# Patent

## Title
Diagnosis of liver pathology through assessment of anti-gal IgG glycosylation

## Abstract

Methods for diagnosing pathology of the liver in subject suspected of having such pathology by measuring the glycosylation of anti-gal IgG in various biological fluids of the subject.

## Background
GOVERNMENT SUPPORT
Research leading to the disclosed inventions was funded, in part, with funds from the National Cancer Institute under grants R01 CA120206-01 and U01 CA084951. Accordingly, the United States government has certain rights in the inventions described therein.


FIELD OF THE INVENTION
The invention relates generally to the field of immunodiagnostics for detecting alterations in the glycosylation of the immunoglobulin that is reactive towards the alpha-gal epitope, which is a marker for liver diseases such as hepatocellular carcinoma, hepatitis, and cirrhosis.
BACKGROUND OF THE INVENTION
In this application we disclose a specific aspect of the invention disclosed in U.S. patent application Ser. No. 11/418,598, filed May 5, 2006, the contents of which are incorporated by reference. Further, all references cited herein are incorporated herein by reference in their entireties.
SUMMARY OF THE INVENTION
The present invention features a method for diagnosing pathologies of the liver or biliary system by obtaining biological fluid from the subject, quantifiably detecting glycosylation on immunoglobulin reactive for the antig-gal epitope (“anti-gal IgG”) in the biological fluid, and comparing the detected glycosylation with reference values for glycosylation of anti-gal IgG in subjects with no liver pathology, with known liver pathologies, or both; said glycosylation relative to the reference values being indicative of the presence or absence of the pathology of the liver, and preferably for liver fibrosis, cirrhosis, or hepatocellular carcinoma. The preferred glycosylation that is a reduction in galactosylation that correlates with an increase association with fucose binding lectins. Suitable biological fluids include whole blood, serum, urine, saliva, tears, or mucous.
The detection of glycosylated anti-gal IgG can proceed via any assay suitable in the art. The detection reagent can directly label the glycosyl moieties, for example, via carbohydrate specific chemicals or dyes, or via labeled lectins, labeled carbohydrate binding proteins, or labeled antibodies. The detection reagent can be a secondary reagent, for example, by first capturing anti-gal IgG and then contacting the capture reagent-anti-gal IgG complex with a labeled secondary reagent. Detection can proceed by separating glycosyl moieties from anti-gal IgG prior to the quantifiable detection of glycosylation. Detection can proceed by separating glycosylated anti-gal IgG from the test sample prior to the quantifiable detection of glycosylation.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 are a series of HPLC graphs showing total serum desialyated glycan profiles from control samples (top), HCV samples with stage 1&2 fibrosis (middle) or HCV patients with biopsy confirmed cirrhotic (stage 5-6) samples (bottom). The major peaks of interest are indicated and include a simple biantennary glycan (A2G2), a core fucosylated biantennary glycan (FcA2G2) and a core fucosylated bisected biantennary glycan (FcA2BG2). Cirrhotic samples were from patients with stage 6 fibrosis. Squares represent N-acetylglucosamine monosaccharides (GlcNAc); circles represent mannose; triangles represent galactose and the star represents the fucose.
FIG. 2 is a photograph, liquid chromatography graph, and chart identifying the major lectin reactive protein(s) in the serum of patients with fibrosis/cirrhosis. FIG. 2A is a photograph of a one dimensional electrophoretic gel of the major fucosylated proteins in the serum of cirrhotic patients were identified by extracting fucosylated proteins from serum using fucose specific lectins. In lane 1 are the lectin reactive proteins from healthy individuals; in lane 2 are the lectin reactive proteins from cirrhotic individuals. The 50 kd species, which is altered in the two patient groups is indicated with the asterisks. FIG. 2B are LC MS/MS graphs of the 50 kd species was digested with trypsin and the protein identified via LC MS/MS analysis. The MS2 spectra for the three major peptides identified are shown. FIG. 2C is a chart describing the major identified fucosylated proteins observed in cirrhotic samples corresponded to IgG reactive towards the alpha-gal epitope.
FIG. 3 is a photograph of the detection of altered anti-gal IgG glycosylation in samples from patients with cirrhosis. Either human serum albumin (HSA, column 1) or synthetic Gal α-1-3Galβ1-(3)4GlcNAc linked HSA (columns 2&3) was plated onto 96 well plates and incubated with human serum from four healthy control individuals or from three individuals with cirrhosis. Captured IgG was either detected using anti-human IgG conjugated secondary antibody (columns 1 &2) or using the fucose specific lectin AAL (column 3).
FIG. 4 is a photograph of the detection of altered anti-gal IgG fucosylation in samples from patients with cirrhosis. Removal of heterophilic alpha-gal antibodies prevents lectin reactivity of IgG from cirrhotic patients. IgG from either healthy individuals, or individuals with cirrhosis were captured from serum using a mouse anti-human IgG antibody. The amount of captured IgG (lane 1) or the level of AAL lectin reactivity (lane 2) was determined as in FIG. 3. Samples were either pre-cleared with HSA (lanes 1&2) or with alpha-gal HSA (lanes 3&4 before analysis. (H), pre-cleared with HSA; (A), pre-cleared with alpha-gal HSA.
FIG. 5 is a photograph and HPLC graph of glycan sequencing of anti-gal IgG from healthy or cirrhotic individuals. FIG. 5A is a photograph of a one dimensional electrophorectic gel after Coomassie staining of anti-gal IgG purified from either healthy individuals (H) or cirrhotic individuals (C). The Marker lane is also indicated (M). The heavy (H) and light chains (L) are indicated. FIG. 5 is an HPLC graph depicting the glycan analysis of the desialylated N-linked glycans associated with the heavy chain from healthy individuals (FIG. 5B) or a cirrhotic individual (FIG. 5C). For structures presented in panels B-C: FcA2G0, core fucosylated (1,6) agalactosylated biantennary glycan; FcA2BG0, core fucosylated (1,6) agalactosylated biantennary glycan with a bisecting N-acetylglucosomine (GlcNac); FcA2G1 (1,6) core fucosylated (1,6) biantennary glycan with a single galatose residue on the 1,6 arm; FcA2BG1 (1,6) core fucosylated (1,6) biantennary glycan with a single galatose residue on the 1,6 arm and a bisecting GlcNac; FcA2G2G1 (1,3), core fucosylated (1,6) biantennary glycan with a single galatose residue on the 1,3 arm; FcA2G2, core fucosylated biantennary N-glycan (FcA2G2); FcA2BG2, bisected core fucosylated biantennary N-glycan.
FIG. 6 is a photograph, HPLC graph, and graph depicting the reactivity of human IgG to the fucose binding lectin AAL is dependent by the type of N-linked glycan attached. FIG. 6A is a photograph of the detection of FcA2G0 glycan structure in IgG from healthy individuals (purchased from Sigma Chemicals) was digested overnight with Sialidase (Arthrobacter ureafaciens) and beta(1-4)-Galactosidase (Jack Bean) to create IgG molecules with the degalactosylated glycans observed in patients with cirrhosis. As a control, a mock sample treated identically, but without enzyme, was used. While IgG purified from healthy individuals has low reactivity with the fucose binding lectin, IgG purified from either cirrhotic individuals or IgG from healthy serum that has been treated with the sialidase and beta galactosidase has much greater reactivity, indicating that the lectin reactivity is directly associated with the FcA2G0 glycan structure. FIG. 6B contains two HPLC graphs depicting the glycan analysis of IgG from either mock treated samples (top) or enzyme treated samples (bottom) to confirm enzymatic digestion. The major peaks are indicated, and structures comprise A2G2S1, mono-sialylated biantennary glycan; FCA2G2S1, mono-sialylated core fucosylated biantennary N-glycan; FCA2BG2S1, mono-sialylated core fucosylated biantennary glycan with a bisecting GlcNac; A2G2S2, disialyated biantennary glycan; FCA2BG2S2, disialyated core fucosylated biantennary glycan; FcA2BG2S2, disialyated core fucosylated biantennary glycan with a bisecting GlcNac. FIG. 6C is a graph quantifying the results shown in FIG. 6B. X-axis is the glycan structure as detailed in panel B, Y-axis is the relative contribution of each glycan structure in the total N-linked glycan profile.
FIG. 7 contains graphs that quantitating lectin reactive IgG in patients with HCV induced fibrosis and cirrhosis. FIG. 7A is a scatter-plot of control individuals, individuals with HCV induced cirrhosis and individuals with HCV induced cirrhosis plus HCC. The n value, mean and standard error, are provided for each group below the graph. X-axis represents the patient group. Y-axis is fold increase in lectin reactive IgG as compared to commercially purchased sera. Statistical difference was obtained between the HCV induced cirrhosis group and control subjects (P<0.0001, Mann-Whitney test). FIG. 7B is a graph depicting the level of lectin reactive anti-gal IgG was measured in patients with HBV, autoimmune, alcohol or cryptogenic induced cirrhosis. The n value, mean, and standard error are provided for each group below the graph. X-axis represents the patient group. Y-axis is fold increase in fucosylated IgG as compared to commercially purchased sera. Values for each group are provided as mean and standard error of the mean. Statistical difference was obtained for all cirrhosis groups and control subjects (p<0.0001, Mann-Whitney test) but not between individual cirrhotic groups.
FIG. 8 contains graphs depicting the Increase in lectin reactive anti-gal IgG with the development of liver fibrosis. FIG. 8A is a graph depicting the level of lectin reactive anti-gal IgG in the control group, people with stage 1-2 fibrosis, stage 3-5 fibrosis and cirrhosis. The mean value for each group is plotted along with the 95% confidence interval for the mean. For graph, X-axis represents the patient group. Y-axis is fold increase in lectin reactive IgG as compared to commercially purchased serum. The n value, mean, and standard error are provided for each group below the graph. In this figure, all patients with cirrhosis (from FIGS. 5A & 5B) are included. FIG. 8B is a graph depicting the ROC analysis comparing people with Stage 1&2 fibrosis with control group. Using an optimal cutoff of 3 fold above commercially purchased sera, fucosylated immunoglobulin has a sensitivity of 84% and a specificity of 87%. FIG. 8C is a graph depicting the ROC analysis comparing Stage 1-2 fibrosis with 3-6 fibrosis/cirrhosis. Using an optimal cutoff of 5 fold above commercially purchased sera, fucosylated immunoglobulin has a sensitivity of 91% and a specificity of 79%.



DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Various terms relating to the methods and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein.
The following abbreviations may be used in the specification and examples: HBV, hepatitis B virus; HCV, hepatitis C virus; AFP, alpha-fetoprotein; HCC, hepatocellular carcinoma; HPLC, high performance liquid chromatography; Fc, fucosylated.
As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.
“Anti-gal IgG” refers to the immunoglobulins of the G class that recognize a specific sugar linkage on glycolipids and glycoproteins, that is present in non-human antigens. This sugar linkage (Gal α-1-3Galβ1-(3)4GlcNAc-R), referred to as the alpha-gal epitope, is absent in humans but is abundantly synthesized by bacteria and nonprimate mammals. Anti-gal antibodies are naturally occurring antibodies that, in healthy subjects, constitute  ̃1% of total serum IgG.
The following examples are provided to describe the invention in more detail. They are intended to illustrate, not to limit the invention.
EXAMPLE 1
General Experimental Procedures
Patients:
Patients for the analysis were obtained from the University of Michigan under a study protocol that was approved by the University of Michigan's Institutional Review Board. In addition, written informed consent was obtained from each subject. Demographic and clinical information was obtained, and a blood sample was collected from each subject. The blood sample from patients with chronic HCV infection was obtained at the time of liver biopsy and antiviral therapy. HCV was defined as the presence of HCV RNA with a lower limit of detection of <50 IU/mL at the University of Michigan Clinical Laboratory. All liver biopsies were at least 30 mm long and 1.4 mm wide and graded by three hepatic pathologists in a blinded fashion and the amount of fibrosis was graded using the Ishak scoring system. (Ishak, et al., 1995. J Hepatol 22:696-9). Details on all patients are provided in Table 1.







TABLE 1







Description of control subjects and patients with liver disease.









Stage of Disease (Ishak)1



Stage of Disease (Ishak)1

















6
Non-







(complete
HCV
HCC +


Variables
Controls
1-2
3-5
cirrhosis)
Cirrhosis
Cirrhosis





Sample Size
113
24
19
57
34
87


Age2
 51 ± 11
50 ± 6
51 ± 4
53 ± 8 
55 ± 8 
56 ± 7 


% NHW/AA/H/Asian3
90/10/0/0
98/1/1/0
96/2/1/1
85/11/2/2
98/0/1/0
88/4/2/6


ALT (IU/mL)4
28 ± 5
75 ± 7
 71 ± 11
79 ± 19
85 ± 26
75 ± 12


AST (IU/mL)5
26 ± 3
73 ± 5
69 ± 8
93 ± 11
108 ± 24 
101 ± 18 


Total Bilirubin6
 0.3 ± 0.1
 0.3 ± 0.2
 0.3 ± 0.4
1.2 ± 1.3
1.4 ± 0.7
1.6 ± 1.3


(mg/dL)


% HCV Genotype 17
NA
78
72
76
NA
75


HCV Viral load8
 0
1.3 ± .8
1.6 ± 1 
1.7 ± 1.5
NA
1.4 ± 1.3


(copies/mL)


serum gamma-globulin
0.54 ± 0.1
0.85 ± 0.2
 1.1 ± 0.4
1.3 ± 0.8
1.4 ± 0.8
1.1 ± 0.3


(g/dL)9





1Fibrosis staging based upon ISHAK scoring system. For this study, stage 5 or greater is considered cirrhotic.


2Mean age in years.


3NHW = non-Hispanic White; AA = African American; H = Hispanic;


4ALT = alanine aminotransferase;


5AST = aspartate aminotransferase.


6P = 0.02 HCC vs. controls, fibrosis stage 1 and 2; otherwise no differences.


7HCV = hepatitis C. Non-HCV cirrhosis = 5 hepatitis B, 6 autoimmune hepatitis, 14 cryptogenic cirrhosis, and 9 alcoholic. NA = not applicable.


3,7,8No significant differences among groups


4,5,9P < 0.0001 Fibrosis stage 1-6 and HCC vs. controls






A group of individuals with no history of liver disease, alcohol consumption less than 40 gm a week, and no risk factors for viral hepatitis were enrolled from the General Internal Medicine clinics. All subjects in this control group were documented to have normal liver biochemistry and negative HCV antibodies. Patients with HCV induced cirrhosis plus HCC, were enrolled from the Liver Clinic during this period. The diagnosis of HCC was made by histopathology (n=87, including all T1 lesions), and if histopathology was not available by two imaging modalities (dynamic ultrasound [US], magnetic resonance imaging [MRI], or computed tomography). All patients with HCC were determined to have underlying cirrhosis based on histopathology (85%) and by clinical parameters (15%). Each of the patients with a histological diagnosis of cirrhosis had a normal US and, if serum AFP was elevated, a MRI of the liver within 3 months prior to enrollment and another one 6 months after enrollment that showed no liver mass, in order to confirm that they have not developed HCC. The cirrhotic controls have been followed for a median of 12 months (range 7-18 months) after enrollment, and no one has developed HCC. The etiology of the liver disease for the patients without HCV infection was determined as previously described (Marrero et al., 2005. J. Hepatology 43:1007-12) and the definition of cirrhosis in these patients was also determined by histology.
Glycan Analysis of Total Serum.
Total serum glycan analysis was performed on composite samples from 10 healthy patients, 10 patients with mild fibrosis, and 10 patients with cirrhosis to determine the glycan changes that occur with the development of liver cirrhosis. Briefly, 5 μl of serum was absorbed into a dehydrated 12% tris-glycine gel plug. The gel plug was reduced and alkylated and the proteins fixed using 10% methanol and 7% acetic acid. The N-linked glycans were removed using N-Glycanase Plus (Prozyme, San Leandro, Calif. USA) as done previously (Block, et al., 2005. Proc Natl Acad Sci USA 102:779-84; Comunale, et al., 2006 J Proteome Research. 6:308-315; Comunale, et al., 2004 Proteomics 4:826-38) and labeled with 2-aminobenzoic acid (Ludger LTD, Abingdon, UK) according to the manufacture's directions (Guile, et al., 1996. Anal Biochem 240:210-26; Rudd, et al., 1999 Biotechnol Genet Eng Rev 16:1-21). Desialation of labeled N-glycan was performed via incubation of dried glycan with 1 μnit/ML of Arthrobacter ureafaciens sialidase (Prozyme,) as per manufacturer's directions. Glycan structures were identified by the calculation of GU value, through comparison to known standards and by sequential exoglycosidase digestion as before (Guile, et al., 1998. Eur J Biochem 258:623-56; Guile, et al., 1996. Anal Biochem 240:210-26).
Proteomic Identification of Fucosylated Glycoproteins:
To determine the identity of those proteins that contained core fucosylated glycans, we utilized specific sugar (glycan) binding proteins (lectins) to enrich for fucosylated proteins prior to proteomic analysis via mass spectrometry. Briefly, lectin extraction was performed using agarose bound Aleuria Aurantia Lectin (AAL) (Vector Laboratories, Ventura, Calif.) and Affi Sep-AAL Absorption Buffer and Elution Buffers (GALAB, Germany). Fucosylated proteins from 10 healthy individuals and 10 cirrhotic individual (serum was combined) were extracted and the fucosylated proteins dissolved in loading buffer and resolved by electrophoresis through 12% SDS/polyacrylamide gels (SDS/PAGE). Proteins of interested were excised from the gel and identified via LC MS/MS. Samples were de-N-glycosylated prior to LC MS/MS analysis. Peptide identification was preformed on a ThermoFinnigan LCQ ion trap mass spectrometer (Thermo-Finnigan, San Jose, Calif.) equipped with on-line microcapillary HPLC (Eldex, Napa, Calif.) and microspray ionization source as done previously (Comunale, et al., 2006. J Proteome Research. 6:308-315). Peptide searches were performed using Sequest (Thermo-Finnigan Corp.) against the Swiss-Prot human database. Xcorr versus change state parameters were set to 1.50, 2.0 and 2.50.
Lectin Fluorophore-Linked Immunosorbent Assay (FLISA):
For the analysis of the glycan modification of total IgG and anti-gal IgG, we utilized a lectin-FLISA based approach. Briefly, to remove the fucosylation of the capture antibody (mouse anti-human IgG, Bethyl Laboratories, Montgomery, Tex.), antibody was incubated with 11 mM sodium periodate for 1 hour at 4° C. An equal volume of ethylene glycol was added and the oxidized antibody brought to a concentration of 10 μg/mL with sodium carbonate buffer, pH 9.5. Antibody (5 μg/well) or human serum albumin attached to Galα1-3Galβ1-3GlcNAc (HSA-alpha-gal; Dextra Labs) or HSA alone (Sigma-Aldrich), was added to the plate and following incubation washed with 0.1% Tween 20/PBS 7.4 and blocked overnight with 3% BSA/PBS. For analysis, 3 μl of serum was diluted in 97 μL pf 3% BSA/PBS and added to the plates for 2 hours and washed 5 times in lectin incubation buffer (10 mM Tris pH 8.0, 0.15M NaCl, 0.1% Tween 20) before fucosylated IgG detected with a biotin conjugated Aleuria aurantia (AAL) lectin (Vector Laboratories, Burlingame, Calif.). Bound lectin was detected using IRDyeTM 800 Conjugated streptavidin and signal intensity measured using the Odyssey® Infrared Imaging System (LI-COR Biotechnology, Lincoln, Nebr.). In all cases sample intensity was compared to commercially purchased human serum (Sigma Inc., St Louis, Mo.) (Kaneko, et al., 2006 Science 313:670-3; Kweon, et al., 2001 B. J Hepatol 35:749-55). All samples were run in triplicate and inter sample variation was less than 1%.
Purification and Glycan Analysis of Anti-Gal IgG.
For analysis of anti-gal IgG, the same samples described above were utilized. Synthetic Galα1-3Galβ1-3GlcNAc-HSA (Dextra Labs, Reading, United Kingdom) was coupled to a NHS-activated Sepharose 4 Fast Flow affinity column (GE Healthcare, Piscataway, N.J.) as per manufacturer's directions. Briefly, 50 μl of serum was incubated with the column and the column washed with five column volumes of TBS-T (Tris buffered saline with 0.1% Tween-20), followed by one column volume was with TBS. The Galα1-3Galβ1-3GlcNAc specific IgG was eluted using 0.1M NaCl2/0.1M Glycine pH 2.8 and immediately neutralized. For this study, an equal amount of anti-gal IgG (1 μg) was reduced, alkylated and separated on a 12% tris-glycine acrylamide gel and stained using Colloidal Coomassie. Anti-gal IgG bands were excised, destained and glycan analysis preformed as done previously and as described above (Block, et al., 2005 Proc Natl Acad Sci USA 102:779-84; Comunale, et al., 2006 J of Proteome Research. 6:308-315).
Exoglycosidase Treatment of Purified IgG.
IgG was purified from commercially purchased human serum (Sigma Chemicals) using the Melon Gel IgG Purification Kit (Pierce, Rockland, Ill.). Purified IgG was subsequently concentrated and buffered exchanged TBS. Half of the sample was incubated with 5 units/ML Arthrobacter ureafaciens sialidase (Prozyme), 5 units/ML Jack Bean beta-galactosidase (Prozyme) and 5× exoglycosidase buffer (0.5 M Citrate Phosphate buffer, pH 4.5, 0.2 mM Zn(O2CCH3)2, 150 mM NaCl2) at 37° C. overnight. The other half of the IgG sample was incubated with buffer alone at 37° C. overnight. The next day, half of the sample was tested for lectin reactivity as described above and the other half was used for HPLC glycan analysis to confirm exoglycosidase digestion.
Statistical Analysis:
Descriptive statistics for stage patients were compared by scatter plots that included the outliers. All values were reported as mean values +/− standard error unless otherwise stated. As the data did not follow typical Gaussian distribution, a non-parametrical test (two-tailed, 95% confidence, Mann-Whitney Test) was used to determine statistical difference between groups. A two-tailed P-value of 0.05 was used to determine statistical significance. All analyses were performed using GraphPad Prism (San Diego, Calif., USA).
EXAMPLE 2
Alterations in the Human Serum Glycome and Identification of Anti-Gal IgG as the Predominant Altered Glycoprotein in Patients with Cirrhosis
FIG. 1 shows the desialylated N-linked glycan analysis of total serum from either control patients (left), HCV patients with stage 1-2 fibrosis (middle) or patients with HCV and biopsy confirmed cirrhosis (right) as performed by normal phase HPLC (Guile, et al., 1996. Anal Biochem 240:210-26; Rudd, et al., 1997 Curr Opin Biotechnol 8:488-97). Each peak corresponds to a different glycan structure and as FIG. 1A shows significant changes in N-linked glycosylation can be seen in patient serum with the development of cirrhosis. Peaks that are significantly altered are indicated and correspond to an agalactosylated core fucosylated biantennary N-glycan (FcA2G0), a core fucosylated biantennary N-glycan (FcA2G2) and a bisected core fucosylated biantennary N-glycan (FcA2BG2).
The identity of the major glycoproteins to which the fucosylated glycan were attached was determined by two approaches. The first was the observation that the difference in glycosylation as observed in FIG. 1 was abolished if immunoglobulin was depleted from serum, prior to N linked glycan analysis. Although the serum depletion approach provided circumstantial evidence that immunoglobulins were the source of elevated fucose in the serum, a second method was used to positively identify the fucosylated proteins. Undenatured proteins containing fucosylated N-linked glycans were extracted from serum by passing over columns with the fucose binding lectin, AAL. Polypeptides binding to the lectin represent the serum “fucome” or “fucosylated proteome”. In this way, the fucosylated proteome was analyzed by one dimensional gel electrophoresis from healthy individuals or from patients with cirrhosis. As FIG. 2A shows, several difference can be seen between the fucosylated proteome from healthy patients and patients with cirrhosis. A species at 50 kd was highly elevated in patients with cirrhosis and was further examined by trypsination of the protein followed by LC MS/MS analysis for protein identification (FIG. 2B). Three of the peptides identified are shown and were used to identify the 50 kd species as IgG with reactivity to the sugar Gal α-1-3Galβ1-(3)4GlcNAc, commonly referred to as the alpha-gal epitope (FIGS. 2B and 2C).
EXAMPLE 3
Confirmation of Alteration in the Glycosylation of Alpha-Gal Reactive IgG
Confirmation of specificity to the alpha-gal epitope was performed through a plate based assay to measure the level of lectin reactive IgG specific for the epitope. As shown in FIG. 3, synthetic alpha-gal linked to human serum albumin (HSA) was plated onto 96 well plates and incubated with human serum from four healthy control individuals, or 3 individuals with cirrhosis. Captured IgG was detected using anti-human IgG conjugated secondary antibody or using the fucose specific lectin AAL. As a control, unconjugated human serum albumin (HSA) was plated on adjacent wells to measure specificity of binding. Columns 1 and 2 of FIG. 3 show the binding of human IgG to wells containing either HSA (column 1) or HSA-alpha-gal (column 2) detected using an anti-human IgG secondary antibody. As this Figure shows, and not surprisingly, while no human IgG molecules bind to the HSA (column 1), strong binding is observed to the HSA conjugated with the alpha-gal sugar (column 2). The binding is present in all serum samples and is consistent with the report of anti-gal antibodies being present in the serum of all patients (Galili, et al., 1993 Blood 82:2485-93). In column 3 is the lectin reactivity of the anti-gal antibody. As column 3 shows, only the captured IgG from patients with cirrhosis were reactive to the lectin, suggesting that the alpha-gal antibody becomes lectin reactive with the development of severe fibrosis or cirrhosis. The relative increase in anti-gal IgG, total IgG, and AAL reactive anti-gal IgG is shown in Table 2.







TABLE 2







The relative levels of total IgG, anti-gal IgG and AAL reactive anti-gal


IgG in patients with limited fibrosis, severe fibrosis, or cirrhosis.









Fold greater than healthy purchased control2













AAL reactive anti-gal


Patient Group1
Total IgG3
Anti-gal IgG4
IgG5





Experimental
  1 +/− 0.1
  1 +/− 0.1
1 +/− 0.1


control samples


F1-F2
1.8 +/− 0.1
  2 +/− 0.2
3 +/− 0.2


F3-F5
2.1 +/− 0.2
3.5 +/− 0.1
9 +/− 0.2


Cirrhotic
2.4 +/− 0.1
3.7 +/− 0.1
15 +/− 0.2 





1Fibrosis staging based upon ISHAK scoring system. Samples were combined for analysis. 10 samples per group were used for analysis.


2All values are provided as relative fold increases over commercially purchases sera.


3The relative increase in IgG as compared to commercially purchased sera. Numbers are fold increase +/− standard deviation.


4The relative increase in anti-gal IgG as compared to commercially purchased sera. Numbers are fold increase +/− standard deviation


5The relative increase in AAL reactive anti-gal IgG as compared to commercially purchased sera. Numbers are fold increase +/− standard deviation.






EXAMPLE 4
Alpha-Gal Antibodies Become Selectively Reactive to the Fucose Binding Lectin, in People with Fibrosis and Cirrhosis
Increased binding of the AAL lectin to IgG from cirrhosis patients could be abolished if samples were pre-cleared of anti-alpha-gal antibodies prior to analysis. FIG. 4 shows the results of a human IgG lectin-FLISA using either sera from four healthy individuals or sera from three patients with cirrhosis. In this assay, a mouse anti-human IgG monoclonal antibody is used to capture all IgG molecules. For both serum samples, samples were pre-cleared with human serum albumin (HSA) or with Gal α-1-3Galβ1-(3)4GlcNAc (alpha-gal) linked to HSA. Captured samples were either probed with a secondary mouse anti human IgG antibody (column 1) or with the fucose binding lectin AAL (column 2). As columns 1 & 3 of FIG. 4 show, no significant change in IgG binding was observed from the healthy and cirrhotic patient as a function of blocking agents. Column 2 of FIG. 4 shows the lectin reactivity of the captured IgG molecules. As before, no signal is detected from the healthy samples regardless of pre-treatment. In contrast, strong AAL binding of captured human IgG from patients with cirrhosis was observed when samples were pre-cleared with HSA (column 2). However, when alpha-gal reactive antibodies have removed, even though large amounts of IgG are still captured on the plate (column 3) greatly reduced binding of the lectin is observed (column 4), indicating that alpha-gal antibodies specifically are altered with the development of cirrhosis. Additionally, when the IgG is denatured before analysis in an IgG lectin-FLISA, all patient samples are reactive to the fucose specific lectin, suggesting that the lectin reactivity IgG is conformation dependent.
EXAMPLE 5
Glycosylation of Human IgG Changes with the Development of Cirrhosis
We performed glycan analysis on anti-gal IgG via N-linked glycan sequencing. Purified anti-gal IgG was isolated from either control patients or patients with HCV induced cirrhosis and 1 μg resolved via SDS-PAGE (FIG. 5A). Subsequently, structural N-linked glycan analysis of the heavy chains was performed and is shown in FIGS. 5B and 5C. Significant differences can be seen on the glycans from patients with cirrhosis as compared to the healthy patients (compare FIG. 5B with 5C). While healthy subjects contained primarily a FcA2G2 glycan structure, the heavy chain from patients with cirrhosis contained truncated structures containing either a single galactose residue (FcA2G1) or no galactose residues (FcA2G0). The agalactosylated core fucosylated biantennary N-glycan (FcA2G0) was also observed in the glycomic analysis of total serum as shown in FIG. 1A. N-linked glycans were not observed on light chains, either via lectin blot or by glycan sequencing. There thus, appears to be a consistent structural difference between the IgG isolated from healthy individuals and those with cirrhosis, and this change (agalactose) has been associated with a pro-inflammatory structure (Kaneko, et al., 2006. Science 313:670-3; Lastra, et al., 1998. Autoimmunity 28:25-30).
EXAMPLE 6
The Change in Glycosylation of Human IgG Allows for Interaction with Fucose Binding Lectins
The relationship between the change in glycosylation observed in cirrhosis (FIGS. 5B and 5C) and the increased reactivity with fucose binding lectins (FIGS. 1-4) was tested by altering the glycan structure on IgG from healthy individuals to mimic that observed in patients with fibrosis/cirrhosis. This was done by treating purified IgG from healthy individuals, which does not interact with the fucose binding lectin AAL, with sialidase (Arthrobacter ureafaciens) and beta-galactosidase (Jack Bean), to create the degalactosylated N-linked glycan normally found on IgG patients with cirrhosis (Nimmerjahn, et al., 2007 Proc Natl Acad Sci USA 104:8433-7). The reactivity of these samples with the fucose binding lectins was tested using a lectin-FLISA for captured IgG. Consistent with the results obtained in FIGS. 2-5, while IgG from control serum is not reactive with fucose binding lectins, IgG from cirrhotic patients is highly reactive (FIG. 6A). Reactivity with fucose binding lectin was obtained when IgG was degalactosylated enzymatically, suggesting that the increased reactivity of IgG to the fucose binding lectins is partially associated with the degalactosylated glycan structure (FIGS. 6A & 6B). Treatment of samples with just sialidase alone had no effect on binding, while treatment with or beta-galactosidase alone had only a minor effect. Glycan sequencing on treated IgG was performed to confirm glycan modification (FIG. 6C). Thus, the increased reactivity of the IgG from cirrhotic individuals with fucose binding lectins is partially related to the truncated glycosylation of the IgG molecule.
EXAMPLE 7
Increased Reactivity of a Fucose Specific Lectin with Human IgG Appears to Correlate with the Development of Cirrhosis
Using the same lectin FLISA shown in FIGS. 2-4, the relative amount of lectin reactive IgG was determined in 257 coded serum samples from either control subjects, those with HCV induced cirrhosis and those with HCV induced cirrhosis plus HCC. The results are shown in FIG. 7A and are expressed as “fold increase” over commercially purchased human serum.
The signal detected in the samples from the 113 control subjects was similar to that observed in commercially purchased serum, with a mean relative value of 0.94 (FIG. 7A). In contrast, 57 patients with HCV induced cirrhosis had values greater than 3 fold above commercially purchased serum with a mean increase of 13 fold. This difference was statistically significant (P<0.0001; Two-tailed Mann-Whitney test) confirming our results in FIGS. 2-6.
As the fucosylation of many proteins has been shown to be increased in HCC (Comunale, et al., 2006. J Proteome Research. 6:308-315; Drake, et al., 2006 Mol Cell Proteomics 5:1957-1967), it was of interest to see if greater lectin reactivity would be observed in patients with cirrhosis plus HCC. Therefore the level of lectin reactive IgG was measured in 87 patients with HCV induced cirrhosis plus HCC. As FIG. 7A shows, patients with cirrhosis plus HCC had a mean 14 fold increase in lectin reactive IgG, which was not statistically different than the increase observed in patients with cirrhosis alone. Thus, no further increases in lectin binding could be observed with the development of HCC.
EXAMPLE 8
Fucose Detection of Anti-Gal IgG is Observed in Patients with Cirrhosis from Multiple Etiologies
To determine if the lectin reactivity with IgG was a phenomenon that correlated with the development of cirrhosis, independent of the etiology, we examined the level of lectin reactive IgG in patients with cirrhosis induced from excessive alcohol consumption, autoimmune disease, HBV infection or from patients with an unknown underlying disease. As FIG. 7B shows, although the sample size is limited, the trend is very clear: IgG from patients with cirrhosis, regardless of the underlying etiology, have greater reactivity to fucose specific lectins as compared to healthy controls. Similar to the results obtained in FIGS. 3-5, all patients with cirrhosis had >3 fold elevations in lectin reactive IgG. Statistical difference was observed for all individual cirrhotic groups (from non HCV induced cirrhosis) when compared to control subjects (P<0.0001; Mann Whitney test). However, no statistical difference was observed between individual cirrhotic groups (One-way ANOVA; Kruskal-Wallis test).
EXAMPLE 9
Increased Fucose Detection of Anti-Gal IgG is Observed in Patients with Fibrosis
As the level of lectin reactive anti-gal IgG was increased in patients with cirrhosis, it was of interest to determine if similar changes could be observed in patients with fibrosis. Thus we examined the level of lectin reactive anti-gal IgG in patients with either limited fibrosis (stage 1-2) or more severe fibrosis (stage 3-4). These data are shown in FIG. 8A along with the data from patients from the control or cirrhosis groups. As this Figure shows, stage 1&2 fibrotic patients had a mean increase of 3.5 fold over commercially purchased sera, compared to only a 0.94 fold increase in the control patients. Even with the small sample size (n=24) this increase was statistically different from the control group (P<0.0001; Two-tailed Mann-Whitney test) implying that the change in IgG starts with the development of liver fibrosis. Patients with bridging fibrosis and incomplete cirrhosis (stage 3-5) had an even greater level of lectin reactive IgG with a mean increase of 9.3 fold over commercially purchased sera. Although the sample size was limited (n=19), significant statistical difference (P<0.0001; Two-tailed Mann-Whitney test) was observed between those patients with limited fibrosis (stage 1&2) and those with more severe fibrosis (stage 3&5). No statistical difference was observed between patients with bridging fibrosis (Stage 3&5) and those patients with cirrhosis (P>0.5).
Receiver operator characteristic (ROC) curves were plotted to help define the optimal cutoff values for differentiating the different stages of liver fibrosis and cirrhosis. ROC analysis was performed between stage 1&2 fibrosis and control subjects and between stage 1 & 2 fibrosis and stage 3-6 fibrosis/cirrhosis. FIG. 8B shows the AUROC for the analysis of control subjects versus stage 1&2 fibrosis was 0.90 with significant statistical difference between the observed curve and no-discrimination line (P<0.0001). Using a cutoff of 3 fold above commercially purchased sera, lectin reactive anti-gal IgG had a sensitivity of 84%, a specificity of 87%, a positive predictive value (PPV) of 70% and a negative predictive value (NPV) of 94%. The ROC analysis for the differentiation of those patients with limited fibrosis (stage 1 & 2) and those with stage 3 or greater fibrosis is shown in FIG. 8C. In such an analysis, the AUROC curve for fucosylated IgG was 0.93 with a sensitivity of 91% and a specificity of 79%, using an optimal cutoff of 5 relative units.

## Claims

1. A method for detecting a liver disease in a subject suspected of having the liver pathology comprising:
obtaining biological fluid from the subject;
separating anti-gal IgG from said biological fluid using a separation agent;
contacting said separated anti-gal IgG from said biological fluid with a fucose-binding lectin, IgG, and allowing said lectin to bind to said anti-gal IgG; quantifiably detecting lectin bound to said anti-gal IgG to obtain a detected bound lectin value;
comparing the detected bound lectin value with a reference value for lectin binding on said anti-gal IgG from a comparable biological fluid of subjects without said liver disease, said detected bound lectin value relative to the reference value being indicative of the presence or absence of said liver disease, wherein a detected bound lectin value that deviates from said reference value to a statistically significant degree is indicative of the presence of said liver disease, or,
comparing the detected bound lectin value with a reference value for lectin binding on said anti-gal IgG from a comparable biological fluid of subjects in which said liver disease is known to be present, said detected bound lectin value relative to the reference value being indicative of the presence or absence of said liver disease, wherein a detected bound lectin value that deviates from said reference value to a statistically significant degree is indicative of the absence of said liver disease.

2. The method according to claim 1, wherein said liver disease is cirrhosis.

3. The method according to claim 1, wherein said liver disease is liver fibrosis.

4. The method according to claim 1, wherein said liver disease is hepatocellular carcinoma.

5. The method according to claim 1, wherein said liver disease is hepatitis.

6. The method of claim 1 wherein the biological fluid is whole blood, serum, urine, saliva, tears, or mucous.

7. The method of claim 1, wherein said separation agent comprises lectins, antibodies, or polypeptides by carbohydrate-recognition domains.

8. The method of claim 1, wherein the lectin is coupled to a detectable moiety.

9. An assay for detecting a liver disease in a subject suspected of having the liver disease comprising: quantifiably detecting fucosylated anti-gal IgG from a biological fluid of said subject to provide a detected fucosylated anti-gal IgG value, wherein said detecting step utilizes an assay comprising a fucose-binding lectin, wherein a detected fucosylated anti-gal IgG value that deviates from a reference value for fucosylated anti-gal IgG from a comparable biological fluid of subjects without the liver disease is indicative of the presence of said liver disease, and wherein a detected fucosylated anti-gal IgG value that deviates from a reference value for fucosylated anti-gal IgG from a comparable biological fluid of subjects with the liver disease is indicative of the absence of said liver disease.

10. The assay according to claim 9 further comprising utilizing a separation agent to separate said fucosylated IgG from the biological fluid prior to quantifiably detecting the fucosylated anti-gal IgG.

11. The assay according to claim 10, wherein said separation agent comprises lectins, antibodies, or polypeptides by carbohydrate-recognition domains.