31 amino acids in Intron 3 of lacritin-c.

[0035] The term lacritin as used herein refers to the naturally-occurring human monomeric protein having the following full 138 amino acid sequence, SEQ ID NO. 3:

TABLE-US-00003 1 MKFTTLLFLAAVAGALVYAEDASSDSTGADPAQEAGTSKPNEEISGPAEPASPPETTTTA 61 QETSAAAVQGTAKVTSSRQELNPLKSIVEKSILLTEQALAKAGKGMHGGVPGGKQFIENG 121 SEFAQKLLKKFSLLKPWA

[0036] Upon secretion by acinar cells in the lacrimal gland, the first 19 amino acids are cleaved from the lacritin polypeptide as transcribed to produce mature lacritin.

[0037] The term immunoassay as used herein refers broadly to ELISA-based methods, whether micro-plate ELISA-based, fully-automated or robotic immunoassay, latex agglutination assay, sandwich ELISA using a capture antibody and a detection antibody, or other similar ELISA-based methods known in the art. Preferably, the immunoassay system of the present inventive subject matter is based upon the infrared excitation/detection of fluorescent secondary antibodies with increased sensitivity, improved reproducibility, and quantification of banding patterns. Such a system offers the advantage of multiplex detection enabling analysis of more than one protein on the same blot, in particular providing a single test for lacritin and lacritin-c. The LI-COR Odyssey CLx Imaging System is one example of such a system.

[0038] The term antibody as used herein refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes, and encompasses polyclonal antibodies, monoclonal antibodies, and fragments thereof, as well as molecules engineered from immunoglobulin gene sequences.

[0039] The term tear sample refers to the biological material being tested for and/or suspected of containing analyte(s) of interest. A tear sample may be used directly as obtained from the biological source or following a pretreatment to modify the character of the sample. If pretreatment is employed with respect to the tear sample, such pretreatment methods are such that the analyte of interest remains in the sample at a concentration proportional to that in an untreated sample. According to one aspect of the present inventive subject matter, the biological fluid is preferably obtained directly from the eye of a subject. A tear sample is obtained using any technique known in the art, preferably using Schirmer Tear Test strips such as those available from the Sjgren's International Collaborative Clinical Alliance.

[0040] A heathy ocular surface is maintained in large part by a complex mixture of biologically active proteins found in tear film. For example, tears collected on Schirmer Tear Test (SST) strips during routine clinical examinations provides an opportunity for biochemical analysis of the tear proteome to determine relative concentrations of human proteins secreted and collected in a cell-free environment. Applicants expect that tear proteins, such as lacritin and lacritin-c, accurately prognosticate ocular diseases and function as tear biomarkers for the diagnosis of Sjgren's Syndrome.

B. Inventive Methods for Detecting Lacritin-c

[0041] Lacritin is a 12.3 kDa endogenous tear prosecretory mitogen reported to be decreased in patients with various forms of dry eye disease. Lacritin is detected in tears as an active monomer of 25 kDa, an active proteolytically cleaved fragment of 12 to 15 kDa, and inactive, tissue transglutaminase-generated dimers, trimers, and larger polymers >40 kDa. The proteolytically cleaved fragment of lacritin releases a potent gram negative and positive bactericidal activity that functions under physiological conditions. Lacritin promotes basal tearing when topically applied to eyes of normal rabbits and rescues cultured human corneal epithelial cells from inflammatory cytokine stress, including stress induced by tears from dry eye patients. Topical administration of lacritin may be a novel therapy for aqueous-deficient dry eye disease.

[0042] Additionally, lacritin has two splice variants, lacritin-b and lacritin-c. Lacritin-b is a deletion of DNA coding for 13 amino acids in exon 4, which is then normally spliced in frame to exon 5. Lacritin-c is composed of coding sequences for the first three lacritin exons, into which is spliced an open reading frame coding for 39 amino acids derived from intron 3 (13), followed by a stop codon. The lacritin splice variants are translated into proteins and are present in human tears. The unique 39 amino acid sequence found in lacritin-c is not found in lacritin, which has provided Applicants an opportunity to create unique antibodies that will detect only lacritin-c in tear samples, for example using Western blot analysis as in the Examples herein.

[0043] Applicants used synthetic peptides to produce lacritin and lacritin-c specific polyclonal antibodies in rabbits, have determined that their anti-lacritin-c antibodies detect putative lacritin-c proteins in human tears, have determined that their anti-lacritin antibodies detect lacritin proteins in human tears, and have determined that the protein banding patterns in tears from Sjgren's Syndrome patients are different compared to normal tears.

[0044] The inventive subject matter thus relates to a method for detecting lacritin, lacritin-c, or both in a subject, comprising detecting whether lacritin or lacritin-c is present in a tear sample from a human subject by contacting the tear sample with an anti-lacritin antibody or an anti-lacritin-c antibody and detecting binding between lacritin or lacritin-c and the respective antibody. Lacritin and lacritin-c can be detected in the same assay, as lacritin-c lacks the C-terminus of full length lacritin associated with the biological activities of that tear protein, and has a different N-terminus from full length lacritin.

C. Inventive Methods for Diagnosing Sjgren's Syndrome

[0045] Applicants have found that reduced levels of monomeric lacritin and increased levels of cross-linked lacritin dimers and other polymers in tears from Sjgren's Syndrome (SS) patients compared to age-matched controls are highly correlated with clinical signs of dry eye. In addition, Applicants have shown that measurement of tear lacritin levels are equivalent or better than standard clinical measures for diagnosing Sjgren's Syndrome-associated dry eye. FIG. 1 shows representative Western blot and Schirmer Tear Test data obtained from SS patients and age-matched controls, showing the inactive, tissue transglutaminase-generated complexes of lacritin >40 kDa labeled WB40, a proteolytically cleaved fragment of 15 kDa labeled WB15, and the active lacritin monomer of 26 kDa labeled WB26. As the Western blot analysis demonstrates, bands for monomeric lacritin are either missing or reduced in intensity in SS patients compared to age-matched controls. The Schirmer Tear Test values correlated with reduced tear production in SS patients, while quantitation of tear lacritin in the lacritin ELISA corresponds with reduced monomeric lacritin visualized by Western blot analysis.

[0046] In addition to the lacritin work discussed above, Applicants used a synthetic peptide to produce lacritin-c specific polyclonal antibodies in rabbits and determined that their anti-lacritin-c antibodies detect putative lacritin-c proteins in human tears. Additionally, Applicants have determined that the protein banding patterns in tears from Sjgren's Syndrome patients are different compared to normal tears when exposed to anti-lacritin-c antibodies, providing novel methods and reagents for detecting lacritin-c as a biomarker for Sjgren's Syndrome.

[0047] Thus, the inventive subject matter relates to a method for diagnosing Sjgren's Syndrome in a subject, comprising: (a) detecting whether lacritin-c is present in a tear sample obtained from a human subject by contacting the tear sample with a non-human animal anti-lacritin-c antibody and detecting binding between lacritin-c and said antibody; and (b) diagnosing the subject with Sjgren's Syndrome when lacritin-c in the tear sample is not detected.

[0048] The inventive subject matter relates to an additional method for diagnosing Sjgren's Syndrome in a subject, comprising: (a) obtaining a tear sample from a human subject; (b) detecting whether lacritin is present in the tear sample by contacting the tear sample with antibody to SEQ ID NO. 1, and detecting binding between lacritin and said antibody to SEQ ID NO. 1; (c) detecting whether lacritin-c is present in the tear sample by contacting the tear sample with antibody to SEQ ID NO. 2 and detecting binding between lacritin-c and said antibody to SEQ ID NO. 2; and (d) diagnosing the subject with Sjgren's Syndrome when lacritin or lacritin-c in the tear sample is not detected.

[0049] The inventive subject matter relates to a further method for diagnosing Sjgren's Syndrome in a subject, comprising: (a) in a tear sample of a subject suspected to suffer from Sjgren's Syndrome, determining the amount of lacritin-c present in said tear sample with an immunoassay, wherein said immunoassay comprises: (i) contacting the sample with an anti-lacritin-c antibody; (ii) measuring the amount of bound lacritin-c in the sample; and (b) comparing the amount of lacritin-c with reference amount(s) obtained from one or more sample(s) of subject(s) known not to suffer from Sjgren's Syndrome, wherein Sjgren's Syndrome is diagnosed if the amount of lacritin-c is decreased as compared to said reference amount(s).

[0050] Finally, the inventive subject matter relates to an additional method for diagnosing Sjgren's Syndrome in a subject, comprising: (a) in a tear sample of a subject suspected to suffer from Sjgren's Syndrome, determining the amount of lacritin and the amount of lacritin-c present in said tear sample with an immunoassay, wherein said immunoassay comprises: (i) contacting the sample with antibody microarray comprising an anti-lacritin antibody and an anti-lacritin-c antibody; (ii) measuring the amounts of bound lacritin and lacritin-c in the sample; and (b) comparing the amounts of lacritin and lacritin-c with reference amounts obtained from samples from subject(s) known not to suffer from Sjgren's Syndrome, wherein Sjgren's Syndrome is diagnosed if the amounts of both lacritin and lacritin-c are decreased as compared to said reference amounts.

D. Inventive Compounds for Detecting Latritin-c

[0051] Applicants have developed novel compounds, antibodies termed the I3 antibody herein, which selectively bind to SEQ ID NO 2, a synthetic peptide derived from Intron 3 of Lacritin-c. Such compounds can further be labeled, radiographically, using chemiluminescence, or with binding sites of flourescent secondary antibodies, in order to make detection of Lacritin-c binding possible. Such novel compounds are an additional embodiment of the present inventive subject matter.

EXAMPLES

[0052] The following examples are illustrative of the inventive subject matter and are not intended to be limitations thereon.

Example 1

[0053] Production of Antibodies.

[0054] The synthetic peptide EDASSDSTGADPAQEAGTS was used to make anti-lacritin polyclonal antibodies in New Zealand white rabbits, also called the N-Term Antibody herein. The synthetic peptide SKSLSLCQINNLEKSLAAGPHHTSTHRDKPG was used to make anti-lacritin-c polyclonal antibodies in New Zealand white rabbits, also called the I3 Antibody herein. A synthetic peptide of 19 amino acids in the N-94/C-6 C-terminus of lacritin was used to make anti-lacritin polyclonal antibodies in goats, also called the Lacripep Antibody herein. All antibodies were produced using standard techniques well known in the art.

Example 2

[0055] Western Blot Analysis of Human Tear Samples for Lacritin.

[0056] Human tear samples from 10 patients with Sjgren's Syndrome (SS) and 10 age-matched controls (C) were analyzed by Western blot, Schirmer Tear Test, and ELISA.

[0057] ELISA was used to assess total tear lacritin. For assay of tear samples, 100 ng total tear protein was coated in each well. To generate a standard curve of recombinant lacritin, each plate contained triplicate wells to which 2, 4, 6, 8, 10, 12, 14, or 16 ng protein was added. Wells were washed, blocked with PBS-Tween (PBS with 0.3% Tween-20 [PBS-T]), and then incubated for 1 hour at 37 C. with 100 L anti-Pep Lac N-Term antiserum diluted 1:3,000 in PBS-T. After washing three times with PBS-T, horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (MP Biomedicals, Solon, Ohio) diluted 1:1,000 in PBS-T was added for 1 hour (37 C.). Plates were washed three times with PBS-T, and then bound antibody was measured after incubation for 10 minutes with 100 L OPD substrate (Acros Organics, Geel, Belgium) by absorbance at 415 nm (model 680; Bio-Rad, Hercules, Calif.).

[0058] Western blot was used to detect active lacritin monomer (25 kDa), active lacritin fragment (12-15 kDa), and inactive, tissue transglutaminase-generated lacritin (>40 kDa). Tear samples were loaded on Any kD Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad), electrophoresed at 200 V, and transferred to nitrocellulose (Protran BA 83; Whatman, Dassel, Germany). Blots were blocked with PBS-T, incubated with anti-Lac Pep N-Term (1:1,000 dilution in PBS-T) for 1 hour at room temperature, rinsed with PBS-T, and incubated for 1 hour at room temperature with HRP-138 conjugated goat anti-rabbit IgG (MP Biomedicals) diluted 1:5,000 in PBS-T.

[0059] Western blots shown the drawings herein in black and white were rinsed with PBS-T and developed via chemiluminescence with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific Inc., Rockford, Ill.). Western blots shown in the drawings herein in color were developed with fluorescent secondary antibodies and imaged by a LI-COR Odyssey CLx Imaging System.

[0060] The signal intensity of lacritin bands was determined by multiplying the area of the band with the average pixel intensity, which provided volume-sum intensity for each band. The signal intensity of the 12-15 kDa, 25 kDa and >40 kDa bands from each individual's tear sample was quantified using Image J. The signal intensity value of each band was expressed as area pixel density. Finally, in vivo confocal microscopy was used to assess nerve fiber density and nerve fiber length.

[0061] As shown in FIG. 1, the WB26 bands are the active lacritin monomer and the WB40 bands are inactive >40 kDa dimer and larger complexes of lacritin. The WB15 bands are a proteolytically cleaved fragment of lacritin. Tears eluted from Schirmer strips were obtained from 10 SS patients and 10 age-matched controls. Applicants found that active tear lacritin was significantly reduced in SS patients (4.315.73 ng/100 ng [SS] vs. 18.174.57 ng/100 ng total tear protein [controls]; P<0.001), while inactive lacritin was increased. Nerve fiber density (21.0010.85 vs. 33.609.29; P=0.02) and nerve fiber length (4.312.09 vs. 7.462.62; P<0.001) were significantly decreased in SS patients compared to controls. Both reduced nerve fiber length (r=0.71, p<0.001) and nerve fiber density (r=0.68, p<0.001) were highly correlated with reduced tear lacritin. Similarly, total tear lacritin was highly correlated with tear secretion (r=0.72, p<0.001), ocular staining (r=0.78, p<0.001), corneal sensitivity (r=0.69, p<0.001) and tear breakup time (r=0.49, p=0.03).

[0062] As shown in Table 1, Applicants found that overall levels of tear lacritin were profoundly reduced in the tears of SS patients. Similarly, both the active cleaved fragment and lacritin monomer were significantly decreased, while inactive lacritin was increased in SS tears. Shown graphically, levels of the cleaved fragment, migrating at approximately 15 kDa on Western Blots (WB15), were 14% lower in SS versus control tears (95% CI: 622%), while lacritin monomer, migrating at approximately 26 kDa on Western Blots (WB26), was 26% lower than control eyes (95% CI: 1636%). Perhaps most compelling, inactive lacritin cross-linked by tissue transglutaminase in tears and migrating as multiple bands around 40 kDa on Western Blots (WB40), was 39% higher in SS patients compared to controls (95% CI: 3184%).

TABLE-US-00004 TABLE 1 Comparison of total lacritin and percent expression of lacritin species Control SS Lacritin Mean (SD) Mean (SD) p value ELISA* 18.17 (4.57) 4.31 (5.73) <0.001 WB15.sup. 15.0 (7.0) 2.0 (6.0) <0.01 WB26.sup. 34.0 (11.0) 16.0 (10.0) <0.01 WB40.sup. 51.0 (12.0) 82.0 (10.0) <0.001 *Total tear lacritin measured by ELISA and expressed as ng/100 ng total protein .sup.Percent expression of 15 kDa, 26 kDa and >40 kDa lacritin species assessed by Western blot (WB) and quantified by densitometry

Example 3

[0063] Additional Western Blot Analysis of Human Tear Samples for Lacritin.

[0064] A collection of 46 Schirmer Tear Test strips from the Sjgren's International Collaborative Clinical Alliance (SICCA) in vials labeled UCSF ASB were analyzed. Tear samples were eluted, total protein determined by the BCA assay, and tear protein samples normalized to 200 g/mL. 4 g of each sample was loaded on SDS PAGE, transferred to nitrocellulose, challenged with anti-lacritin antibodies, and developed by chemiluminescence. FIG. 2 is a summary panel of the Western blots challenged with anti-lacritin antibodies for 46 samples from the SICCA collection along with 5 normal tear samples, where N denotes normal tear samples.

[0065] As shown in FIG. 2, higher molecular weight complexes of inactive lacritin can be seen in all 46 SS samples while monomeric lacritin is not detected in 36 (78%) of the SS samples. Reduced levels of monomeric lacritin can be seen in 4 (9%) of the SS samples and 6 (13%) of the SS samples showed normal monomeric levels. In a preliminary analysis of tear lacritin from 46 patients diagnosed with Sjgren's Syndrome, 87% exhibited reduced levels of active lacritin while 13% showed normal levels of monomeric lacritin.

Example 4

[0066] Comparison to Prior Art Diagnostic Tests.

[0067] Compared to current clinical readouts used to assess the signs and symptoms of SS dry eye, lacritin and its isoforms performed as well or better than traditional diagnostic tests to differentiate SS dry eye from controls. As shown in Table 2, using optimized cutoff values for total lacritin, lacritin monomer and inactive lacritin species, SS cases were correctly identified with a sensitivity of 100.00% and specificities of 87.50% (95% CI: 60.00100.00%), 70.00% (95% CI: 40.00100.00%), and 87.5% (95% CI: 60.00100.00%), respectively. The OSS, currently the primary objective clinical test used to diagnose the ocular component of SS, had a sensitivity of 90.00% (73.86100.00%) and corresponding specificity of 89.47% (72.23100.00%) when using the cutoff of >3, as specified by the current American College of Rheumatology diagnostic criteria. As such, the sum of sensitivity and specificity for OSS was 8.03% lower than that of total tear lacritin and its inactive isoform (WB40), which had sums equal to 187.50%. It was also noted that the AUCs of lacritin ELISA (AUC=0.98) and inactive lacritin WB40 (AUC=0.97) were slightly higher than any of the clinical parameters used to differentiate SS patients from the healthy controls.

TABLE-US-00005 TABLE 2 Diagnostic performance of tear lacritin and clinical test using standard and optimized cutoffs Tested AUC Cutoff Sensitivity (%) Specificity (%) Sensitivity + variables (95% CI) (95% CI) (95% CI) (95% CI) Specificity Diagnostic Performance of Tear Lacritin Lacritin ELISA 0.98 14.50 100.00 87.50 187.50 (0.94~1.00) (5.05~16.50) ()* (60.00~100.00) WB15 0.90 3% 83.33 100.00 183.33 (0.75~1.00) (2%~20%) (58.85~100.00) () WB26 0.95 30% 100.00 75.00 175.00 (0.84~1.00) (11%~41%) () (40.00~100.00) WB50 0.97 62% 100.00 87.50 187.50 (0.90~1.00) (60%~74%) () (60.00~100.00) Diagnostic Performance of Traditional Clinical Tests OSDI Standard 0.86 20 66.67 78.95 145.62 (0.74~0.98) (42.86~87.50) (57.91~95.24) Optimized 18.05 77.78 78.95 156.73 (12.03~53.10) (57.14~94.74) (57.91~95.24) Schir Standard 0.95 5 50.00 100.00 150.00 (0.88~1.00) (26.32~71.41) () Optimized 8.50 85.00 94.74 179.74 (6.50~15.00) (66.70~100.00) (83.33~100.0) OSS Standard 0.96 3 90.00 89.47 179.47 (0.74~0.98) (73.68~100.00) (72.23~100.0) Optimized 4.50 80.00 100 180.00 (59.28~95.24) () TBUT Standard 0.75 10 100.00 0.00 (0.59~0.92) () () Optimized 2.5 65.00 94.74 159.74 (2.50~3.00) (45.00~85.00) (84.21~100) CST Optimized 0.88 4.25 80.00 88.89 168.89 (0.77~0.99) (3.75~5.25) (55.00~100.00) (55.56~100)

Example 5

[0068] Lacritin Splice Variants and Antibodies Thereto.

[0069] FIG. 3 illustrates the genomic DNA of lacritin and the splice variant lacritin-c. Genomic lacritin contains four Intron DNA sequences (I) separating five Exon DNA sequences (E) coding for amino acids. Following transcription, the five Exons within the mRNA are spliced together to produce a continuous coding sequence for lacritin protein. Upon secretion by acinar cells in the lacrimal gland, the signal peptide sequences in Exon 1 are cleaved off to produce mature lacritin found in human tears.

[0070] A splice variant of lacritin containing the first three exons of lacritin and DNA sequences coding for 39 amino acids in Intron 3 is known as lacritin-c. Lacritin-c is composed of coding sequences for the first three lacritin exons, which is then spliced into an open reading frame coding for 39 new amino acids derived from intron 3 (I3), followed by a stop codon.

[0071] The unique amino acid sequences in lacritin-c are not found in lacritin or other known splice variants, which provided Applicants with an opportunity to create unique antibodies that detect only lacritin-c in tear samples using Western blot analysis. A synthetic peptide, SEQ ID NO. 2, present in lacritin-c but not other lacritin species, was used to make lacritin-c specific polyclonal antibodies in New Zealand white rabbits.

Example 6

[0072] Western Blot Analysis of Tear Samples for Splice Variant Lacritin-c.

[0073] Human tear samples from patients with Sjgren's Syndrome (SS) and age-matched controls (N) were eluted, loaded on SDS PAGE, transferred to nitrocellulose, challenged with anti lacritin-c antibodies, and developed by chemiluminescence. The Western blot shown in FIG. 4 shows the results for tear samples challenged with anti-lacritin-c antibodies, which reveals a dramatic difference in banding patterns in tear samples from Sjgren's Syndrome patients compared to normal tears. Tears from SS patients show a major band migrating at approximately 50 kDa while normal tears show a predominant band migrating at approximately 15 kDa, the expected molecular weight of monomeric lacritin-c. The monomeric lacritin-c is missing in all tested SS patients, and the Schirmer Tear Test values for the same SS patients correlate with reduced tear production, providing a second biomarker for the diagnosis of Sjgren's Syndrome-associated Dry Eye Disease.

Example 7

[0074] Western Blot Analysis of Human Tears with Lacritin Antibodies.

[0075] Tears collected from healthy individuals using Schirmer strips were eluted by centrifugation and total protein concentrations determined by the BCA assay as described above. Tear proteins normalized to 200 g/ml were separated by SDS PAGE, transferred to nitrocellulose and challenged by N-Term lacritin antibodies and 13 lacritin-c antibodies. Western blots shown in FIG. 5 were prepared as described above, developed by fluorescent secondary antibodies, and imaged by a LI-COR Odyssey CLx Imaging System. The N-Term lacritin antibodies detected lacritin monomers, cross-linked lacritin multimers, and lacritin-c. On the other hand, 13 lacritin-c antibodies detected lacritin-c and not lacritin monomers.

Example 8

[0076] Western Blot Quantitative Analysis.

[0077] Tears collected from healthy individuals using Schirmer strips were eluted by centrifugation and total protein concentrations determined by the BCA assay. Tear proteins normalized to 200 g/ml and purified recombinant lacritin of known concentrations were separated by SDS PAGE, transferred to nitrocellulose and challenged by N-Term lacritin antibodies. Western blots shown in FIG. 6 were prepared as described above, developed by fluorescent secondary antibodies, and imaged by a LI-COR Odyssey CLx Imaging System.

[0078] Specific protein bands for lacritin, lacritin-c, and cross-linked polymers of lacritin were quantified by pixel density with the Odyssey CLx Imaging System. Decreasing concentrations of purified recombinant lacritin were run on each blot and quantified to generate the standard curve shown in FIG. 7, which was then used to determine concentrations of tear lacritin proteins. Table 3 shows the quantitation of several samples using the standard curve generated as described above.

TABLE-US-00006 TABLE 3 Exemplary quantitation of tear lacritin proteins. Tear Lacritin Lacritin Lacritin-c Lacritin-c Sample g % Protein g % Protein 1 N/D N/D N/D N/D 2 0.078 4.7 0.161 9.64 3 0.041 2.5 0.050 3.0 4 0.110 6.6 0.329 19.7 5 0.183 11.0 0.194 11.8 N/D = Not Detected; negative control

Example 9

[0079] A Diagnostic Test for Sjgren's Syndrome.

[0080] Applicants expect that the predominant band migrating at approximately 15 kDa found in Example 6, which has the expected molecular weight of monomeric lacritin-c, is in fact lacritin-c. A diagnostic reagent comprising an anti-lacritin-c antibody raised in rabbit, goat, or other animal is found to accurately and specifically detect lacritin-c in tear samples. Lacritin-c is found to be an effective biomarker for diagnosing Sjgren's Syndrome, as well as determining the prognosis and severity of Sjgren's Syndrome disease when quantified.

Example 10

[0081] A Kit for Diagnosing Sjgren's Syndrome.

[0082] Tear proteins are collected on modified Schirmer strips and incubated with a predetermined titer of antibodies embedded on the strips, and developed with a colorimetric reagent to provide a qualitative positive/negative test for threshold concentrations of active tear lacritin and/or lacritin-c. A non-obvious and unique attribute of this diagnostic test is that lacritin antibodies embedded on the strips under non-denaturing conditions do not detect the inactive >40 kDa cross-linked polymers of lacritin. Further, the same Schirmer strip used for the Schirmer Tear Test is used for the lacritin dipstick immunoassay, minimizing patient intrusions and cost.

Example 11

[0083] Multiplex Western Blot Analysis.

[0084] In order to analyze both lacritin and lacritin-c more efficiently and cost-effectively on the same blot, Lacripep anti-lacritin antibodies were produced in goat and used with rabbit 13 anti-lacritin-c antibodies to enable detection with secondary antibodies labeled with spectrally-distinct NIR fluorescent dyes. It was expected that the fluorescent bands would be quantified and differential expression of the two proteins in tears from Sjgren's Syndrome patients would be quantified and compared to control tears. Applicants prepared recombinant lacritin in decreasing concentrations, which were run in the first three lanes and decreasing concentrations of lacritin-c in the middle three lanes. Both proteins were mixed at the same decreasing concentrations and were run together in the last three lanes. The protein samples were separated by SDS PAGE, transferred to nitrocellulose and challenged by a mixture of Lacripep lacritin and 13 lacritin-c antibodies at the same time on the same blot. A Western blot as shown in FIG. 8 was prepared as described above, developed by fluorescent secondary antibodies unique to lacritin and lacritin-c, and imaged by a LI-COR Odyssey CLx Imaging System. In FIG. 8, the red bands depict 13 lacritin-c antibody binding, while the green bands depict Lacripep lacritin antibody binding, demonstrating specificity of the respective antibodies: Lacripep antibodies detected only lacritin and 13 antibodies detected only lacritin-c.

REFERENCES

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[0127] The inventive subject matter being thus described, it will be obvious that the same may be modified or varied in many ways. Such modifications and variations are not to be regarded as a departure from the spirit and scope of the inventive subject matter, and all such modifications and variations are intended to be included within the scope of the following claims.