WO2006058385A1 - Multiplex-bead complex for determination of lysosomal storage disorders - Google Patents

Multiplex-bead complex for determination of lysosomal storage disorders Download PDF

Info

Publication number
WO2006058385A1
WO2006058385A1 PCT/AU2005/001821 AU2005001821W WO2006058385A1 WO 2006058385 A1 WO2006058385 A1 WO 2006058385A1 AU 2005001821 W AU2005001821 W AU 2005001821W WO 2006058385 A1 WO2006058385 A1 WO 2006058385A1
Authority
WO
WIPO (PCT)
Prior art keywords
seqid
microsphere
purified
epitope
lsd
Prior art date
Application number
PCT/AU2005/001821
Other languages
French (fr)
Inventor
Peter John Meikle
John Joseph Hopwood
Douglas Alexander Brooks
Caroline Dean
Original Assignee
Women's And Children's Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Women's And Children's Hospital filed Critical Women's And Children's Hospital
Publication of WO2006058385A1 publication Critical patent/WO2006058385A1/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • the present invention generally pertains to a process of population screening that leads to the detection of Lysosomal Storage Disorders ("LSDs") and related diseases in a subject. More particularly, this invention pertains to compounds, reagents, and methods for identifying and quantifying the levels and ratios of multiple target antigens that are used to identify individuals likely to be affected with an LSD.
  • the target antigens are naturally present in biological fluids or tissues of either LSD or non-LSD patients.
  • LSDs represent a group of over 40 distinct genetic diseases that generally affect young children. Individuals that are affected with a LSD present a wide range of clinical symptoms that depend upon the specific disorder or a particular genotype involved. The clinical symptoms associated with LSDs can have a devastating impact on both the child and the family of affected individuals. For example, central nervous system dysfunction, behavioral problems, and severe mental retardation are characteristic of many LSDs. Other clinical symptoms may include skeletal abnormalities, organomegaly, corneal clouding and dysmorphic features (Neufeld and Muenzer, 1995). Patients are usually born without the visible features of a LSD, but early stage symptoms can quickly develop into a progressive clinical concern. In severe cases, the affected children require constant medical management but still often die before adolescence.
  • the next stage of diagnosis involves a urine screen, wherein a "positive" urine screen is then followed by specific enzymatic analysis.
  • chromatographic-based screening methods are simple to perform, they are relatively labor-intensive and often require experience to accurately interpret results.
  • One example includes a method of identifying and quantifying biochemical markers ("biomarkers”) that are present in biological fluids or tissues of a patient having a MPS or related disorders, and comprises determining a target quantity of a target MPS biomarker oligosaccharide from a target biological sample taken from the target animal, and then comparing the target quantity to a reference quantity of a reference MPS biomarker oligosaccharide for the diagnosis, characterization, monitoring, and clinical management of MPS and related disease, as described in PCT Application AU03/00731 entitled "Identification of Oligosaccharides and their Use in the Diagnosis and Evaluation of Mucopolysaccharidoses and Other Related Disorders," filed on June 13, 2003 with Hopwood et al., listed as inventors (the entire content of PCT Application AU03/00731 is hereby incorporated by reference). Consequently, chromatographic-based screening tests for LSDs are not used in some centers. Furthermore, these chromatographic-based screens are not readily amenable to
  • LSDs One common feature of LSDs is the accumulation and storage of materials within lysosomes. It is generally recognized that the accumulation and storage of material in LSD affected individuals results in an increase in the number and the size of lysosomes within a cell from approximately 1% to as much as 50% of total cellular volume. In non-affected individuals, such materials are typically degraded into degradation products within the lysosome and then transported across the lysosomal membrane. Certain lysosomal proteins are present at elevated levels in the lysosomes of affected individuals (Meilde et al, 1997; Hua et al, 1998). These identified proteins are useful biomarkers for an early diagnosis of all LSDs.
  • Multiplexing Bead Technology is built around 3 core technologies.
  • the first is the family of fluorescently dyed microspheres having specific biomolecules bound to the surface of the microsphere.
  • the second is a flow cytometer with 2 lasers and associated optics to measure biochemical reactions that occur on the surface of the microspheres, and the third is a high-speed digital signal processor to efficiently manage the fluorescent output.
  • This type of system has been described in, for example: United States Patents 6,449,562; 6,524,793 and United States Patent Application SN 09/956,857.
  • United States Patent 6,449,562 (“the '562 Patent") entitled "Multiplexed Analysis of Clinical Specimens Apparatus and Method," having Chandler et al.
  • the '562 Patent discloses a method for the multiplexed diagnostic and genetic analysis of enzymes, DNA fragments, antibodies, and other biomolecules comprising the steps of constructing an appropriately labeled beadset, exposing the beadset to a clinical sample, and analyzing the combined sam ⁇ le ⁇ eadset by flow cytometry. Flow cytometric measurements are used to classify, in real-time, beads within an exposed beadset and textual explanations, based on the accumulated data obtained during realtime analysis, are generated for the user.
  • the inventive technology of the '562 Patent enables the simultaneous, and automated, detection and interpretation of multiple biomolecules or DNA sequences in real-time while also reducing the cost of performing diagnostic and genetic assays.
  • the '562 Patent does not describe how to utilize the technology for diagnosing LSD's.
  • United States Patent 6,524,793 entitled “Multiplexed Analysis of Clinical Specimens Apparatus and Method,” having Chandler et al. listed as inventors, was issued on February 25, 2003.
  • the '793 Patent discloses a method for the multiplexed diagnostic and genetic analysis of enzymes, DNA fragments, antibodies, and other biomolecules comprising the steps of constructing an appropriately labeled beadset, exposing the beadset to a clinical sample, and analyzing the combined sample/beadset by flow cytometry. Flow cytometric measurements used to classify, in real-time, beads within an exposed beadset and textual explanations, based on the accumulated data obtained during realtime analysis, are generated for the user.
  • the '793 Patent enables the simultaneous, and automated, detection and interpretation of multiple biomolecules or DNA sequences in real-time while also reducing the cost of performing diagnostic and genetic assays.
  • the '793 Patent does not describe how to utilize the technology for diagnosing LSD's.
  • the '857 Application also provides a composition comprising an addressable microsphere carrying at least two fluorescent reactants capable of forming reactant-analyte pairs distinguishable by their fluorescence intensity, and kits comprising the inventive composition and a plurality of reporter reagents.
  • the '857 Application does not describe how to utilize the technology for diagnosing LSD's. The entirety of each of the applications or patents listed above is hereby specifically incorporated by reference.
  • the present invention provides compounds, reagents, and methods for a LSD diagnostic multiplex assay.
  • Figure 1 shows a microsphere capture sandwich immunoassay having a microsphere with two spectrally distinct fluorophores, the target LSD capture antibody and the unique LSD target protein or target antigen bound to the target LSD capture antibody and a reporter molecule;
  • Figure 2 shows a calibration curve for ⁇ -glucosidase in a microsphere based assay
  • Figure 3 shows multiplexed calibration curves in a microsphere based assay
  • Figure 4 shows the multiplex technology having at least a 10-plex for LSD's
  • Figure 5 shows calibration curves for a 10-plex immune quantification of lysosomal proteins
  • Figure 6 shows calibration curves for a 11-plex immune quantification of lysosomal proteins
  • Figure 7 shows the antibodies and bead regions used for the 10- plex assay
  • Figure 8 shows a list of the precision studies that were conducted on the 11-plex
  • Figure 9 shows the Intra and Inter assay percentage CV for the 11- plex
  • Figure 10 shows protein markers for LSD screening using multiplex assays for LSD
  • Figure 11 shows the LAMP Protein Standard and Adult Controls in Fluorescent Units
  • Figure 12 shows the GAA Protein Standard and Adult Controls in Fluorescent Units
  • Figure 13 shows the beta-Glucosidease Protein Standard and Adult Controls in Fluorescent Units
  • Figure 14 shows retrospective analysis of CTH C 16:0 in newborn blood spots
  • Figure 15 shows retrospective analysis of HNAc-U A-HNAc-UA in newborn blood spots
  • Figure 16 shows Validation of the Multiplex Newborn Screening Assay
  • Figure 18 shows Assay performance during validation studies
  • Figure 19 shows control ranges for protein markers (sulphamidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • protein markers sulphamidase
  • Figure 20 shows control ranges for protein markers (acid sphingomyelinase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • Figure 21 shows control ranges for protein markers (alpha iduronidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • Figure 22 shows control ranges for protein markers (LAMP-I), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • LAMP-I protein markers
  • Figure 23 shows control ranges for protein markers (alpha- glucosidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • protein markers alpha- glucosidase
  • Figure 24 shows control ranges for protein markers (beta- glucosidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • Figure 25 shows control ranges for protein markers (Saposin C), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • Figure 26 shows control ranges for protein markers (alpha- galactosidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • protein markers alpha- galactosidase
  • Figure 27 shows control ranges for protein markers (arylsulphatase A), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • protein markers arylsulphatase A
  • Figure 28 shows control ranges for protein markers (iduronate-2- sulphatase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • protein markers iduronate-2- sulphatase
  • Figure 29 shows control ranges for protein markers (4-sulphatase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • Figure 30 shows LSD affected and control ranges for protein markers (sulphamidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • protein markers sulphamidase
  • Figure 31 shows LSD affected and control ranges for protein markers (acid sphingomyelinase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • Figure 32 shows LSD affected and control ranges for protein markers (alpha-iduronidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • protein markers alpha-iduronidase
  • FIG 33 shows LSD affected and control ranges for protein markers (LAMP-I), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • LAMP-I protein markers
  • Figure 34 shows LSD affected and control ranges for protein markers (alpha-glucosidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • Figure 35 shows LSD affected and control ranges for protein markers (beta-glucosidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • Figure 36 shows LSD affected and control ranges for protein markers (Saposin C), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • Figure 37 shows LSD affected and control ranges for protein markers (alpha-galactosidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • protein markers alpha-galactosidase
  • Figure 38 shows LSD affected and control ranges for protein markers (arylsulphatase A), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • protein markers arylsulphatase A
  • Figure 39 shows LSD affected and control ranges for protein markers (iduronate-2-sulphatase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • protein markers iduronate-2-sulphatase
  • Figure 40 shows LSD affected and control ranges for protein markers (4-sulphatase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • Figure 41 shows control and I-cell ranges for protein markers (sulphamidase; acid sphingomyelinase; alpha-iduronidase; LAMP-I; alpha- glucosidase; beta-glucosidase; Saposin C; alpha-galactosidase; arylsulphatase A; iduronate-2-sulphatase; and 4-sulphatase);
  • protein markers sulphamidase; acid sphingomyelinase; alpha-iduronidase; LAMP-I; alpha- glucosidase; beta-glucosidase; Saposin C; alpha-galactosidase; arylsulphatase A; iduronate-2-sulphatase; and 4-sulphatase
  • Figure 42 shows control, MLD and pseudo MLD ranges for arylsulphatase A, wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • Figure 43 shows control, and multiple sulphatase deficiency values for selected protein markers in Adult, newborn and MSD, wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • Figure 44 shows a table having the percent identification of LSD using single protein MLD and pseudo MLD ranges for arylsulphatase A;
  • Figure 45 shows a discrimination plot of Gaucher, newborn control, and adult patients using protein ratios (beta-glucosidase and alpha- glucosidase / alpha-galactosidase);
  • Figure 46 shows a discrimination plot of Pompe, newborn control, and adult patients using protein ratios (alpha-glucosidase and alpha-iduronidase);
  • Figure 47 shows a discrimination plot of Pompe, and newborn control patients using protein ratios (alpha-glucosidase and alpha-iduronidase);
  • Figure 48 shows a discrimination plot of MPSII, newborn control, and adult patients using protein ratios (iduronate-2-sul ⁇ hatase and LAMP-I);
  • Figure 49 shows a discrimination plot of MPSII, newborn control, and adult patients using protein ratios (iduronate-2-sulphatase and acid sphingomyelinase); .
  • Figure 50 shows a table indicating the percentage identification of LSD using protein ratios
  • Figure 51 shows a retrospective analysis of newborns from Guthrie card samples (alpha-galactosidase in Denmark, Fabry, Gaucher, MPSII, MPSIIIA, and Pompe), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths torn the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • Figure 52 shows a retrospective analysis of newborns from Guthrie card samples (alpha-glucosidase in Denmark, Fabry, Gaucher, MPSII, MPSIIIA, and
  • Pompe wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • Figure 53 shows a retrospective analysis of newborns from Guthrie card samples (beta-glucosidase in Denmark, Fabry, Gaucher, MPSII, MPSIIIA, and Pompe), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • Figure 54 shows a retrospective analysis of newborns from Guthrie card samples (sulphamidase in Denmark, Fabry, Gaucher, MPSII, MPSIIIA, and Pompe), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • Figure 55 shows a retrospective analysis of newborns from Guthrie card samples (iduronate-2-sulphatase in Denmark, Fabry, Gaucher, MPSII, MPSIIIA, and Pompe), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
  • Figure 56 shows a retrospective analysis of newborns from Guthrie card samples (sulphamidase; acid sphingomyelinase; alpha-iduronidase; LAMP-I; alpha-glucosidase; beta-glucosidase; Saposin C; alpha-galactosidase; arylsulphatase A; iduronate-2-sulphatase; and 4-sulphatase); [0069] Figure 57 shows a retrospective analysis plot of Denmark, Fabry, Gaucher, MPSII, MPSIIIA, and Pompe newborn Guthrie card samples (alpha- glucosidase and alpha-iduronidase);
  • Figure 58 shows a retrospective analysis plot of Denmark, Fabry, Gaucher, MPSII, MPSIIIA, and Pompe newborn Guthrie card samples (beta- glucosidase and saposin C);
  • Figure 59 shows a retrospective analysis plot of Denmark, Fabry, Gaucher, MPSII, MPSIIIA, and Pompe newborn Guthrie card samples (beta- glucosidase and acid sphingomyelinase);
  • Figure 60 shows a table of the percentage of total identified LSD
  • Figure 61 shows a table of the percentage of identified LSD using single proteins
  • Figure 62 shows a table of the percentage of identified LSD affected newborns using retrospective analysis
  • Figure 63 shows the multiplex neonatal screening strategy for LSD
  • Figure 64 shows a table of proposed structure of MPS II Oligosaccharides derived from dermatan sulfate
  • Figure 65 shows a table of proposed structure of MPS II Oligosaccharides derived from heparan sulfate
  • Figure 66 shows Elution profile of control and MPS II urine from
  • Bio-Gel P4. GAGs were isolated from control and MPS II patient urine by a combination of anion exchange and size exclusion chromatography. Fractions from the Bio-Gel P4 column were assayed for UA equivalents. The solid and broken lines represent elution from the Bio-Gel P4 column of control and MPS II urine, respectively.
  • Figure 61 shows ESI-MS of selected oligosaccharides. Eluate fractions from the Bio-Gel P4 column were lyophilised, derivatized with PMP and analyzed by ESI-MS.
  • Panel a shows ESI-MS of heptasaccharides; [UA-HN] 2 -UA- HNAc-UA with 3-6 S of [M-3H] "3 ions 605.3, 631.9, 658.5, 685.5 and with 4-6 S [M- 4H] "4 ions 473.8, 493.7, 513.7; and UA-HN-[UA-HNAC] 2 -UA with 2-3 S of [M-2H] "2 ions 888.9, 929.3 and with 3-4 S [M-3H] '3 ions 619.1, 645.7.
  • Panel b shows ESI-MS of an octasaccharide; [UA-HNAc] 4 with 4-6 S [M-3H] "3 ions 727.8, 754.2, 780.8 and [M-4H] "4 ions 545.5, 565.4, 585.5, and with 5-7 S [M-5H] -5 ions 452.2, 468.5, 484.4; a nonasaccharide UA-HN-(UA-HNAC) 3 -UA with 5-6 S [M-3H] "3 ions 797.9, 824.7, with 5-8 S [M-4H] "4 ions 598.3, 618.2, 638.4, 658.1 and with 4-6 S [M-5H] "5 ions 494.5, 510.3, 526.5.
  • Panel c shows ESI-MS of decasaccharides; (UA-HNAc) 5 with 5- 7 S [M-4H] "4 ions 660.6, 680.3, 700.2, with 4-8S [M-5H] "5 ions 511.4, 528.2, 544.3, 560.3, 576.4, with 6-9 S [M-6H] "6 ions 453.3, 466.6, 480, 492.9; UA-HN-[UA- HNAc] 4 with 3-5 S [M-4H] '4 ions 609.9, 629.6, 649.8.
  • Figure 68 shows Product ion spectra of oligosaccharides from a
  • Panel b shows the product ion spectra of a pentasaccharide UA- HN-UA-HNAc-UA with 4S.
  • Figure 69 shows Mass spectra of oligosaccharides following recombinant enzyme digests. Negative ion ESI-MS of pooled hexasaccharide and heptasaccharide isolated from MPS II urine.
  • Panel a shows ESI-MS of a heptasaccharide (UA-HN-UA-HN-UA-HNAc-UA); with 3-5 S producing [M-2H] "2 ions at m/z 908, 947.8, 988, with 3-6 S producing [M-3H] "3 ions at m/z 605.2, 631.8, 658.5, 685.3 and with 4-6 S producing [M-4H] "4 ions at m/z 473.8, 494.6.
  • a hexasaccharide (UA-HNAc-UA-HNAc-UA-HNAc); with 1-4 S producing [M-2H] "2 ions at m/z 782.3, 822.5, 861.7 and [M-3H]- 3 ions at m/z 520.5, 546.8, 573.8, 600.8.
  • Panel b shows the same pooled heptasaccharide and hexasaccharide from MPS II urine following treatment with recombinant I2S and IDUA.
  • Figure 70 shows Generation of HS-derived oligosaccharides in MPS II. Following endohydrolase action by heparanase on the regions of low sulphation, exoenzymes involved in the breakdown of HS act sequentially until a IdoA-2S residue is reached at the end of a region of high sulphation. Further degradation of the oligosaccharide is prevented by the deficiency of the iduronate-2- sulphatase. An analogous scheme where the endohydrolase acts on the GlcNAc-GlcA bonds in the low sulphation region is also proposed to account for the oligosaccharides with HNAc at the reducing terminus. A similar scheme involving an endo-N-acetylglucoasminidase activity is also proposed to account for the HS derived oligosaccharides in Table 2 series 4 (in Figure 65).
  • LSDs Lysosomal Storage Disorders
  • the present invention is generally related to a multiple screening diagnostic for LSD and related diseases. More particularly, this invention pertains to compounds, reagents, and methods for identifying and quantifying multiple target enzymes and proteins that are used to accurately diagnose a LSD. These target enzymes and proteins are naturally present in biological fluids or tissues of patients. The invention also pertains to a
  • One aspect of the current invention is a composition used for detection of a LSD.
  • the composition comprises a capture antibody capable of binding an epitope of a target antigen, and a microsphere having the capture antibody conjugated to the microsphere.
  • the target antigen is selected from the group consisting of: saposin (SeqID No. 1), LAMP-I (SeqID No. 2), ⁇ -iduronidase (SeqID No. 3), ⁇ -glucosidase (SeqID No. 4), ⁇ -glucosidase (SeqID No. 5), 2-sulphatase (SeqID No. 6), 4-sulphatase (SeqID No.
  • the microsphere having the conjugated capture antibody has a preferred diameter of about 5 ⁇ m and at least a first fluorophore and a second fluorophore, the first fluorophore being spectrally distinct from the second fluorophore.
  • the composition may further comprise a detection antibody, wherein the detection antibody is capable of binding the target antigen, and the detection antibody may be different from the capture antibody.
  • the detection antibody is conjugated to any detectable label known in the art (e.g. a fluorescent label).
  • animal refers to any species of the animal kingdom. In preferred embodiments it refers more specifically to humans.
  • biomolecule as used herein is understood to represent the target molecule, such as a protein, an antibody, a metabolite, a DNA sequence, an RNA sequence, a biologic with activities used or measured for the purposes of multiplexing and profiling of target biomolecules, or a combination thereof, for the composition and method of determining LSD, used in administering, monitoring, or modifying an LSD therapy.
  • Clinical status refers to patients that are being studied or treated by physicians for a LSD.
  • fluorophore refers to any fluorescent compound or protein that can be used to quantify the LSD antigens.
  • the term "normalize” as used herein refers to bringing a target, reference, or other samples into conformity with a standard, pattern, model, etc. For example, in one embodiment, urine samples from LSD patients and non-LSD patients were normalized by using a 1 ⁇ mol equivalent of creatinine from each sample.
  • phenotype refers to the manifest characteristics of an organism collectively, including anatomical and psychological traits that result from both its heredity and its environment.
  • preclinical status refers to the period of a disease before any of the clinical symptoms appear.
  • LSD associated biomolecule refers to any biomolecule that has been linked to any LSD.
  • a LSD associated biomolecule includes, but is not limited to: saposin (SeqID No. 1), LAMP-I (SeqID No. 2), ⁇ -iduronidase (SeqID No. 3), ⁇ -glucosidase (SeqID No. 4), ⁇ -glucosidase (SeqID No. 5), 2-sulphatase (SeqID No. 6), 4-sulphatase (SeqID No. 7), ⁇ -galactosidase (SeqID No.
  • sphingomyelinase (SeqID No. 9), sulphamidase, or 3-sulphatase (SeqID No. 10), or sulphamidase, LAMP-2, ⁇ -galactosidase A, iduronate-2-sulphatase, ⁇ -iduronidase, N- acetylgalactosamine 4-sulphatase, galactose 6-sulphatase, acid sphingomyelinase, galactocerebrosidase, arylsulphatase A, saposin B, heparan-N-sulphatase, ⁇ -N- acetylglucosaminidase, acetylCoA: glucosamine N-acetyltransferase, N- acetylglucosamine 6-sulphatase, ⁇ -galactosidase, ⁇ -glucu
  • the term "reference quantity" as used herein refers to a known, normalized amount of a LSD biomarker in a biological fluid.
  • the reference quantity is determined from an animal, or group of animals having a defined clinical status, preclinical status, or phenotype of a LSD disease.
  • the reference quantity may refer to a table compiled from various animals or groups of animals having correlations between relative amounts of LSD biomarkers in a biological fluid, and a known clinical status, preclinical status, or phenotype.
  • MRS mucopolysaccharidosis
  • GAG glycosaminoglycans
  • HS heparan sulfate
  • DS dermatan sulfate
  • IDUA ⁇ -L- iduronidase
  • I2S iduronate-2-sulfatase
  • ESI-MS/MS electrospray ionization- taiidem mass spectrometry
  • IdoA ⁇ -L-iduronic acid
  • HN hexosamine
  • U uronic acid
  • GcN glucosamine
  • GIcNAc N-acetylglucosamine
  • GaINAc N- acetylgalactosamine
  • HNAc N-acetylhexosamine
  • MRM multiple-reaction monitoring.
  • LSD LYSOSOMAL STORAGE DISORDERS
  • the LSD's represent a group of over 40 distinct genetic diseases that generally affect young children. Patients are usually born without the visible features of a LSD, but early stage symptoms can quickly develop into a progressive clinical concern. Although some effective LSD therapies have been developed, it is paramount that therapy be started as soon as the LSD has been diagnosed. Unfortunately, a clinical diagnosis of a LSD often requires multiple visits to a range of specialists requiring time-consuming, invasive, complex, inconvenient, and expensive assays. The current process for an accurate diagnosis of LSD for a patient not having a family history of LSD can take months to years, which is unacceptable when effective LSD therapies are needed earlier.
  • Recombinant Lamp-1 (minus tail) was isolated from CHO-Kl cells as detailed in Isaac et al [Isaac EL, Karageorgous LE, Brooks DA, Hopwood JJ and Meikle PJ. Experimental Cell Research 2000, 254: 204-209].
  • Recombinant Saposin C was a gift from Dr GA Grabowski and was prepared by the method of Qi and Grabowski [Qi TL and Grabowski GA J Biol Chem 1994, 269: 16746-16753].
  • the ami Lamp-1 monoclonal antibody (BB6) was generated using intact Lamp-1 protein by the method of Carlsson and Fukada [Carlsson SR and Fukada M JBC (1989) 264(34): 20526-20531] and 7B2 (ami Saposin C) monoclonal antibody was produced using the recombinant protein by the method described in [Zola H and Brooks D. Techniques for the production and characterization of monoclonal hybridoma antibodies. In: Hurrell JGR, ed. Monoclonal hybridoma antibodies: techniques and applications. Boca Raton, FL: CRC Press, 1982:1-57].
  • Polyclonal antibodies were generated for both Lamp-1 and Saposin C by immunizing separate rabbits with 200 ⁇ g of each recombinant protein per inoculation (four inoculations in total) based upon the method of Leonova et al, 1996, [JBC 271:17312- 20]. All antibodies were purified using 5ml HitrapTM Protein G affinity column (Pharmacia, Uppsala, Sweden). The polyclonal antibodies were affinity purified further by column chromatography using their respective recombinant proteins coupled to Affi-Gel ® 10 Gel (Bio-Rad #153-6046, CA, USA) according to manufacturers instructions.
  • Blood spot calibrators containing final concentrations of 2000, 1000, 500, 250, 62.5 and 0 ⁇ g/L for Lamp-1 and saposin C were prepared as detailed in Umapathysivam et al [Umapathysivam K, Whittle AM, Ranieri E, Bindloss C, Ravenscroft EM, van Diggelen OP, Hopwood JJ and Meikle PJ Clin Chem 46(9): 1318-1325 2000].
  • Two blood spot controls containing low (Lamp-1 400 ⁇ g/L; saposin 200 ⁇ g/L) and high (Lamp-1 800 ⁇ g/L; saposin C 500 ⁇ g/L) protein concentrations were similarly prepared.
  • Lamp-1 and Saposin C were measured in dried blood spots using one step three tier, time-delayed fluorescence immunoassays. Microtiter plates (Labsytems, Helsinki, Finland #95029180) were coated with either BB6 or 7B2 at a concentration of 5 ⁇ g/L in O.lmol/1 NaHCO3, pH 8.3 and incubated covered for approximately l ⁇ hrs at 4°C.
  • Standard calibrators, controls and patient dried blood spots were placed in duplicate into the coated microtiter wells with 200 ⁇ l of either polyclonal antibody diluted in assay buffer (0.15mol/l NaCl, 0.05mol/L Tris, 20 ⁇ mol/L Diethylene triamine-penta-acetic acid, containing 0.01% Tween 40, 0.5% bovine serum albumin (A-9647), 0.05% bovine ⁇ -globulin (G-7516), and 0.05% sodium azide, pH 7.8).
  • the antibodies were used at a final concentration of 200 ⁇ g/L and 400 ⁇ g/L for the anti-Lamp- 1 and anti saposin C polyclonal respectively.
  • LAMPs lysosome-associated membrane proteins
  • LAMP-2 lysosome-associated membrane proteins
  • the first is the family of fluorescently dyed microspheres having bound biomolecules.
  • the second is a flow cytometer with 2 lasers and associated optics to measure biochemical reactions that occur on the surface of the microspheres, and the third is a high-speed digital signal processor to efficiently manage the fluorescent output.
  • Bio-Rad (Hercules, CA), provides a commercially available protein array system called the "Bio- PlexTM”.
  • the Bio-PlexTM protein array system includes fluorescently dyed microspheres, a flow cytometer with 2 lasers and associated optics, and a high-speed digital signal processor.
  • neither the Bio-PlexTM protein array system nor any other commercially available systems include any specific biomolecules, methods, compounds, or reagents needed for the simultaneous screening of specific LSD enzymes.
  • the Bio-PlexTM protein array system uses multiplexing technology to enable the simultaneous quantitation of up to 100 different analytes.
  • This technology uses polystyrene microspheres internally dyed with differing ratios of 2 spectrally distinct fluorophores.
  • Each fluorophore can have any of 10 possible levels of fluorescent intensity, thereby creating a family of 100 spectrally distinct bead sets.
  • the dyed microspheres are conjugated with antibodies specific for a target LSD protein or peptide thereof.
  • each of the 100 spectrally distinct bead sets can contain a capture antibody specific for a unique LSD target protein.
  • FIG. 1 shows a drawing of a complete microsphere capture sandwich immunoassay having a polystyrene microsphere (110) with 2 spectrally distinct fluorophores; the target LSD capture antibody (120) bound to the microsphere; a unique LSD target protein or target antigen (130) bound to the target LSD capture antibody; a detection LSD antibody (140); and a detection molecule (e.g. phycoerythrin, "PE” or Biotin) (150) .
  • a detection molecule e.g. phycoerythrin, "PE” or Biotin
  • the immunoassay solution is then drawn into the Bio-PlexTM array reader, which illuminates and reads the sample.
  • the Bio-PlexTM array reader which illuminates and reads the sample.
  • Bio-PlexTM Manager software correlates each bead set to the assay reagent that has been coupled to it (for example, a first LSD capture antibody coupled to bead set #22, and a second LSD capture antibody coupled to bead set #42). hi this way the Bio-PlexTM protein array system can distinguish between the different assays combined within a single microtiter well.
  • a green "reporter” laser (532 nm) in the array reader simultaneously excites a third fluorescent dye (phycoerythrin, "PE") bound to the detection LSD antibody in the assay.
  • PE phytoerythrin
  • the amount of green fluorescence is proportional to the amount of target analyte captured in the immunoassay. Extrapolating the captured amount of target analyte to a standard curve allows quantitation of each LSD analyte in the sample.
  • BioPlex Protein Array System was used as one embodiment to demonstrate the type and nature of the reagents necessary for a LSD multiplex diagnostic assay.
  • Capture antibodies and detection antibodies have been purified that selectively bind epitopes of: saposin, LAMP-I, ⁇ -iduronidase, ⁇ -glucosidase, ⁇ -glucosidase, 2-sulphatase, 4- sulphatase , ⁇ -galactosidase, sphingomyelinase, and 3-sulphatase.
  • the full or partial sequences of the following proteins that were used to generate the purified antibodies are as follows: saposin (SeqID No.
  • mice using standard immunisation protocols (Harlow et ah, 1988). Mice were immunized with recombinant enzyme using established protocols. Plasma cells from these immunized mice were fused with P3.653 myeloma cells (Zola et ah, 1982) and the resulting hybridoma cell lines screened for antibodies against the recombinant protein by direct ELISA (Harlow et al., 1988). Monoclonal antibodies were purified from cell culture supernatants by ammonium sulfate precipitation followed by affinity purification on HitrapTM Protein G affinity column (Pharmacia Biotech, Uppsala,
  • a monoclonal capture antibody for LAMP-I was BB6 developed and provided by Sven Carlsson (Carlsson et ah, 1989).
  • the monoclonal reporter antibody for ⁇ -glucosidase (43Dl) was obtained from Pharming, Inc. and has been described (Fransen et ah, 1988).
  • the polyclonal reporter antibody for LAMP-I, the rabbit polyclonal reporter antibody for saposin C, the sheep polyclonal capture antibody for ⁇ -glucosidase, and the monoclonal capture antibody ("7B2") for saposin C were prepared within the Lysosomal Diseases Research Unit at the WCH in Sydney, Australia using standard techniques, known in the art, and briefly described below.
  • the availability and production of specific monoclonal and polyclonal antibodies are know to one of ordinary skill in the art. Production of the specific antibodies uses in the current examples are given below:
  • Sheep polyclonal antibodies were produced against recombinant proteins (e.g. saposin (SeqID No. 1) , LAMP-I (SeqID No. 2), ⁇ -iduronidase (SeqID No. 3), ⁇ -glucosidase (SeqID No. 4), ⁇ -glucosidase (SeqID No. 5), 2-sulphatase (SeqID No. 6), 4-sulphatase (SeqID No. 7), ⁇ - galactosidase (SeqID No. 8), sphingomyelinase (SeqID No.
  • recombinant proteins e.g. saposin (SeqID No. 1) , LAMP-I (SeqID No. 2), ⁇ -iduronidase (SeqID No. 3), ⁇ -glucosidase (SeqID No. 4), ⁇ -glucosidase (
  • a sheep was injected sub-cutaneously with 2mg of protein in 1 mL of an emulsion of phosphate buffered saline (pH 7.4) and complete Freunds adjuvant, followed by four booster injections (2mg each) with incomplete Freunds adjuvant, each three weeks apart. One week after the last injection the sheep was bled out and serum collected. Rabbit polyclonal antibody was produced in the same manner, except 0.2-1.0 mg of protein was used per immunisation.
  • Sheep polyclonal antibody was purified on a 5 mL Hitrap Protein G affinity column (Pharmacia Biotech, Uppsala, Sweden) followed by an affinity column prepared from the recombinant protein used for the immunisation.
  • Recombinant protein affinity columns were prepared by coupling 5 mg of the recombinant protein to 2.5 mL of Affi-gel 10 (Bio-Rad, Hercules, CA, USA) as per manufacturer's instructions.
  • the eluate was diluted four fold and then loaded on to the appropriate recombinant protein affinity column at the same flow rate.
  • the column was washed and eluted as described for the Protein G column.
  • the target capture antibodies were coupled to Bio-Rad carboxylated (“COOH") beads as follows: anti- saposin to bead No. 42 , anti-LAMP-1 to bead No. 25, anti- ⁇ -iduronidase to bead No. 24, anti- ⁇ -glucosidase to bead No. 26, anti- ⁇ -glucosidase to bead No. 28, anti-2- sulphatase to bead No. 45, anti-4-sulphatase to bead No. 46, anti- ⁇ -galactosidase to bead No. 43, anti-sphingomyelinase to bead No.
  • COOH Bio-Rad carboxylated
  • the coupling of the target capture antibodies to the polystyrene microspheres was performed using the BioRad bead coupling kit (Catalog number 171-406001, BioRad, Hercules, CA).
  • the Bio-PlexTM amine coupling kit includes 4 ml bead wash buffer, 85 ml bead activation buffer, 135 ml PBS, pH 7.4, 10 ml blocking buffer, 25 ml storage buffer, 105 ml staining buffer, 40 coupling reaction tubes.
  • the Bio-PlexTM amine coupling kit provides the buffers necessary to covalently couple 6-150 IcD proteins to 5.5 ⁇ m dyed carboxylated polystyrene beads in under 5 hr.
  • the covalent couple of the target capture antibody to the carboxylated polystyrene bead is achieved via carbodiimide reactions involving the protein primary amino groups and the carboxyl functional groups bound on the surface of polystyrene beads.
  • the covalent attachment is permanent, leaving no unbound protein after cleanup, even after months of storage.
  • the protein-coupled beads can then be used in multiplex protein-protein binding studies or in the development of multiplex assays that can be analyzed with the Bio-PlexTM protein array system.
  • the bead yield per coupling reaction is approximately 80%, or enough protein-coupled beads for two 96- well microtiter plates using 5,000 beads per well.
  • the target capture antibody-coupled beads were enumerated and the efficiency of the protein coupling reaction was validated, according to the manufacturer's protocol with modifications.
  • the protein-coupled beads were reacted with a phycoerythrin ("PE")-labeled antibody that binds to the coupled protein, which was then analyzed using the Bio-PlexTM protein array system.
  • PE phycoerythrin
  • This procedure was performed by reacting the beads with a PE-labeled antibody.
  • a reaction using a biotinylated antibody followed by streptavidin-PE may be used.
  • the intensity of the fluorescent signal of this reaction is directly proportional to the amount of protein on the surface of the beads.
  • a successful coupling typically yields a mean fluorescent intensity (“MFI") signal that is greater than 2,000.
  • MFI mean fluorescent intensity
  • the detection antibodies that specifically bind epitopes of: saposin; LAMP-I; ⁇ -iduronidase; ⁇ -glucosidase; ⁇ -glucosidase; 2-sulphatase; 4- sulphatase; ⁇ -galactosidase; sphingomyelinase; and 3-sulphatase were biotinylated according to the manufacturer's instructions (Molecular Probes, FluroReporter Biotin- XX protein labelling kit F-2610). There are several published methods known in the art for preparation of biotinylated or phycobiliprotein conjugates with antibodies and other proteins.
  • the coupling chemistry used to crosslink a phycobiliprotein to another protein includes: (a) treating the antibody or other protein with a succinimidyl ester maleimide derivative at pH 7.5, which converts some lysine residues of the antibody to thiol-reactive maleimides; (b) preparing a thiolated phycobiliprotein by reducing the appropriate SPDP-modified phycobiliprotein with dithiothreitol ("DTT") or with tris-(2-carboxyethyl) ⁇ hosphine (“TCEP”); (c) mixing the above two dialyzed protein conjugates to yield a stable thioether crosslink; and (d) chromatographically separating the phycobiliprotein conjugates from the unreacted proteins.
  • DTT dithiothreitol
  • TCEP tris-(2-carboxyethyl) ⁇ hosphine
  • a calibration curve was generated using liquid calibrator proteins in a microsphere based assay using calibrator protein capture antibodies and bead sets #25, #42, #26, #28, #43, #46, #45, #44, and #22 (BioRad, Hercules, CA 3 USA).
  • Figure 2 shows a calibration curve for a single assay for ⁇ -glucosidase.
  • the detection capability for the amount of calibrator protein present in each well reaction was linear in the range of 0 to 4 ng/well of the assay.
  • the MFI was the average of the total fluorescence detected for the beads in the defined bead region.
  • Figure 4 shows a drawing of a microsphere collection of capture sandwich immunoassays for the 10-plex having: 10 spectrally distinct polystyrene microsphere (510-519); 10 target LSD capture antibody (520-529) bound to the microsphere; 10 unique LSD target proteins or target antigens and representing: saposin, LAMP-I 3 ⁇ -iduronidase, ⁇ -glucosidase, ⁇ -glucosidase, 2- sulphatase, 4-sulphatase, ⁇ -galactosidase, sphingomyelinase, and 3 -sulphatase (530- 539) bound to the corresponding target LSD capture antibody; 10 unique detection LSD antibody (540-549); and a detection molecule (550).
  • 10 spectrally distinct polystyrene microsphere 510-519
  • 10 target LSD capture antibody 520-529) bound to the microsphere
  • 10 unique LSD target proteins or target antigens and representing:
  • the plates were incubated for one-hour at room temperature with shaking and then placed at 4°C overnight. After a further one-hour incubation at room temperature with shaking, the plates were washed three-times with filtered (0.2 ⁇ m) PBS containing 0.05% Tween 20, pH 7.2 (wash buffer) under vacuum. Streptavidin conjugated to phycoerythrin (Molecular Probes S-866) was diluted in assay buffer (1.2 ⁇ g/mL) and added to the wells (125 ⁇ l/well); the plates were then incubated at room temperature with shaking for 10 minutes. The plates were then read on the Bio-Plex suspension array system (Bio-Rad) using version 3.0 software and counting 100 beads/region.
  • Bio-Plex suspension array system Bio-Rad
  • Figure 5 shows the resulting calibration curves for saposin C (asterisk), LAMP-I (X-mark), ⁇ - iduronidase (solid diamond), ⁇ -glucosidase (open square), ⁇ -glucosidase (solid square), 2-sulphatase (open triangle), 4-sulphatase (solid square), ⁇ -galactosidase (solid circle), sphingomyelinase (open diamond), and 3 -sulphatase (open circle) of the 10-plex assay.
  • Figure 6 shows an 11-plex assay with an added sulphamidase (asterisk).
  • the box length is the interquartile range that covers 25th to 75th percentile
  • the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box
  • the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box.
  • the box length is the interquartile range that covers 25th to 75th percentile
  • the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box
  • the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box.
  • Figure 44 shows a table having the percent identification of LSD using single proteins that allowed many of the LSD's to be 100% identified
  • Figure 50 shows a table having the percent identification of LSD using ratios as having all but Gaucher identified as
  • Figures 51-55 show the retrospective analysis of: alpha- galactosidase; alplia-glucosidase; beta-glucosidase; sulphamidase; and iduronate-2- sulphatase.
  • the box length is the interquartile range that covers 25th to 75th percentile
  • the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box
  • the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box.
  • Figure 62 shows that Fabry, ML WUI, MPS II, MPS IIIA and Pompe were identified from retrospective LSD affected newborns at 100%, and Gaucher was identified in retrospective LSD affected newborns at 80%. The other disorders were not identified in this retrospective analysis.
  • a defect in exo-degradative pathways provides insight into endo-degradation of heparan and dermatan sulfates.
  • Maria Fuller, Ally Chau, Rachael C. Nowak, John J. Hopwood' and Peter J. Meikle contributed to the invention of Example 3.
  • dermatan sulfate and heparan sulfate are degraded in two steps. The initial endohydrolysis of these polysaccharides is followed by the sequential action of lysosomal exoenzymes to reduce the resulting oligosaccharides to monosaccharides and inorganic sulfate.
  • Mucopolysaccharidosis types II is a lysosomal storage disorder caused by a deficiency of the exoenzyme iduronate-2- sulfatase. Consequently, partially degraded fragments of dermatan sulfate and heparan sulfate, have been shown to accumulate in the lysosomes of affected cells and are excreted in the urine. Di- to hexadecasaccharides, isolated from the urine of a mucopolysaccharidosis type II patient using anion exchange and gel filtration chromatography, were identified using electrospray ionization-tandem mass spectrometry.
  • oligosaccharides were shown to have non-reducing terminal iduronate 2-sulfate residues by digestion with recombinant iduronate 2-sulfatase.
  • a pattern of growing oligosaccharide chains composed of alternating uronic acid and N- acetylhexosamine residues was identified and suggested to originate from dermatan sulfate.
  • a series of oligosaccharides consisting of hexosamine/N-acetylhexosamine alternating with uronic acid residues were also identified and based on the presence of unacetylated hexosamine, these oligosaccharides are proposed to derive from heparan sulfate.
  • HS glycosaminoglycans heparan sulfate
  • DS dermatan sulfate
  • HS has repeating disaccharide units consisting of uronic acid (UA) alternating with ⁇ -lmked (1,4) glucosamine (GIcN) residues. Biosynthesis of HS occurs in the Golgi.
  • the base polymer consisting of the disaccharide repeat (GIcA 1-4 GIcNAc)
  • a series of enzymatic reactions occurs, deacetylating the GIcNAc, epimerising the glucuronic acid (GIcA) to iduronic acid (IdoA) and adding sulfate groups to both O and N positions on IdoA and GIcN. Consequently, the UA residue may be ⁇ -linked (1,4) D- GIcA, or ⁇ -linked (1,4) L-IdoA, unsulfated or with O-sulfation of the C2-hydroxyl.
  • the amino group of GIcN may be N-sulfated, N-acetylated or occasionally unsubstituted.
  • the GIcN may also be sulfated on the C6-hydroxyl and sometimes on the C3-hydroxyl.
  • the proportion of GIcA and IdoA varies considerably, not only between different species of HS but also within a particular HS chain. Likewise, the degree and type of sulfation is not stoichiometric (3).
  • the modification reactions that are responsible for the structural diversity are not complete, producing a final HS molecule with a domain structure (4).
  • HS chains typically contain regions rich in GIcA and N-acetylated GIcN (GIcNAc) disaccharides with no sulfation (NA domains), contiguous variable length sequences containing IdoA and GIcNS derivatives with high sulfation (NS domains), and bridging these domains are mixed sequences in which GIcNAc disaccharides and GIcNS disaccharides alternate (NA/NS domains) (5).
  • GIcNAc N-acetylated GIcN
  • DS is composed of repeating disaccharide units consisting of UA alternating with ⁇ -lmked (1,4) D-N-acetylgalactosamine (GaINAc) residues that may be sulfated on the C4-and/or C ⁇ -position.
  • Some DS chains have predominantly (1,3) ⁇ -linked IdoA residues with some C2-sulfation, while others have primarily (1,3) ⁇ - linked GIcA.
  • DS forms block structures of lowly sulfated GIcA- GaINAc disaccharides alternating with blocks of highly sulfated IdoA-GalNAc disaccharides.
  • Hyaluronidases endo- ⁇ -N-acetylhexosaminidases
  • chondroitin sulfate and DS are a family of enzymes that degrade hyaluronan, as well as chondroitin sulfate and DS.
  • Hyaluronidases cleave internal ⁇ -linked (1,4) glycosidic bonds between GaINAc and GIcA in DS (7).
  • Heparanase endo- ⁇ -glucuronidase
  • Heparanase activity has been shown to be associated with cell invasion, angiogenesis, inflammation and tissue remodelling through a number of cell-matrix interactions.
  • HS proteoglycans The action of heparanase on cell surface and extracellular matrix HS proteoglycans is thought to be a highly regulated process involving the binding and internalisation of heparanse to specific cell surface proteoglycans (syndecans), thereby limiting the extracellular accumulation and action of this endoglycosidase. Endosulfatase action on heparin has also been reported and it is likely that similar activity will be shown to the sulfated domains of HS.
  • HS and DS are degraded from their non-reducing termini by the sequential action of highly specific lysosomal exoenzymes. At least 10 lysosomal exoenzymes act to reduce these oligosaccharides to monosaccharides and inorganic sulfate to enable exit out of the lysosome. A deficiency in any one of these exoenzyme activities may result in lysosomal storage of the GAG substrates with the resulting clinical manifestation of the mucopolysaccharidoses (MPS).
  • MPS mucopolysaccharidoses
  • MPS II results from a deficiency of iduronate-2- sulfatase (I2S; EC 3.1.6.13), which hydrolyzes the C2-sulfate ester bond of non- reducing terminal ⁇ -L-iduronic acid residues in HS and DS.
  • I2S iduronate-2- sulfatase
  • the catabolism of HS and DS is blocked. Consequently, partially degraded fragments of HS and DS accumulated in the lysosomes of affected cells and are excreted in the urine.
  • These oligosaccharide fragments are presumably produced by endohydrolase activities, followed by exohydrolase activities that terminate at the substrate for I2S.
  • Recombinant human IDUA and recombinant human I2S were each prepared from CHO-Kl expression systems. MPS II patient urine and control urine was supplied with consent.
  • Urine samples from an MPS II patient and an age-matched control 500 ml were clarified by centrifugation and passed over a 30 ml column of DEAE-Sephacel previously equilibrated with 0.1 M NaCOOCH 3 buffer, pH 5. The column was washed with 10 column volumes of the equilibration buffer and urinary GAG eluted in the same buffer containing 1.2 M NaCl. Fractions were assayed for UA and the GAG-containing fractions (20 ml) were pooled, lyophilized and reconstituted in 4 ml of H 2 O.
  • Samples were made up to 500 ⁇ l with H 2 O and then extracted with an equal volume of CHCl 3 to remove excess l-phenyl-3-methyl-5 -pyrazolone and centrifuged at 13,000 xg for 5 min. Chloroform extraction was repeated a further 2 times and the aqueous layer was lyophilised and the derivatized oligosaccharides resuspended in 500 ⁇ L of an aqueous solution of 50 % (v/v) CH 3 CN/0.025 % (v/v) HCOOH prior to analysis by mass spectrometry.
  • Enzymatic cleavage Column fractions containing 10 ⁇ g of UA were lyophilized prior to digestion with recombinant I2S and IDUA. Enzymatic cleavage was performed at 37 0 C for 16hr in 50 ⁇ L of 50 mM NaCOOCH 3 buffer (pH 4.5) supplemented with 0.5 mg/ml BSA and 20 ⁇ g recombinant enzyme (IDUA and/or I2S). Following digestion, the oligosaccharides were derivatized as described above and analyzed by ESI-MS in negative ion mode.
  • Electrospray ionisation-tandem mass spectrometry was also used for further characterisation of these oligosaccharides.
  • Precursor ion scan of m/z 173, corresponding to an ionised l-phenyl-3-methyl-5-pyrazolone (PMP) fragment was used to support the assignments made from the ESI-MS scans. Strong signals were observed for oligosaccharide ions in higher charged states (> [M-3H] "3 ), whereas signals from the [M-2H] "2 were weak or not detectable (data not shown).
  • CAD-MS/MS Collisionally activated dissociation-tandem mass spectrometry
  • FIG. 68a A representative product ion spectra of a tetrasaccharide (UA-HN- UA-HNAc 4S) with a characteristic product ion m/z 256 is shown in Figure 68a, and Figure 68b shows the product ion spectra from a pentasaccharide (UA-HN-UA- HNAc-UA 4S) with a characteristic product ion of m/z 331.
  • Product ion analysis was performed on all major signals in the ESI-MS and in most instances enabled the identification of the reducing terminal sugar as either UA or HNAc.
  • the first series with an even number of saccharides, had a UA residue on the non- reducing end and a HNAc on the reducing end (Table 1, series 1).
  • the second series consisted of an odd number or residues ranging from tri- to heptasaccharide (Table 1, series 2) with UA at the reducing terminus.
  • oligosaccharides . containing UA, HN and HNAc were identified with different levels of sulfation (Table 2).
  • Series 1-3 consist of an odd number of residues with UA at the reducing terminus, and series 4 contains an even number of residues with HNAc at the reducing terminus.
  • the oligosaccharides in series 1 are composed of one HN with either zero, one or two HNAc.
  • Series 2 contains oligosaccharides with two HN and up to five HNAc.
  • the oligosaccharide in series 3 contains three HN residues and a single HNAc.
  • Series 4 lists oligosaccharides with an even number of residues containing one HN and up to four HNAc residues.
  • the hexasaccharide identified by m/z ratios with the putative structure [UA-HNAc] 3 with 1 to 4 sulfates was shown to lose 176 and 80 amu, representing UA and S respectively, following incubation with recombinant I2S and IDUA.
  • a heptasaccharide with the proposed structure (UA-HN) 2 -UA-HNAc- UA with 3 to 6 S also showed losses of 176 and 80 amu following digestion with recombinant IDUA and I2S ( Figure 69).
  • IdoA2S the substrate for the enzyme deficiency in MPS II.
  • ESI-MS identified oligosaccharides that have a different number of residues but very similar molecular weights, which occurs as a result of the similarity between the mass of HN (161 Da) and 2 S (160 Da).
  • the octasaccharide [UA-HN] 3 -UA-HNAc (4S)
  • the heptasaccharide [UA-HN] 2 -UA-HNAC-UA (6S)
  • the [M-4H] "4 ions are compared the difference is m/z 513.9 compared with m/z 513.6, respectively.
  • the mass accuracy of the instrument used in this study could not positively discriminate these structures.
  • identification of the reducing terminus as either UA or HNAc and the non-reducing terminus as IdoA2S enabled this discrimination.
  • oligosaccharide species in this series.
  • the limitation on the size of the oligosaccharide identified may reflect substrate specificity of the endoglucuronidase activity for terminal regions of the DS chains, although it may also be that larger species are not present in sufficient amounts to be resolved from the more abundant oligosaccharide species terminating in HNAc, seen in the complex spectra of the larger oligosaccharides.
  • the oligosaccharide series containing UA-HN and UA-HNAc disaccharides provides a more complex picture of endohydrolysis of HS.
  • Two series of oligosaccharides (Table 2, series 1 and 2) are likely products of heparanase activity towards HS. These series have a trisaccharide (UA-HN-UA) or a pentasaccharide (UA-HN-UA-HN-UA) base structure with an increasing number (up to five) of HNAc-UA disaccharides.
  • the number of sulfates associated with this series does not increase with the size of the oligosaccharide but remains relatively constant between three and seven.
  • heparanase appears to have specificity for the relatively highly sulphated linkage region between the NS and NA domains (thus producing the base tri and pentasaccharides) and also for the unsulfated region represented by the extending GIcNAc-UA disaccharides (NA domain).
  • Previous studies have indicated multiple substrates for heparanase activity. For example, earlier studies indicated that human heparanase cleaved a sequence within the highly modified NS domains of HS.
  • Heparanase from CHO cells has been proposed to have substrate specificity for the mixed NA/NS domains.
  • Studies on the heparanase from a rat parathyroid cell line identified relatively undersulfated structures at the cleavage site and also proposed that the cleavage occurred at the boundary of highly sulphated and undersulfated domains.
  • studies on recombinant human heparanase with defined tetra and hexasaccharide structures indicated the requirement for a highly sulphated structure containing an unsulfated GIcA at the cleavage site.
  • oligosaccharides larger than a pentasaccharide containing only UA and HN suggests that the NA/NS domain of HS is more resistant to heparanase activity than the NA domains.
  • the larger NS regions are to be found in the high molecular weight structures eluting at V 0 from the Bio-Gel P4 column. The structures observed, appear to arise primarily from the action of heparanase in NA domains of HS, these regions of low sulfation are separated by short (up to three) regions of sulfated UA-HN disaccharide repeats (NS domains).
  • each oligosaccharide is then trimmed back by the action of exoenzymes to the first IdoA2S residue where, as a result of the deficient enzyme, exodegradation is halted.
  • a similar scheme involving an endo-N- acetylglucoasminidase activity is also proposed to account for the HS derived oligosaccharides in Table 2 series 4 ( Figure 70).
  • This invention comprises general and specific compositions for LSD diagnostics, including multiplex bead technology. Although species specific antibodies have been used as illustrative examples, other types of antibodies that bind LSD target proteins are not considered to deviate from the spirit and scope of the claimed invention.
  • United States Patent 6,449,562 (“the '562 Patent”) entitled “Multiplexed Analysis of Clinical Specimens Apparatus and Method,” having Chandler et al. listed as inventors was issued on September 10, 2002.
  • United States Patent 6,524,793 (“the '793 Patent”) entitled “Multiplexed Analysis of Clinical Specimens Apparatus and Method,” having Chandler et al. listed as inventors, was issued on February 25, 2003.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Genetics & Genomics (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Multiplexing bead technology is used for simultaneous screening of multiple LSDs and normalizing measured enzyme activity or protein levels against other lysosomal proteins, enzymes, or enzyme activities. Diagnostic compositions include microspheres conjugated to purified antibodies that specifically bind LSD target antigens: saposin, LAMP-I, α-iduronidase, α-glucosidase, β-glucosidase, 2- sulphatase, 4-sulphatase, α-galactosidase, sphingomyelinase, 3-sulphatase or sulphamidase. The target antigens are naturally present in biological fluids or tissues of either LSD or non-LSD patients.

Description

MULTIPLEX-BEAD COMPLEX FOR DETERMINATION
OF LYSOSOMAL STORAGE DISORDERS RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application, Serial Number 60/632,610, entitled "Multiplex-Bead Complex for
Determination of Lysosomal Storage Disorders," filed on December 02, 2004, having
Meikle et si., listed as the inventor(s), the entire content of which is hereby incorporated by reference.
BACKGROUND: [0002] The present invention generally pertains to a process of population screening that leads to the detection of Lysosomal Storage Disorders ("LSDs") and related diseases in a subject. More particularly, this invention pertains to compounds, reagents, and methods for identifying and quantifying the levels and ratios of multiple target antigens that are used to identify individuals likely to be affected with an LSD. The target antigens are naturally present in biological fluids or tissues of either LSD or non-LSD patients.
[0003] LSDs represent a group of over 40 distinct genetic diseases that generally affect young children. Individuals that are affected with a LSD present a wide range of clinical symptoms that depend upon the specific disorder or a particular genotype involved. The clinical symptoms associated with LSDs can have a devastating impact on both the child and the family of affected individuals. For example, central nervous system dysfunction, behavioral problems, and severe mental retardation are characteristic of many LSDs. Other clinical symptoms may include skeletal abnormalities, organomegaly, corneal clouding and dysmorphic features (Neufeld and Muenzer, 1995). Patients are usually born without the visible features of a LSD, but early stage symptoms can quickly develop into a progressive clinical concern. In severe cases, the affected children require constant medical management but still often die before adolescence. [0004] The significance of LSDs to health care becomes obvious when comparing the group incidence rate for a LSD (1:5,000 births) to the group incidence rate of other well-known and intensively studied genetic disorders, such as phenylketonuria (1:14,000) and cystic fibrosis (1:2,500), wherein these figures reflect incidence rates for Caucasian populations.
[0005] Once an individual begins to present the symptoms of a LSD, the actual clinical diagnosis of the disease is still a complex process. A clinical diagnosis of a LSD often requires multiple visits to a range of specialists, which can take months or even years. This long process is extremely stressful on the patient and family. Fortunately, there has been considerable progress in the diagnosis of LSDs over the past 20 years. For example, the development and introduction of chromatographic-based urine screens for a specific group of LSDs called mucopolysaccharidoses ("MPS") and oligosaccharidoses has facilitated screening of clinically selected patients for these disorders. Following a clinical index of suspicion for the disorders, the next stage of diagnosis involves a urine screen, wherein a "positive" urine screen is then followed by specific enzymatic analysis. Although the chromatographic-based screening methods are simple to perform, they are relatively labor-intensive and often require experience to accurately interpret results. One example includes a method of identifying and quantifying biochemical markers ("biomarkers") that are present in biological fluids or tissues of a patient having a MPS or related disorders, and comprises determining a target quantity of a target MPS biomarker oligosaccharide from a target biological sample taken from the target animal, and then comparing the target quantity to a reference quantity of a reference MPS biomarker oligosaccharide for the diagnosis, characterization, monitoring, and clinical management of MPS and related disease, as described in PCT Application AU03/00731 entitled "Identification of Oligosaccharides and their Use in the Diagnosis and Evaluation of Mucopolysaccharidoses and Other Related Disorders," filed on June 13, 2003 with Hopwood et al., listed as inventors (the entire content of PCT Application AU03/00731 is hereby incorporated by reference). Consequently, chromatographic-based screening tests for LSDs are not used in some centers. Furthermore, these chromatographic-based screens are not readily amenable to automation, which has further limited their utilization in screening strategies for newborns.
[0006] The production of specific substrates and antibody capture assays has made the enzymatic analyses for LSDs more accurate. Although not wanting to be bound by theory, the majority of LSDs result from a reduction in levels of a particular enzyme(s) involved in a specific LSD, and the identification of the specific enzyme(s) steady state in normal individuals will help identify the particular form of LSD in the affected individual. The ability to quickly and accurately determine the levels of the more than 40 enzymes known to be involved with LSDs will assist in the development of better and more economical screening assays. Unfortunately, many of the chromatographic-based screens and enzyme assays mentioned above are time- consuming, invasive, complex, and require cultured cells, or tissue biopsies, which tends to make such assays inconvenient and expensive. As a result, testing for a LSD is often not a first line strategy for an affected child with early stage symptoms. Newborn screening for LSDs promises to provide early detection of the LSD, but all newborns must be screened in order to detect the disease early. Patients having a family history of LSDs may have a justifiable reason to perform an early screen for a LSD. However, the cost of an early screen of the LSD in individuals not having a family history may not be justified economically. Therefore, it would be beneficial that any LSD screening process be capable of economically screening large numbers of newborns.
[0007] One common feature of LSDs is the accumulation and storage of materials within lysosomes. It is generally recognized that the accumulation and storage of material in LSD affected individuals results in an increase in the number and the size of lysosomes within a cell from approximately 1% to as much as 50% of total cellular volume. In non-affected individuals, such materials are typically degraded into degradation products within the lysosome and then transported across the lysosomal membrane. Certain lysosomal proteins are present at elevated levels in the lysosomes of affected individuals (Meilde et al, 1997; Hua et al, 1998). These identified proteins are useful biomarkers for an early diagnosis of all LSDs. For example, sensitive immunoquantification assays have been developed to monitor the level of useful biomarkers such as the lysosome-associated membrane proteins ("LAMPs"), saposins, and α-glucosidase. Although the determination of either LAMP-I or LAMP-2 levels alone in an 'at-increased-risk' group will identify up to 65% of LSD affected individuals, the combination of a LAMP with one of the saposins increase identification of LSD affected individuals to approximately 85%. Therefore, a method to identify two or more biomarkers simultaneously would increase the accuracy of diagnosing a specific LSD as compared to any single assay. An automated multiplex assay that could perform a simultaneous screen on each of the known LSD deficient enzymes would reduce time and cost for accurate LSD diagnosis.
[0008] Multiplexing Bead Technology is built around 3 core technologies. The first is the family of fluorescently dyed microspheres having specific biomolecules bound to the surface of the microsphere. The second is a flow cytometer with 2 lasers and associated optics to measure biochemical reactions that occur on the surface of the microspheres, and the third is a high-speed digital signal processor to efficiently manage the fluorescent output. This type of system has been described in, for example: United States Patents 6,449,562; 6,524,793 and United States Patent Application SN 09/956,857. United States Patent 6,449,562 ("the '562 Patent") entitled "Multiplexed Analysis of Clinical Specimens Apparatus and Method," having Chandler et al. listed as inventors was issued on September 10, 2002. The '562 Patent discloses a method for the multiplexed diagnostic and genetic analysis of enzymes, DNA fragments, antibodies, and other biomolecules comprising the steps of constructing an appropriately labeled beadset, exposing the beadset to a clinical sample, and analyzing the combined samρle^eadset by flow cytometry. Flow cytometric measurements are used to classify, in real-time, beads within an exposed beadset and textual explanations, based on the accumulated data obtained during realtime analysis, are generated for the user. The inventive technology of the '562 Patent enables the simultaneous, and automated, detection and interpretation of multiple biomolecules or DNA sequences in real-time while also reducing the cost of performing diagnostic and genetic assays. However, the '562 Patent does not describe how to utilize the technology for diagnosing LSD's.
[0009] United States Patent 6,524,793 ("the '793 Patent") entitled "Multiplexed Analysis of Clinical Specimens Apparatus and Method," having Chandler et al. listed as inventors, was issued on February 25, 2003. The '793 Patent discloses a method for the multiplexed diagnostic and genetic analysis of enzymes, DNA fragments, antibodies, and other biomolecules comprising the steps of constructing an appropriately labeled beadset, exposing the beadset to a clinical sample, and analyzing the combined sample/beadset by flow cytometry. Flow cytometric measurements used to classify, in real-time, beads within an exposed beadset and textual explanations, based on the accumulated data obtained during realtime analysis, are generated for the user. The '793 Patent enables the simultaneous, and automated, detection and interpretation of multiple biomolecules or DNA sequences in real-time while also reducing the cost of performing diagnostic and genetic assays. However, the '793 Patent does not describe how to utilize the technology for diagnosing LSD's.
[0010] United States Patent Application Serial No. 09/956,857 ("the '857 Application") entitled "Multiple Reporter Read-out for Bioassays" was published on March 20, 2003. The '857 Application describes a method for detecting a plurality of reactive sites on an analyte, comprising allowing reactants on an addressable microsphere and the reactive sites to react, forming reactant-reactive site pairs distinguishable by fluorescence intensity. The '857 Application also provides a method for detecting a plurality of analytes in a sample using addressable microspheres in combination with one or more reporter reagents. Also provided is a method for determining allele zygosity of a genetic locus having two alleles or more alleles using microparticles, and a method for detecting a plurality of SNPs in nucleic acid molecules. The '857 Application also provides a composition comprising an addressable microsphere carrying at least two fluorescent reactants capable of forming reactant-analyte pairs distinguishable by their fluorescence intensity, and kits comprising the inventive composition and a plurality of reporter reagents. However, the '857 Application does not describe how to utilize the technology for diagnosing LSD's. The entirety of each of the applications or patents listed above is hereby specifically incorporated by reference.
[0011] Accordingly, there is a need for the development of a fast, accurate and economical screen for early diagnosis of LSDs, which is amenable to automation. The ability to identify specific LSD enzymes in an automated multiplex assay will have a significant impact on the development of a newborn screening programs, as well as the ability to address a number of other issues associated with the early diagnosis and treatment of LSDs. The present invention provides compounds, reagents, and methods for a LSD diagnostic multiplex assay.
[0012] FIGURES
[0013] Figure 1 shows a microsphere capture sandwich immunoassay having a microsphere with two spectrally distinct fluorophores, the target LSD capture antibody and the unique LSD target protein or target antigen bound to the target LSD capture antibody and a reporter molecule;
[0014] Figure 2 shows a calibration curve for α-glucosidase in a microsphere based assay;
[0015] Figure 3 shows multiplexed calibration curves in a microsphere based assay;
[0016] Figure 4 shows the multiplex technology having at least a 10-plex for LSD's;
[0017] Figure 5 shows calibration curves for a 10-plex immune quantification of lysosomal proteins;
[0018] Figure 6 shows calibration curves for a 11-plex immune quantification of lysosomal proteins;
[0019] Figure 7 shows the antibodies and bead regions used for the 10- plex assay;
[0020] Figure 8 shows a list of the precision studies that were conducted on the 11-plex;
[0021] Figure 9 shows the Intra and Inter assay percentage CV for the 11- plex;
[0022] Figure 10 shows protein markers for LSD screening using multiplex assays for LSD;
[0023] Figure 11 shows the LAMP Protein Standard and Adult Controls in Fluorescent Units; [0024] Figure 12 shows the GAA Protein Standard and Adult Controls in Fluorescent Units;
[0025] Figure 13 shows the beta-Glucosidease Protein Standard and Adult Controls in Fluorescent Units;
[0026] Figure 14 shows retrospective analysis of CTH C 16:0 in newborn blood spots;
[0027] Figure 15 shows retrospective analysis of HNAc-U A-HNAc-UA in newborn blood spots;
[0028] Figure 16 shows Validation of the Multiplex Newborn Screening Assay;
[0029] Figure 17 shows Validation of the Multiplex Newborn Screening
Assay;
[0030] Figure 18 shows Assay performance during validation studies;
[0031] Figure 19 shows control ranges for protein markers (sulphamidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0032] Figure 20 shows control ranges for protein markers (acid sphingomyelinase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box; [0033] Figure 21 shows control ranges for protein markers (alpha iduronidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0034] Figure 22 shows control ranges for protein markers (LAMP-I), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0035] Figure 23 shows control ranges for protein markers (alpha- glucosidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0036] Figure 24 shows control ranges for protein markers (beta- glucosidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0037] Figure 25 shows control ranges for protein markers (Saposin C), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0038] Figure 26 shows control ranges for protein markers (alpha- galactosidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0039] Figure 27 shows control ranges for protein markers (arylsulphatase A), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0040] Figure 28 shows control ranges for protein markers (iduronate-2- sulphatase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0041] Figure 29 shows control ranges for protein markers (4-sulphatase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0042] Figure 30 shows LSD affected and control ranges for protein markers (sulphamidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0043] Figure 31 shows LSD affected and control ranges for protein markers (acid sphingomyelinase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0044] Figure 32 shows LSD affected and control ranges for protein markers (alpha-iduronidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0045] Figure 33 shows LSD affected and control ranges for protein markers (LAMP-I), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0046] Figure 34 shows LSD affected and control ranges for protein markers (alpha-glucosidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box; [0047] Figure 35 shows LSD affected and control ranges for protein markers (beta-glucosidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0048] Figure 36 shows LSD affected and control ranges for protein markers (Saposin C), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0049] Figure 37 shows LSD affected and control ranges for protein markers (alpha-galactosidase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0050] Figure 38 shows LSD affected and control ranges for protein markers (arylsulphatase A), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0051] Figure 39 shows LSD affected and control ranges for protein markers (iduronate-2-sulphatase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0052] Figure 40 shows LSD affected and control ranges for protein markers (4-sulphatase), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0053] Figure 41 shows control and I-cell ranges for protein markers (sulphamidase; acid sphingomyelinase; alpha-iduronidase; LAMP-I; alpha- glucosidase; beta-glucosidase; Saposin C; alpha-galactosidase; arylsulphatase A; iduronate-2-sulphatase; and 4-sulphatase);
[0054] Figure 42 shows control, MLD and pseudo MLD ranges for arylsulphatase A, wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0055] Figure 43 shows control, and multiple sulphatase deficiency values for selected protein markers in Adult, newborn and MSD, wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0056] Figure 44 shows a table having the percent identification of LSD using single protein MLD and pseudo MLD ranges for arylsulphatase A; [0057] Figure 45 shows a discrimination plot of Gaucher, newborn control, and adult patients using protein ratios (beta-glucosidase and alpha- glucosidase / alpha-galactosidase);
[0058] Figure 46 shows a discrimination plot of Pompe, newborn control, and adult patients using protein ratios (alpha-glucosidase and alpha-iduronidase);
[0059] Figure 47 shows a discrimination plot of Pompe, and newborn control patients using protein ratios (alpha-glucosidase and alpha-iduronidase);
[0060] Figure 48 shows a discrimination plot of MPSII, newborn control, and adult patients using protein ratios (iduronate-2-sulρhatase and LAMP-I);
[0061] Figure 49 shows a discrimination plot of MPSII, newborn control, and adult patients using protein ratios (iduronate-2-sulphatase and acid sphingomyelinase); .
[0062] Figure 50 shows a table indicating the percentage identification of LSD using protein ratios;
[0063] Figure 51 shows a retrospective analysis of newborns from Guthrie card samples (alpha-galactosidase in Denmark, Fabry, Gaucher, MPSII, MPSIIIA, and Pompe), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths torn the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0064] Figure 52 shows a retrospective analysis of newborns from Guthrie card samples (alpha-glucosidase in Denmark, Fabry, Gaucher, MPSII, MPSIIIA, and
Pompe), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0065] Figure 53 shows a retrospective analysis of newborns from Guthrie card samples (beta-glucosidase in Denmark, Fabry, Gaucher, MPSII, MPSIIIA, and Pompe), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0066] Figure 54 shows a retrospective analysis of newborns from Guthrie card samples (sulphamidase in Denmark, Fabry, Gaucher, MPSII, MPSIIIA, and Pompe), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0067] Figure 55 shows a retrospective analysis of newborns from Guthrie card samples (iduronate-2-sulphatase in Denmark, Fabry, Gaucher, MPSII, MPSIIIA, and Pompe), wherein the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box;
[0068] Figure 56 shows a retrospective analysis of newborns from Guthrie card samples (sulphamidase; acid sphingomyelinase; alpha-iduronidase; LAMP-I; alpha-glucosidase; beta-glucosidase; Saposin C; alpha-galactosidase; arylsulphatase A; iduronate-2-sulphatase; and 4-sulphatase); [0069] Figure 57 shows a retrospective analysis plot of Denmark, Fabry, Gaucher, MPSII, MPSIIIA, and Pompe newborn Guthrie card samples (alpha- glucosidase and alpha-iduronidase);
[0070] Figure 58 shows a retrospective analysis plot of Denmark, Fabry, Gaucher, MPSII, MPSIIIA, and Pompe newborn Guthrie card samples (beta- glucosidase and saposin C);
[0071] Figure 59 shows a retrospective analysis plot of Denmark, Fabry, Gaucher, MPSII, MPSIIIA, and Pompe newborn Guthrie card samples (beta- glucosidase and acid sphingomyelinase);
[0072] Figure 60 shows a table of the percentage of total identified LSD;
[0073] Figure 61 shows a table of the percentage of identified LSD using single proteins;
[0074] Figure 62 shows a table of the percentage of identified LSD affected newborns using retrospective analysis;
[0075] Figure 63 shows the multiplex neonatal screening strategy for LSD;
[0076] Figure 64 shows a table of proposed structure of MPS II Oligosaccharides derived from dermatan sulfate;
[0077] Figure 65 shows a table of proposed structure of MPS II Oligosaccharides derived from heparan sulfate;
[0078] Figure 66 shows Elution profile of control and MPS II urine from
Bio-Gel P4. GAGs were isolated from control and MPS II patient urine by a combination of anion exchange and size exclusion chromatography. Fractions from the Bio-Gel P4 column were assayed for UA equivalents. The solid and broken lines represent elution from the Bio-Gel P4 column of control and MPS II urine, respectively. [0079] Figure 61 shows ESI-MS of selected oligosaccharides. Eluate fractions from the Bio-Gel P4 column were lyophilised, derivatized with PMP and analyzed by ESI-MS. Panel a shows ESI-MS of heptasaccharides; [UA-HN]2-UA- HNAc-UA with 3-6 S of [M-3H]"3 ions 605.3, 631.9, 658.5, 685.5 and with 4-6 S [M- 4H]"4 ions 473.8, 493.7, 513.7; and UA-HN-[UA-HNAC]2-UA with 2-3 S of [M-2H]"2 ions 888.9, 929.3 and with 3-4 S [M-3H]'3 ions 619.1, 645.7. Panel b shows ESI-MS of an octasaccharide; [UA-HNAc]4 with 4-6 S [M-3H]"3 ions 727.8, 754.2, 780.8 and [M-4H]"4 ions 545.5, 565.4, 585.5, and with 5-7 S [M-5H]-5 ions 452.2, 468.5, 484.4; a nonasaccharide UA-HN-(UA-HNAC)3-UA with 5-6 S [M-3H]"3 ions 797.9, 824.7, with 5-8 S [M-4H]"4 ions 598.3, 618.2, 638.4, 658.1 and with 4-6 S [M-5H]"5 ions 494.5, 510.3, 526.5. Panel c shows ESI-MS of decasaccharides; (UA-HNAc)5 with 5- 7 S [M-4H]"4 ions 660.6, 680.3, 700.2, with 4-8S [M-5H]"5 ions 511.4, 528.2, 544.3, 560.3, 576.4, with 6-9 S [M-6H]"6 ions 453.3, 466.6, 480, 492.9; UA-HN-[UA- HNAc]4 with 3-5 S [M-4H]'4 ions 609.9, 629.6, 649.8.
[0080] Figure 68 shows Product ion spectra of oligosaccharides from a
MPS II patient. Eluate fractions from the Bio-Gel P4 column were lyophilised, derivatized with PMP and analyzed by ESI-MS/MS. Panel a shows the product ion spectra of a tetrasaccharide UA-HN-UA-HNAc with 4S. Products of the [M-2H]2", m/z 691.4, were obtained. Major product ions at m/z 651.2 [M-S-2H]2', m/z 611.4 [M- 2S-2H]2", m/z 173 [PMP-H]" and m/z 256 corresponding to a PMP molecule with a fragmented HNAc. Panel b shows the product ion spectra of a pentasaccharide UA- HN-UA-HNAc-UA with 4S. Products of the [M-2H]2", m/z 779.6, were obtained. Major product ions at m/z 739.8 [M-S-2H]2", m/z 699.2 [M-2S-2H]2", m/z 173 [PMP- H]" m/z 331 [PMP-UA -H]" and 269 corresponding to a PMP molecule with a fragmented UA.
[0081] Figure 69 shows Mass spectra of oligosaccharides following recombinant enzyme digests. Negative ion ESI-MS of pooled hexasaccharide and heptasaccharide isolated from MPS II urine. Panel a shows ESI-MS of a heptasaccharide (UA-HN-UA-HN-UA-HNAc-UA); with 3-5 S producing [M-2H]"2 ions at m/z 908, 947.8, 988, with 3-6 S producing [M-3H]"3 ions at m/z 605.2, 631.8, 658.5, 685.3 and with 4-6 S producing [M-4H]"4 ions at m/z 473.8, 494.6. A hexasaccharide (UA-HNAc-UA-HNAc-UA-HNAc); with 1-4 S producing [M-2H]"2 ions at m/z 782.3, 822.5, 861.7 and [M-3H]-3 ions at m/z 520.5, 546.8, 573.8, 600.8. Panel b shows the same pooled heptasaccharide and hexasaccharide from MPS II urine following treatment with recombinant I2S and IDUA. The loss of S (80 amu) and IdoA (176 amu) from the heptasaccharide results in a hexasaccharide (HN-UA- HN-UA-HNAc-UA); with 2-4 S producing [M-2H]"2 ions at m/z 779.7, 820, 860.8 and [M-3H]'3 ions 546.7, 573, 599.8. The loss of S and IdoA from the hexasaccharide results in a pentasaccharide (HNAc-UA-HNAc-UA-HNAc); with 1-3 S producing [M-2H]"2 ions at m/z 654.3, 693, 733.2 and [M-3H]'3 ions at m/z 461.5, 488.5.
[0082] Figure 70 shows Generation of HS-derived oligosaccharides in MPS II. Following endohydrolase action by heparanase on the regions of low sulphation, exoenzymes involved in the breakdown of HS act sequentially until a IdoA-2S residue is reached at the end of a region of high sulphation. Further degradation of the oligosaccharide is prevented by the deficiency of the iduronate-2- sulphatase. An analogous scheme where the endohydrolase acts on the GlcNAc-GlcA bonds in the low sulphation region is also proposed to account for the oligosaccharides with HNAc at the reducing terminus. A similar scheme involving an endo-N-acetylglucoasminidase activity is also proposed to account for the HS derived oligosaccharides in Table 2 series 4 (in Figure 65).
SUMMARY
[0083] Lysosomal Storage Disorders ("LSDs") represent a group of over
40 distinct genetic diseases that generally affect young children. Individuals that are affected with a LSD present a wide range of clinical symptoms that depend upon the specific disorder or a particular genotype involved. The present invention is generally related to a multiple screening diagnostic for LSD and related diseases. More particularly, this invention pertains to compounds, reagents, and methods for identifying and quantifying multiple target enzymes and proteins that are used to accurately diagnose a LSD. These target enzymes and proteins are naturally present in biological fluids or tissues of patients. The invention also pertains to a
Multiplexing Bead Technology for simultaneous screening of specific LSD enzymes.
[0084] One aspect of the current invention is a composition used for detection of a LSD. The composition comprises a capture antibody capable of binding an epitope of a target antigen, and a microsphere having the capture antibody conjugated to the microsphere. The target antigen is selected from the group consisting of: saposin (SeqID No. 1), LAMP-I (SeqID No. 2), α-iduronidase (SeqID No. 3), α-glucosidase (SeqID No. 4), β-glucosidase (SeqID No. 5), 2-sulphatase (SeqID No. 6), 4-sulphatase (SeqID No. 7), α-galactosidase (SeqID No. 8), sphingomyelinase (SeqID No. 9), 3-sulphatase (SeqID No. 10) and sulphamidase. The microsphere having the conjugated capture antibody has a preferred diameter of about 5 μm and at least a first fluorophore and a second fluorophore, the first fluorophore being spectrally distinct from the second fluorophore. The composition may further comprise a detection antibody, wherein the detection antibody is capable of binding the target antigen, and the detection antibody may be different from the capture antibody. The detection antibody is conjugated to any detectable label known in the art (e.g. a fluorescent label). DETAILED DESCRIPTION
Terms:
[0085] The term "a" or "an" as used herein in the specification may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.
[0086] The term "animal," "subject," or "patient" as used herein may be used interchangeably and refers to any species of the animal kingdom. In preferred embodiments it refers more specifically to humans.
[0087] The term "biomolecule" as used herein is understood to represent the target molecule, such as a protein, an antibody, a metabolite, a DNA sequence, an RNA sequence, a biologic with activities used or measured for the purposes of multiplexing and profiling of target biomolecules, or a combination thereof, for the composition and method of determining LSD, used in administering, monitoring, or modifying an LSD therapy.
[0088] The term "clinical status" as used herein refers to patients that are being studied or treated by physicians for a LSD.
[0089] The term "comprise," or variations such as "comprises" or "comprising," as used herein may be used to imply the inclusion of a stated element or integer or group of elements or integers, but not the exclusion of any other element or integer or group of elements or integers.
[0090] The term "fluorophore" as used herein refers to any fluorescent compound or protein that can be used to quantify the LSD antigens.
[0091] The term "normalize" as used herein refers to bringing a target, reference, or other samples into conformity with a standard, pattern, model, etc. For example, in one embodiment, urine samples from LSD patients and non-LSD patients were normalized by using a 1 μmol equivalent of creatinine from each sample. [0092] The term "phenotype" as used herein refers to the manifest characteristics of an organism collectively, including anatomical and psychological traits that result from both its heredity and its environment.
[0093] The term "preclinical status" as used herein refers to the period of a disease before any of the clinical symptoms appear.
[0094] The term "lysosomal storage disorder ("LSD") associated biomolecule" as used herein refers to any biomolecule that has been linked to any LSD. In preferred embodiments, a LSD associated biomolecule includes, but is not limited to: saposin (SeqID No. 1), LAMP-I (SeqID No. 2), α-iduronidase (SeqID No. 3), α-glucosidase (SeqID No. 4), β-glucosidase (SeqID No. 5), 2-sulphatase (SeqID No. 6), 4-sulphatase (SeqID No. 7), α-galactosidase (SeqID No. 8), sphingomyelinase (SeqID No. 9), sulphamidase, or 3-sulphatase (SeqID No. 10), or sulphamidase, LAMP-2, α-galactosidase A, iduronate-2-sulphatase, α-iduronidase, N- acetylgalactosamine 4-sulphatase, galactose 6-sulphatase, acid sphingomyelinase, galactocerebrosidase, arylsulphatase A, saposin B, heparan-N-sulphatase, α-N- acetylglucosaminidase, acetylCoA: glucosamine N-acetyltransferase, N- acetylglucosamine 6-sulphatase, β-galactosidase, β-glucuronidase, aspartylglucosaminidase, acid lipase, β-hexosamindase A, β-hexosarnindase B, GM2- activator, acid ceramidase, α-L-fucosidase, α-D-mannosidase, β-D-mannosidase, neuraminidase, phosphotransferase, phosphotransferase g-subunit, palmitoyl protein thioesterase, tripeptidyl peptidase I, cathepsin K, α-galactosidase B, or sialic acid transporter. As shown below, Table 1 indicates some enzyme deficiencies for LSDs.
[0095] The term "reference quantity" as used herein refers to a known, normalized amount of a LSD biomarker in a biological fluid. The reference quantity is determined from an animal, or group of animals having a defined clinical status, preclinical status, or phenotype of a LSD disease. The reference quantity may refer to a table compiled from various animals or groups of animals having correlations between relative amounts of LSD biomarkers in a biological fluid, and a known clinical status, preclinical status, or phenotype. [0096] Other Abbreviations: "MPS," mucopolysaccharidosis; "GAG," glycosaminoglycans; "HS," heparan sulfate; "DS," dermatan sulfate; "IDUA," α-L- iduronidase; "I2S," iduronate-2-sulfatase; "ESI-MS/MS," electrospray ionization- taiidem mass spectrometry; "IdoA," α-L-iduronic acid; "HN," hexosamine; "UA," uronic acid; "GIcN," glucosamine; "GIcNAc," N-acetylglucosamine; "GaINAc," N- acetylgalactosamine; "HNAc," N-acetylhexosamine; "MRM," multiple-reaction monitoring.
LYSOSOMAL STORAGE DISORDERS [0097] The LSD's represent a group of over 40 distinct genetic diseases that generally affect young children. Patients are usually born without the visible features of a LSD, but early stage symptoms can quickly develop into a progressive clinical concern. Although some effective LSD therapies have been developed, it is paramount that therapy be started as soon as the LSD has been diagnosed. Unfortunately, a clinical diagnosis of a LSD often requires multiple visits to a range of specialists requiring time-consuming, invasive, complex, inconvenient, and expensive assays. The current process for an accurate diagnosis of LSD for a patient not having a family history of LSD can take months to years, which is unacceptable when effective LSD therapies are needed earlier.
[0098] It is generally recognized that the accumulation of storage materials in the lysosomes of LSD affected individuals will increase from approximately 1% to as much as 50% of the total cellular volume. Certain lysosomal proteins are present at altered levels in the LSD affected individuals (Meikle et al, 1997; Hua et ah, 1998). Unless stated otherwise all regents were of analytical grade and were obtained from Sigma Chemical Company, MO USA. Preparation of recombinant proteins, antibodies and calibration standards for Lamp-1 and saposin C. Recombinant Lamp-1 (minus tail) was isolated from CHO-Kl cells as detailed in Isaac et al [Isaac EL, Karageorgous LE, Brooks DA, Hopwood JJ and Meikle PJ. Experimental Cell Research 2000, 254: 204-209]. Recombinant Saposin C was a gift from Dr GA Grabowski and was prepared by the method of Qi and Grabowski [Qi TL and Grabowski GA J Biol Chem 1994, 269: 16746-16753].
[0099] Identifying "at risk" individuals. In order to demonstrate that identification of two or more LSD biomarkers can increase the accuracy of LSD diagnosis, methods that combined the identification of LAMP-I proteins and saposin C proteins in patients samples were evaluated.
[00100] The ami Lamp-1 monoclonal antibody (BB6) was generated using intact Lamp-1 protein by the method of Carlsson and Fukada [Carlsson SR and Fukada M JBC (1989) 264(34): 20526-20531] and 7B2 (ami Saposin C) monoclonal antibody was produced using the recombinant protein by the method described in [Zola H and Brooks D. Techniques for the production and characterization of monoclonal hybridoma antibodies. In: Hurrell JGR, ed. Monoclonal hybridoma antibodies: techniques and applications. Boca Raton, FL: CRC Press, 1982:1-57]. Polyclonal antibodies were generated for both Lamp-1 and Saposin C by immunizing separate rabbits with 200μg of each recombinant protein per inoculation (four inoculations in total) based upon the method of Leonova et al, 1996, [JBC 271:17312- 20]. All antibodies were purified using 5ml Hitrap™ Protein G affinity column (Pharmacia, Uppsala, Sweden). The polyclonal antibodies were affinity purified further by column chromatography using their respective recombinant proteins coupled to Affi-Gel® 10 Gel (Bio-Rad #153-6046, CA, USA) according to manufacturers instructions.
[00101] Blood spot calibrators containing final concentrations of 2000, 1000, 500, 250, 62.5 and 0 μg/L for Lamp-1 and saposin C were prepared as detailed in Umapathysivam et al [Umapathysivam K, Whittle AM, Ranieri E, Bindloss C, Ravenscroft EM, van Diggelen OP, Hopwood JJ and Meikle PJ Clin Chem 46(9): 1318-1325 2000]. Two blood spot controls containing low (Lamp-1 400μg/L; saposin 200μg/L) and high (Lamp-1 800μg/L; saposin C 500μg/L) protein concentrations were similarly prepared. [0100] Quantification of Lamp-1 and Saposin C in dried blood spots containing EDTA. Lamp-1 and Saposin C were measured in dried blood spots using one step three tier, time-delayed fluorescence immunoassays. Microtiter plates (Labsytems, Helsinki, Finland #95029180) were coated with either BB6 or 7B2 at a concentration of 5 μg/L in O.lmol/1 NaHCO3, pH 8.3 and incubated covered for approximately lόhrs at 4°C. Plates were washed twice with wash buffer (0.25mol/l NaCl, 0.02mol/l Tris containing 0.005% Tween 20 (BDH, Poole, England) and 0.002% Thiomerosal, pH7.8) Non-specific binding sites on the plates were blocked by the addition of lOOμl of 0.25M NaCl, 0.02M Tris containing 0.5% skim milk powder (Diploma, Bonlac Foods, Victoria, Australia), pH 7.8, per well. After a two hour incubation at room temperature, the microtiter plates were washed twice with 0.25M NaCl, 0.02M Tris pH 7.8 and tapped dry before being lyophilized and stored desiccated at 40C prior to use.
[0101] Standard calibrators, controls and patient dried blood spots were placed in duplicate into the coated microtiter wells with 200μl of either polyclonal antibody diluted in assay buffer (0.15mol/l NaCl, 0.05mol/L Tris, 20μmol/L Diethylene triamine-penta-acetic acid, containing 0.01% Tween 40, 0.5% bovine serum albumin (A-9647), 0.05% bovine γ-globulin (G-7516), and 0.05% sodium azide, pH 7.8). The antibodies were used at a final concentration of 200μg/L and 400μg/L for the anti-Lamp- 1 and anti saposin C polyclonal respectively. The plates were covered and incubated at room temperature for one hour with shaking, then placed overnight at 4°C, followed by an hour incubation with shaking at room temperature. The blood spots were removed by suction and the plates washed six times with wash buffer. After dilution in assay buffer to final concentration of O.lμg/ml, lOOμl of anti rabbit europium labeled antibody (Wallac, Finland #AD0105), was added to every well and incubated for one hour at room temperature with shaking. After washing the plates a final six times with wash buffer, 200μl of DELFIA® Enhancement solution (Wallac, Finland) was added per well and the plates incubated at room temperature for ten minutes with shaking. Fluorescence was measured on a DELFIA® 1234 Research Fluorometer (Wallac, Finland). The concentrations of Lamp-1 and Saposin C in the blood spots were calculated using spline fit curves generated by Multicalc Data Analysis software (version 2.4 Wallac, Finland).
[0102] Although certain lysosomal target proteins are present at altered levels in the affected individuals, the current individual screening assays may be inaccurate due to variations among individual samples. For example, a given sample is assumed to contain an average number of lysosomes or white blood cells ("WBC"), however variations in these values between individual samples are not typically considered. Thus, variations in an individual having a deficiency in a particular LSD biomolecule (e.g. lysosomal target protein), but also having an unusually high WBC count or high numbers of lysosomes in the test sample may return an assay result that is consistent for individuals that do not have a LSD. Consequently, if WBC or high numbers of lysosomes were controlled in the sample preparation a large inaccuracy could be avoided, and a proper diagnosis could be made during the first round of LSD screening.
[0103] Determining the quantities of multiple target enzymes increases the accuracy of diagnosing a specific LSD as compared to any single assay. For example, using immunoquantification assays directed toward identifying the levels of the lysosome-associated membrane proteins ("LAMPs"), such as LAMP-I or LAMP-2, in an "at-increased-risk" group will identify up to 65% of LSD affected individuals. However, the combination of LAMP'S with one of the saposins increases identification of LSD affected individuals to approximately 85%. Therefore, a method to identify two or more biomarkers simultaneously would increase the accuracy of LSD diagnosis and reduce the time and cost for each assay. A Multiplexing Bead Technology is used to simultaneously detect specific at least 2 LSD target antigens is described below or in Table 1.
Table 1 Enzymes deficient in some common lysosomal storage disorders
Disease Clinical Phenotype Enzyme Deficiency Australian
Prevalence
Gaucher disease types I / II / III Gaucher disease Glucocerebrosidase 1 in 57,000
(β-glucosidase) Cystinosis Cystine transporter 1 in 192,000
Fabry disease Fabry disease α-Galactosidase A 1 in 117,000
Glycogen storage disease II Pompe disease α-Glucosidase 1 in 146,000
Mucopolysaccharidosis type I Hurler/Scheie syndrome α-L-Iduronidase 1 in 88,000
Mucopolysaccharidosis type II Hunter syndrome Iduronate-2-sulphatase 1 in 136,000
Mucopolysaccharidosis type VI Maroteaux-Lamy syndrome N-acetylgalactosamine 4- 1 in 235,000 sulphatase
Mucopolysaccharidosis type IVA Morquio syndrome Galactose 6-sulphatase 1 in 169,000
Niemann-Pick disease types A / B Niemann-Pick disease Acid sphingomyelinase 1 in 248,000
Globoid cell leucodystrophy Krabbe disease Galactocerebrosidase 1 in 201,000
Metachromatic leucodystrophy Arylsulphatase A 1 in 92,000
Metachromatic leucodystrophy Saposin B
Mucopolysaccharidosis type IHA Sanfilippo syndrome Heparan-N-sulphatase 1 in 114,000
Mucopolysaccharidosis type UIB Sanfilippo syndrome α-N-Acetylglucosaminidase 1 in 211,000
Mucopolysaccharidosis type IHC Sanfilippo syndrome AcetylCoA:N-acetyltransferase 1 in 1,407,000
Mucopolysaccharidosis type HID Sanfilippo syndrome N-Acetylglucosamine 6- 1 in 1,056,000 sulphatase
Mucopolysaccharidosis type IVB Morquio syndrome β-Galactosidase
Mucopolysaccharidosis type VII Sly β-Glucuronidase 1 in 2, 111,000
Niemann-Pick disease type Cl Niemann-Pick disease Cholesterol trafficking 1 in 211,000
Niemann-Pick disease type C2 Niemann-Pick disease Cholesterol trafficking
Aspartylglucosaminuria Aspartylglucosaminidase 1 in 2,111,000
Cholesterol ester storage disease Wolman disease Acid lipase 1 in 528,000
GMl -Gangliosidosis types I/II/III β-Galactosidase 1 in 384,000
GM2-Gangliosidosis type I Tay Sachs disease β-Hexosaminidase A 1 in 201,000
GM2-Gangliosidosis type II Sandhoff disease β-Hexosaminidase A & B 1 in 384,000
GM2-Gangliosidosis GM2-activator deficiency
Farber Lipogranulomatosis Farber disease Acid ceramidase
Fucosidosis α-L-Fucosidase > 1 in
2,000,000
Galactosialidosis types I / II Protective protein α-Mannosidosis types I / II α-D-Mannosidase 1 in 1,056,000 β-Mannosidosis β-D-Mannosidase
Mucolipidosis type I Sialidosis types I / II Neuraminidase
Mucolipidosis types II / III I-cell disease; Phosphotransferase 1 in 325,000
Mucolipidosis type IHC pseudo-Hurler Phosphotransferase g-subunit polydystrophy
Mucolipidosis type IV Unknown
Multiple sulphatase deficiency Multiple sulphatases 1 in 1,407,000
Neuronal Ceroid Lipofuscinosis, CLNl Batten disease Palmitoyl protein thioesterase
Neuronal Ceroid Lipofuscinosis, CLN2 Batten disease Tripeptidyl peptidase I
Neuronal Ceroid Lipofuscinosis, CLN3 Vogt-Spielmeyer disease Protein function not known
Neuronal Ceroid Lipofuscinosis, CLN5 Batten disease Protein function not known
Neuronal Ceroid Lipofuscinosis, CLN8 Northern Epilepsy Protein function not known
Pycnodysostosis Cathepsin K
Sialic acid storage disease Schindler disease α-Galactosidase B
Sialic acid storage disease Sialuria; salla disease Sialic acid transporter 1 in 528,000
Prevalence figures quoted from Miekle et al., JAMA 281:249-254 (1999). Prevalence and ratio of lysosomal storage disorders may vary from country to country
EXAMPLE 1
[0104] Multiplexing Bead Technology and Target LSD Proteins. The
Multiplexing Bead Technology is built around 3 core technologies. The first is the family of fluorescently dyed microspheres having bound biomolecules. The second is a flow cytometer with 2 lasers and associated optics to measure biochemical reactions that occur on the surface of the microspheres, and the third is a high-speed digital signal processor to efficiently manage the fluorescent output. Bio-Rad (Hercules, CA), provides a commercially available protein array system called the "Bio- Plex™". The Bio-Plex™ protein array system includes fluorescently dyed microspheres, a flow cytometer with 2 lasers and associated optics, and a high-speed digital signal processor. However, neither the Bio-Plex™ protein array system nor any other commercially available systems include any specific biomolecules, methods, compounds, or reagents needed for the simultaneous screening of specific LSD enzymes.
[0105] The Bio-Plex™ protein array system uses multiplexing technology to enable the simultaneous quantitation of up to 100 different analytes. This technology uses polystyrene microspheres internally dyed with differing ratios of 2 spectrally distinct fluorophores. Each fluorophore can have any of 10 possible levels of fluorescent intensity, thereby creating a family of 100 spectrally distinct bead sets. In a preferred embodiment, the dyed microspheres are conjugated with antibodies specific for a target LSD protein or peptide thereof. Although not wanting to be bound by theory, each of the 100 spectrally distinct bead sets can contain a capture antibody specific for a unique LSD target protein. In a multiplexed Bio-Plex™ assay, LSD antibody-conjugated beads are allowed to react with the sample and a secondary LSD antibody, or a detection LSD antibody in a microtiter plate well to form a capture sandwich immunoassay. Figure 1 shows a drawing of a complete microsphere capture sandwich immunoassay having a polystyrene microsphere (110) with 2 spectrally distinct fluorophores; the target LSD capture antibody (120) bound to the microsphere; a unique LSD target protein or target antigen (130) bound to the target LSD capture antibody; a detection LSD antibody (140); and a detection molecule (e.g. phycoerythrin, "PE" or Biotin) (150) . Once the complete microsphere capture sandwich immunoassay has formed in solution, the immunoassay solution is then drawn into the Bio-Plex™ array reader, which illuminates and reads the sample. Although not wanting to be bound by theory, there are many enzyme deficiencies specific for a particular LSD, and some of these enzymes are shown in Table 1.
[0106] When a red diode "classification" laser (635 nm) in the Bio-Plex™ array reader illuminates a dyed bead, the bead's fluorescent signature identifies it as a member of one of the 100 possible bead sets. Bio-Plex™ Manager software correlates each bead set to the assay reagent that has been coupled to it (for example, a first LSD capture antibody coupled to bead set #22, and a second LSD capture antibody coupled to bead set #42). hi this way the Bio-Plex™ protein array system can distinguish between the different assays combined within a single microtiter well. A green "reporter" laser (532 nm) in the array reader simultaneously excites a third fluorescent dye (phycoerythrin, "PE") bound to the detection LSD antibody in the assay. Although not wanting to be bound by theory, the amount of green fluorescence is proportional to the amount of target analyte captured in the immunoassay. Extrapolating the captured amount of target analyte to a standard curve allows quantitation of each LSD analyte in the sample. The digital signal processing algorithms provide simultaneous real-time data acquisition of classification and reporter signal output from thousands of beads per second, supporting up to 100 x 96 = 9,600 analyte measurements from each 96-well plate.
EXAMPLE 2 [0107] Designing and Producing LSD Target Microspheres. The
BioPlex Protein Array System was used as one embodiment to demonstrate the type and nature of the reagents necessary for a LSD multiplex diagnostic assay. Capture antibodies and detection antibodies have been purified that selectively bind epitopes of: saposin, LAMP-I, α-iduronidase, α-glucosidase, β-glucosidase, 2-sulphatase, 4- sulphatase , α-galactosidase, sphingomyelinase, and 3-sulphatase. The full or partial sequences of the following proteins that were used to generate the purified antibodies are as follows: saposin (SeqID No. 1) , LAMP-I (SeqID No. 2), α-iduronidase (SeqID No. 3), α-glucosidase (SeqID No. 4), β-glucosidase (SeqID No. 5), 2-sulphatase (SeqID No. 6), 4-sulphatase (SeqID No. 7), α-galactosidase (SeqID No. 8), sphingomyelinase (SeqID No. 9), 3 -sulphatase (SeqID No. 10), and sulphamidase.
[0108] Monoclonal Antibodies. Monoclonal antibodies were produced in
Balb/C mice using standard immunisation protocols (Harlow et ah, 1988). Mice were immunized with recombinant enzyme using established protocols. Plasma cells from these immunized mice were fused with P3.653 myeloma cells (Zola et ah, 1982) and the resulting hybridoma cell lines screened for antibodies against the recombinant protein by direct ELISA (Harlow et al., 1988). Monoclonal antibodies were purified from cell culture supernatants by ammonium sulfate precipitation followed by affinity purification on Hitrap™ Protein G affinity column (Pharmacia Biotech, Uppsala,
Sweden).
[0109] In some embodiments, a monoclonal capture antibody for LAMP-I was BB6 developed and provided by Sven Carlsson (Carlsson et ah, 1989). The monoclonal reporter antibody for α-glucosidase (43Dl) was obtained from Pharming, Inc. and has been described (Fransen et ah, 1988). The polyclonal reporter antibody for LAMP-I, the rabbit polyclonal reporter antibody for saposin C, the sheep polyclonal capture antibody for α-glucosidase, and the monoclonal capture antibody ("7B2") for saposin C were prepared within the Lysosomal Diseases Research Unit at the WCH in Adelaide, Australia using standard techniques, known in the art, and briefly described below. The availability and production of specific monoclonal and polyclonal antibodies are know to one of ordinary skill in the art. Production of the specific antibodies uses in the current examples are given below:
[0110] Polyclonal Antibodies. Sheep polyclonal antibodies were produced against recombinant proteins (e.g. saposin (SeqID No. 1) , LAMP-I (SeqID No. 2), α-iduronidase (SeqID No. 3), α-glucosidase (SeqID No. 4), β-glucosidase (SeqID No. 5), 2-sulphatase (SeqID No. 6), 4-sulphatase (SeqID No. 7), α- galactosidase (SeqID No. 8), sphingomyelinase (SeqID No. 9), 3 -sulphatase (SeqID No. 10) and sulphamidase). A sheep was injected sub-cutaneously with 2mg of protein in 1 mL of an emulsion of phosphate buffered saline (pH 7.4) and complete Freunds adjuvant, followed by four booster injections (2mg each) with incomplete Freunds adjuvant, each three weeks apart. One week after the last injection the sheep was bled out and serum collected. Rabbit polyclonal antibody was produced in the same manner, except 0.2-1.0 mg of protein was used per immunisation. Sheep polyclonal antibody was purified on a 5 mL Hitrap Protein G affinity column (Pharmacia Biotech, Uppsala, Sweden) followed by an affinity column prepared from the recombinant protein used for the immunisation. Recombinant protein affinity columns were prepared by coupling 5 mg of the recombinant protein to 2.5 mL of Affi-gel 10 (Bio-Rad, Hercules, CA, USA) as per manufacturer's instructions.
[0111] Briefly, 5 mL of sheep serum was diluted with 5 mL of phosphate buffered saline (pH 7.4) and centrifuged (220Og, 10 min, 40C). The centrifuged serum was passed through a 0.2 μm filter, and then loaded on to the Protein G column at a flow rate of 0.5 mL/min. The column was washed with phosphate buffered saline, pH 7.4 and the antibody eluted with 0.1 mol/L H3PO4/NaH2PO4, pH 2.5 and immediately neutralized by adding 1.0 mol/L Na2HPO4 (l/10th vol). The protein content was estimated by absorbance at 280nm (absorbance = 1.4 for 1.0 g/L of protein). The eluate was diluted four fold and then loaded on to the appropriate recombinant protein affinity column at the same flow rate. The column was washed and eluted as described for the Protein G column.
[0112] Coupling Antibodies to Microspheres. The target capture antibodies were coupled to Bio-Rad carboxylated ("COOH") beads as follows: anti- saposin to bead No. 42 , anti-LAMP-1 to bead No. 25, anti-α-iduronidase to bead No. 24, anti-α-glucosidase to bead No. 26, anti-β-glucosidase to bead No. 28, anti-2- sulphatase to bead No. 45, anti-4-sulphatase to bead No. 46, anti-α-galactosidase to bead No. 43, anti-sphingomyelinase to bead No. 22, and anti-3 -sulphatase to bead No. 44), as indicated in Figure 7. The coupling of the target capture antibodies to the polystyrene microspheres was performed using the BioRad bead coupling kit (Catalog number 171-406001, BioRad, Hercules, CA). The Bio-Plex™ amine coupling kit includes 4 ml bead wash buffer, 85 ml bead activation buffer, 135 ml PBS, pH 7.4, 10 ml blocking buffer, 25 ml storage buffer, 105 ml staining buffer, 40 coupling reaction tubes. The Bio-Plex™ amine coupling kit provides the buffers necessary to covalently couple 6-150 IcD proteins to 5.5 μm dyed carboxylated polystyrene beads in under 5 hr. The covalent couple of the target capture antibody to the carboxylated polystyrene bead is achieved via carbodiimide reactions involving the protein primary amino groups and the carboxyl functional groups bound on the surface of polystyrene beads. The covalent attachment is permanent, leaving no unbound protein after cleanup, even after months of storage. The protein-coupled beads can then be used in multiplex protein-protein binding studies or in the development of multiplex assays that can be analyzed with the Bio-Plex™ protein array system. The bead yield per coupling reaction is approximately 80%, or enough protein-coupled beads for two 96- well microtiter plates using 5,000 beads per well.
[0113] Once the coupling reaction was completed, the target capture antibody-coupled beads were enumerated and the efficiency of the protein coupling reaction was validated, according to the manufacturer's protocol with modifications. In this procedure, the protein-coupled beads were reacted with a phycoerythrin ("PE")-labeled antibody that binds to the coupled protein, which was then analyzed using the Bio-Plex™ protein array system. This procedure was performed by reacting the beads with a PE-labeled antibody. Alternatively, a reaction using a biotinylated antibody followed by streptavidin-PE may be used. Although not wanting to be bound by theory, the intensity of the fluorescent signal of this reaction is directly proportional to the amount of protein on the surface of the beads. A successful coupling typically yields a mean fluorescent intensity ("MFI") signal that is greater than 2,000. The protein coupling validation procedure provided a rapid relative assessment of the amount of protein coupled to the beads, but could not verify the functionality of the protein.
[0114] The detection antibodies that specifically bind epitopes of: saposin; LAMP-I; α-iduronidase; α-glucosidase; β-glucosidase; 2-sulphatase; 4- sulphatase; α-galactosidase; sphingomyelinase; and 3-sulphatase, were biotinylated according to the manufacturer's instructions (Molecular Probes, FluroReporter Biotin- XX protein labelling kit F-2610). There are several published methods known in the art for preparation of biotinylated or phycobiliprotein conjugates with antibodies and other proteins. Generally, the coupling chemistry used to crosslink a phycobiliprotein to another protein includes: (a) treating the antibody or other protein with a succinimidyl ester maleimide derivative at pH 7.5, which converts some lysine residues of the antibody to thiol-reactive maleimides; (b) preparing a thiolated phycobiliprotein by reducing the appropriate SPDP-modified phycobiliprotein with dithiothreitol ("DTT") or with tris-(2-carboxyethyl)ρhosphine ("TCEP"); (c) mixing the above two dialyzed protein conjugates to yield a stable thioether crosslink; and (d) chromatographically separating the phycobiliprotein conjugates from the unreacted proteins.
[0115] A calibration curve was generated using liquid calibrator proteins in a microsphere based assay using calibrator protein capture antibodies and bead sets #25, #42, #26, #28, #43, #46, #45, #44, and #22 (BioRad, Hercules, CA3 USA). Figure 2 shows a calibration curve for a single assay for α-glucosidase. The detection capability for the amount of calibrator protein present in each well reaction was linear in the range of 0 to 4 ng/well of the assay. The MFI was the average of the total fluorescence detected for the beads in the defined bead region. Calibration curves were also established, using liquid calibrators, for LAMP-I (open square), saposin C (open circle), and α-glucosidase (open triangle), as shown in Figure 3. Increased MFI for the α-glucosidase protein, when compared to Figure 2, is the result of improvements in the capture antibody labeling of the microspheres and the phycoerythrin reporter labeled antibodies. Figure 3 also indicates that the detection capability for a multiplex assay of three calibrators was linear from 0 to 2 ng/well of the assay.
[0116] As a general illustration, Figure 4 shows a drawing of a microsphere collection of capture sandwich immunoassays for the 10-plex having: 10 spectrally distinct polystyrene microsphere (510-519); 10 target LSD capture antibody (520-529) bound to the microsphere; 10 unique LSD target proteins or target antigens and representing: saposin, LAMP-I3 α-iduronidase, α-glucosidase, β-glucosidase, 2- sulphatase, 4-sulphatase, α-galactosidase, sphingomyelinase, and 3 -sulphatase (530- 539) bound to the corresponding target LSD capture antibody; 10 unique detection LSD antibody (540-549); and a detection molecule (550).
[0117] Development of Ten-plex and Eleven-plex Assays. All bead assays were performed in 96-well filtration plates (Millipore MAB VNS 1250), sealed and protected from light. All standards were performed in duplicate. Standard solutions containing saposin C, LAMP-I, α-iduronidase, α-glucosidase, β- glucosidase, 2-sulphatase, 4-sulphatase, α-galactosidase, sphingomyelinase, and 3 — sulphatase protein (50μl) were added in serial 2-fold dilutions in assay buffer, as indicated. Standards were generated by using the recombinant form of each specific target protein. Biotinylated antibodies (50μl) were added to each well, wherein the final concentration of each antibody was long/well in assay buffer. Antibody-coated beads (5,000/well) for each individual assay were mixed in filtered (0.2μm) PBS containing 0.5% BSA (Sigma A-9647), 0.05% γ-globulin (Sigma G-7516) and 0.05% Tween 20, pH 7.2 (assay buffer), placed into pre-wetted filtration plates and the supernatant removed by vacuum. Diluted pre-mixed standards were added to the beads followed by the 10 pre-mixed biotinylated reporter antibodies. The plates were incubated for one-hour at room temperature with shaking and then placed at 4°C overnight. After a further one-hour incubation at room temperature with shaking, the plates were washed three-times with filtered (0.2μm) PBS containing 0.05% Tween 20, pH 7.2 (wash buffer) under vacuum. Streptavidin conjugated to phycoerythrin (Molecular Probes S-866) was diluted in assay buffer (1.2 μg/mL) and added to the wells (125μl/well); the plates were then incubated at room temperature with shaking for 10 minutes. The plates were then read on the Bio-Plex suspension array system (Bio-Rad) using version 3.0 software and counting 100 beads/region. Figure 5 shows the resulting calibration curves for saposin C (asterisk), LAMP-I (X-mark), α- iduronidase (solid diamond), α-glucosidase (open square), β-glucosidase (solid square), 2-sulphatase (open triangle), 4-sulphatase (solid square), α-galactosidase (solid circle), sphingomyelinase (open diamond), and 3 -sulphatase (open circle) of the 10-plex assay. Figure 6 shows an 11-plex assay with an added sulphamidase (asterisk).
[0118] The control ranges for protein markers in adult, NRL controls, Denmark and newborn samples are shown in Figures 19-29 for each of the specific markers (sulphamidase; acid sphingomyelinase; alpha-iduronidase; LAMP-I; alpha- glucosidase; beta-glucosidase; Saposin C; alpha-galactosidase; arylsulphatase A; iduronate-2-sulphatase; and 4-sulphatase). As shown in Figures 19-29, the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box.
[0119] The ranges for protein markers in adult controls, newborn controls, Fabry, Gaucher, MLD, MPS I, MPS II, MPSIIA MPS VI, NP a/b and Pompe samples are shown in Figures 30-40 for each of the specific markers (sulphamidase; acid sphingomyelinase; alpha-iduronidase; LAMP-I; alpha-glucosidase; beta-glucosidase;
Saposin C; alpha-galactosidase; arylsulphatase A; iduronate-2-sulphatase; and 4- sulphatase). As shown in Figures 30-40, the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box.
[0120] Figure 44 shows a table having the percent identification of LSD using single proteins that allowed many of the LSD's to be 100% identified, and
Gaucher and Pompe were not identified 100%. Figure 50 shows a table having the percent identification of LSD using ratios as having all but Gaucher identified as
100%. [0121] A retrospective analysis of newborn samples from Guthrie cards was also conducted. Figures 51-55 show the retrospective analysis of: alpha- galactosidase; alplia-glucosidase; beta-glucosidase; sulphamidase; and iduronate-2- sulphatase. As shown in Figures 51-55, the box length is the interquartile range that covers 25th to 75th percentile, the outliers are represented by (circles), each of these cases representing values between 1.5 and 3 box lengths from the upper or lower edge of the box, and the extreme outlier (stars) are cases with values more than 3 box lengths from the upper or lower edge of the box. Figure 62 shows that Fabry, ML WUI, MPS II, MPS IIIA and Pompe were identified from retrospective LSD affected newborns at 100%, and Gaucher was identified in retrospective LSD affected newborns at 80%. The other disorders were not identified in this retrospective analysis.
EXAMPLE 3
[0122] A defect in exo-degradative pathways provides insight into endo-degradation of heparan and dermatan sulfates. Maria Fuller, Ally Chau, Rachael C. Nowak, John J. Hopwood' and Peter J. Meikle contributed to the invention of Example 3. Within cells, dermatan sulfate and heparan sulfate are degraded in two steps. The initial endohydrolysis of these polysaccharides is followed by the sequential action of lysosomal exoenzymes to reduce the resulting oligosaccharides to monosaccharides and inorganic sulfate. Mucopolysaccharidosis types II is a lysosomal storage disorder caused by a deficiency of the exoenzyme iduronate-2- sulfatase. Consequently, partially degraded fragments of dermatan sulfate and heparan sulfate, have been shown to accumulate in the lysosomes of affected cells and are excreted in the urine. Di- to hexadecasaccharides, isolated from the urine of a mucopolysaccharidosis type II patient using anion exchange and gel filtration chromatography, were identified using electrospray ionization-tandem mass spectrometry. These oligosaccharides were shown to have non-reducing terminal iduronate 2-sulfate residues by digestion with recombinant iduronate 2-sulfatase. A pattern of growing oligosaccharide chains composed of alternating uronic acid and N- acetylhexosamine residues was identified and suggested to originate from dermatan sulfate. A series of oligosaccharides consisting of hexosamine/N-acetylhexosamine alternating with uronic acid residues were also identified and based on the presence of unacetylated hexosamine, these oligosaccharides are proposed to derive from heparan sulfate. The presence of both odd and even length oligosaccharides suggests both endo-β-glucuronidase and endo-N-acetylhexosaminidase activities toward both glycosaminoglycans. Furthermore, the putative oligosaccharide structures identified indicate that heparanase activities are directed towards regions of both low and high sulfation, whilst the N-acetylhexosaminidase activity acted only in regions of low sulfation.
[0123] The sulfated glycosaminoglycans heparan sulfate (HS) and dermatan sulfate (DS) are present in a wide variety of cell types where they play an intricate role in the extracellular matrix. HS has repeating disaccharide units consisting of uronic acid (UA) alternating with α-lmked (1,4) glucosamine (GIcN) residues. Biosynthesis of HS occurs in the Golgi. Following the synthesis of the base polymer consisting of the disaccharide repeat (GIcA 1-4 GIcNAc), a series of enzymatic reactions occurs, deacetylating the GIcNAc, epimerising the glucuronic acid (GIcA) to iduronic acid (IdoA) and adding sulfate groups to both O and N positions on IdoA and GIcN. Consequently, the UA residue may be β-linked (1,4) D- GIcA, or α-linked (1,4) L-IdoA, unsulfated or with O-sulfation of the C2-hydroxyl. The amino group of GIcN may be N-sulfated, N-acetylated or occasionally unsubstituted. The GIcN may also be sulfated on the C6-hydroxyl and sometimes on the C3-hydroxyl. The proportion of GIcA and IdoA varies considerably, not only between different species of HS but also within a particular HS chain. Likewise, the degree and type of sulfation is not stoichiometric (3). The modification reactions that are responsible for the structural diversity are not complete, producing a final HS molecule with a domain structure (4). HS chains typically contain regions rich in GIcA and N-acetylated GIcN (GIcNAc) disaccharides with no sulfation (NA domains), contiguous variable length sequences containing IdoA and GIcNS derivatives with high sulfation (NS domains), and bridging these domains are mixed sequences in which GIcNAc disaccharides and GIcNS disaccharides alternate (NA/NS domains) (5).
[0124] DS is composed of repeating disaccharide units consisting of UA alternating with β-lmked (1,4) D-N-acetylgalactosamine (GaINAc) residues that may be sulfated on the C4-and/or Cό-position. Some DS chains have predominantly (1,3) α-linked IdoA residues with some C2-sulfation, while others have primarily (1,3) β- linked GIcA. Similar to HS, DS forms block structures of lowly sulfated GIcA- GaINAc disaccharides alternating with blocks of highly sulfated IdoA-GalNAc disaccharides.
[0125] The catabolism of HS and DS begins with endohydrolysis of the polysaccharide chains to oligosaccharides. Two classes of human endoenzymes have been reported that cleave at specific sites within the DS and HS polysaccharides. Hyaluronidases (endo-β-N-acetylhexosaminidases) are a family of enzymes that degrade hyaluronan, as well as chondroitin sulfate and DS. Hyaluronidases cleave internal β-linked (1,4) glycosidic bonds between GaINAc and GIcA in DS (7). Levels of hyaluronidase are elevated in a number of cancers but the identity of the type of hyaluronidase expressed in most cancer tissue and cells is still unknown (8). Heparanase (endo-β-glucuronidase) cleaves at glucuronosyl bonds within HS resulting in smaller saccharide chains. Heparanase activity has been shown to be associated with cell invasion, angiogenesis, inflammation and tissue remodelling through a number of cell-matrix interactions. The action of heparanase on cell surface and extracellular matrix HS proteoglycans is thought to be a highly regulated process involving the binding and internalisation of heparanse to specific cell surface proteoglycans (syndecans), thereby limiting the extracellular accumulation and action of this endoglycosidase. Endosulfatase action on heparin has also been reported and it is likely that similar activity will be shown to the sulfated domains of HS.
[0126] Following partial catabolism by endoenzymes, HS and DS are degraded from their non-reducing termini by the sequential action of highly specific lysosomal exoenzymes. At least 10 lysosomal exoenzymes act to reduce these oligosaccharides to monosaccharides and inorganic sulfate to enable exit out of the lysosome. A deficiency in any one of these exoenzyme activities may result in lysosomal storage of the GAG substrates with the resulting clinical manifestation of the mucopolysaccharidoses (MPS). MPS II results from a deficiency of iduronate-2- sulfatase (I2S; EC 3.1.6.13), which hydrolyzes the C2-sulfate ester bond of non- reducing terminal α-L-iduronic acid residues in HS and DS. In the absence of I2S activity the catabolism of HS and DS is blocked. Consequently, partially degraded fragments of HS and DS accumulated in the lysosomes of affected cells and are excreted in the urine. These oligosaccharide fragments are presumably produced by endohydrolase activities, followed by exohydrolase activities that terminate at the substrate for I2S. Structural characterisation of these oligosaccharides can therefore provide insight into the process of endodigestion of HS and DS and detail of the substrate specificities of the enzymes involved. To this end, we have identified a series of oligosaccharides from MPS II patient urine that provide evidence for novel endohydrolase activities and specificities.
[0127] Experimental Procedures and Materials for Example 3.
Recombinant human IDUA and recombinant human I2S were each prepared from CHO-Kl expression systems. MPS II patient urine and control urine was supplied with consent.
[0128] Isolation of urinary GAG. Urine samples from an MPS II patient and an age-matched control (500 ml) were clarified by centrifugation and passed over a 30 ml column of DEAE-Sephacel previously equilibrated with 0.1 M NaCOOCH3 buffer, pH 5. The column was washed with 10 column volumes of the equilibration buffer and urinary GAG eluted in the same buffer containing 1.2 M NaCl. Fractions were assayed for UA and the GAG-containing fractions (20 ml) were pooled, lyophilized and reconstituted in 4 ml of H2O. The pooled GAG fraction was then size-fractionated on a Bio-Gel P4 column (170 cm x 1.5 cm) in 0.5 M NH4COO. Fractions (4 ml) were collected and assayed for UA. [0129] Derivatization of oligosaccharides. Samples from Bio-Gel P4 column fractions were lyophilized prior to derivatization. Samples were resuspended in 100 μL 250 mM l-phenyl-3-methyl-5-pyrazolone, 400 mM NH4OH, heated at 7O0C for 90 min and then acidified with a 2-fold molar excess of HCOOH. Samples were made up to 500 μl with H2O and then extracted with an equal volume of CHCl3 to remove excess l-phenyl-3-methyl-5 -pyrazolone and centrifuged at 13,000 xg for 5 min. Chloroform extraction was repeated a further 2 times and the aqueous layer was lyophilised and the derivatized oligosaccharides resuspended in 500 μL of an aqueous solution of 50 % (v/v) CH3CN/0.025 % (v/v) HCOOH prior to analysis by mass spectrometry. Derivatization of samples following enzyme digestion was performed in the same way except that the aqueous phase from the first chloroform extraction was treated as follows: copolymeric solid phase extraction cartridges (50 mg, Cl 8 and aminopropyl) (United Chemical Technologies, Bristol, PA, USA) were primed with methanol (1 mL) then water (1 mL), after which the sample was applied and allowed to enter the solid phase completely. Samples were desalted with three consecutive 1 mL water washes, dried on a Supelco, Visiprep24 vacuum manifold (Sigma-Aldrich, St. Louis, MO, USA) and the remaining l-phenyl-3-methyl-5-pyrazolone was removed with two 1 mL chloroform washes. The columns were again dried, and derivatized oligosaccharides were eluted in an aqueous solution of 50 % (v/v) CH3CN/NH4OH pH 11.5, lyophilized and resuspended in 50 % (v/v) CH3CN/0.025 % (v/v) HCOOH.
[0130] Enzymatic cleavage. Column fractions containing 10 μg of UA were lyophilized prior to digestion with recombinant I2S and IDUA. Enzymatic cleavage was performed at 370C for 16hr in 50 μL of 50 mM NaCOOCH3 buffer (pH 4.5) supplemented with 0.5 mg/ml BSA and 20 μg recombinant enzyme (IDUA and/or I2S). Following digestion, the oligosaccharides were derivatized as described above and analyzed by ESI-MS in negative ion mode.
[0131] Mass spectrometry. Oligosaccharide analysis was performed by ESI-MS/MS using a PE Sciex API 3000 triple-quadrupole mass spectrometer with an turboionspray source and Analyst 1.1 data system. Samples were either directly infused using a Harvard Apparatus pump at 10 μL /min or injected with a Gilson 233 autosampler at 80 μL /min using a carrying solvent of 50 % (v/v) CH3CN/0.025 % (v/v) HCOOH in H2O. Oligosaccharides were identified based on mass to charge (m/z) ratios by ESI-MS and further characterized using collision induced dissociation- MS/MS in the negative ion mode.
[0132] Isolation of oligosaccharides in MPS II urine. A combination of anion exchange and gel filtration was used to isolate GAG-derived oligosaccharides from control and MPS II urine. From a volume of 500 niL of MPS II urine, 45 mg of UA equivalents were recovered from the DEAE column and of this material 40 % eluted in the fractionation range of the Bio-Gel P4 column (< hexadecasaccharide). In comparison, from 500 niL of urine from a control individual, only 0.2 mg of UA equivalents were recovered and 100 % of this was excluded from the Bio-Gel P4 fractionation range (> hexadecasaccharide). Urinary oligosaccharides, from the MPS II patient, eluting between 96 and 225 mL (Figure 66) were further characterized by mass spectrometry.
[0133] Mass spectrometry of oligosaccharides. Electrospray ionisation- mass spectrometry (ESI-MS) was performed in negative ion mode on each eluate fraction from the Bio-Gel P4 column. For each oligosaccharide structure, multiple sulfated species could be identified. These oligosaccharides showed characteristic multiply charged ions produced by proton abstraction and as such were identified as [M-H]"1 up to [M-8H]"8 ions. Figure 61 shows representative spectra for oligosaccharides ranging from tetrasaccharide to tetradecasaccharides. Electrospray ionisation-tandem mass spectrometry (ESI-MS/MS) was also used for further characterisation of these oligosaccharides. Precursor ion scan of m/z 173, corresponding to an ionised l-phenyl-3-methyl-5-pyrazolone (PMP) fragment was used to support the assignments made from the ESI-MS scans. Strong signals were observed for oligosaccharide ions in higher charged states (> [M-3H]"3), whereas signals from the [M-2H]"2 were weak or not detectable (data not shown). Collisionally activated dissociation-tandem mass spectrometry (CAD-MS/MS) was performed on all major oligosaccharide signals and used to confirm the assignments made from the ESI-MS spectra and to identify the residue at the reducing terminus. Oligosaccharides containing N-acetylhexosamine (HNAc) at the reducing terminus gave a product ion at m/z 256 and those with a UA at the reducing end gave a product ion of m/z 331. A representative product ion spectra of a tetrasaccharide (UA-HN- UA-HNAc 4S) with a characteristic product ion m/z 256 is shown in Figure 68a, and Figure 68b shows the product ion spectra from a pentasaccharide (UA-HN-UA- HNAc-UA 4S) with a characteristic product ion of m/z 331. Product ion analysis was performed on all major signals in the ESI-MS and in most instances enabled the identification of the reducing terminal sugar as either UA or HNAc.
[0134] The mass spectrometry analysis of the oligosaccharides eluted from the Bio-Gel P4 column enabled the identification of 25 different oligosaccharide species ranging in size from di- to hexadecasaccharide with various numbers of sulfates. Tables 1 and 2 (Figures 64 and 65, respectively) display a summary of the oligosaccharides present in the MPS II patient urine that were classified into series based on their proposed structures. Two series of oligosaccharides containing UA and HNAc disaccharide repeat units were identified with 1-2 sulfates per disaccharide. The first series, with an even number of saccharides, had a UA residue on the non- reducing end and a HNAc on the reducing end (Table 1, series 1). The second series consisted of an odd number or residues ranging from tri- to heptasaccharide (Table 1, series 2) with UA at the reducing terminus.
[0135] Four series of oligosaccharides . containing UA, HN and HNAc were identified with different levels of sulfation (Table 2). Series 1-3 consist of an odd number of residues with UA at the reducing terminus, and series 4 contains an even number of residues with HNAc at the reducing terminus. The oligosaccharides in series 1 are composed of one HN with either zero, one or two HNAc. Series 2 contains oligosaccharides with two HN and up to five HNAc. The oligosaccharide in series 3 contains three HN residues and a single HNAc. Series 4 lists oligosaccharides with an even number of residues containing one HN and up to four HNAc residues.
[0136] Enzymatic characterisation of the non-reducing end. For oligosaccharides present in sufficient quantities the non-reducing end UA was identified as iduronate-2-sulfate (Ido2S) by enzymatic cleavage with recombinant I2S and IDUA. Figure 69 shows ESI-MS of pooled hexa- and heptasaccharide fractions isolated from MPS II urine, before and after I2S and IDUA treatment. The hexasaccharide identified by m/z ratios with the putative structure [UA-HNAc]3 with 1 to 4 sulfates was shown to lose 176 and 80 amu, representing UA and S respectively, following incubation with recombinant I2S and IDUA. As with the hexasaccharide, a heptasaccharide with the proposed structure (UA-HN)2-UA-HNAc- UA with 3 to 6 S, also showed losses of 176 and 80 amu following digestion with recombinant IDUA and I2S (Figure 69). A peak with m/z of 523.3 corresponding to IdoA hydrolysed from the oligosaccharides was also identified in the digested sample (Figure 69b). Treatment of the oligosaccharides with only recombinant IDUA resulted in no change to the spectra, while treatment with I2S resulted in the disappearance of each of the oligosaccharides in the highest sulfation form (spectra not shown).
[0137] Di- to hexa-decasaccharides that would appear to originate from endohydrolase activities on HS and DS, and are therefore the result of incomplete catabolism of HS and DS, were isolated from the urine of an MPS II patient. Mass spectrometry has enabled structural characterisation of these oligosaccharides using a combination of ESI-MS and CAD-MS/MS. CAD-MS/MS was performed on all major oligosaccharide signals and used to confirm the assignments made from the ESI-MS spectra and to identify the residue at the reducing terminus. Enzyme treatment with recombinant I2S and IDUA identified the non-reducing residue as
IdoA2S, the substrate for the enzyme deficiency in MPS II.
[0138] ESI-MS identified oligosaccharides that have a different number of residues but very similar molecular weights, which occurs as a result of the similarity between the mass of HN (161 Da) and 2 S (160 Da). For example, the octasaccharide ([UA-HN]3-UA-HNAc (4S)) has a molecular weight of 2059.4 whereas the heptasaccharide ([UA-HN]2-UA-HNAC-UA (6S)) has a molecular weight of 2058.4 and when the [M-4H]"4 ions are compared the difference is m/z 513.9 compared with m/z 513.6, respectively. The mass accuracy of the instrument used in this study could not positively discriminate these structures. However, identification of the reducing terminus as either UA or HNAc and the non-reducing terminus as IdoA2S enabled this discrimination.
[0139] Two series of oligosaccharides that are likely derived from DS (Table 1 - Figure 64) and four series of oligosaccharides likely derived from HS (Table 2 — Figure 65), based on the presence of unacetylated HN residues in the HS- derived oligosaccharides, were identified. The formation of DS-derived oligosaccharides terminating in HNAc (Table 1, series 1) is consistent with the reported action of hyaluronidase to act on the GaINAc-UA linkages of DS. The second series of DS-derived oligosaccharides was composed of an odd number of saccharides with UA at both the reducing and non-reducing termini (Table 1, series 2). As with series 1, these oligosaccharides had a concomitant increase in the number of sulfate residues with oligosaccharide length. Although not wanting to be bound by theory, we would suggest that the sulfate residues are distributed along the DS oligosaccharides, consistent with the reported action of hyaluronidase to GalNAc-4-S residues. Although the smaller oligosaccharides (di- to octasaccharide) had up to 2 sulfates per disaccharide, the larger (up to hexadecasaccharide) had fewer sulfates. This may result from the loss of sulfate residues in the ion source of the mass spectrometer as has been previously reported. However, these oligosaccharides may also arise in part from the structural heterogeneity present in DS. We have previously reported tri- and pentasaccharides, isolated from MPS I urine, and identified them as being derived from DS by the presence of GalNAc-4-S as the penultimate residue at the non-reducing end. These oligosaccharides were suggested to derive from endohydrolase activity against the UA-GaINAc bond in DS. This concept is further supported here in MPS II, with the identification of tri-, penta- and heptasaccharides derived from DS. However, we did not observe any significant signals from larger oligosaccharide species in this series. The limitation on the size of the oligosaccharide identified may reflect substrate specificity of the endoglucuronidase activity for terminal regions of the DS chains, although it may also be that larger species are not present in sufficient amounts to be resolved from the more abundant oligosaccharide species terminating in HNAc, seen in the complex spectra of the larger oligosaccharides.
[0140] The oligosaccharide series containing UA-HN and UA-HNAc disaccharides provides a more complex picture of endohydrolysis of HS. Two series of oligosaccharides (Table 2, series 1 and 2) are likely products of heparanase activity towards HS. These series have a trisaccharide (UA-HN-UA) or a pentasaccharide (UA-HN-UA-HN-UA) base structure with an increasing number (up to five) of HNAc-UA disaccharides. Interestingly, the number of sulfates associated with this series does not increase with the size of the oligosaccharide but remains relatively constant between three and seven. In light of the reported domain structure of HS with regions of high (NS) and low (NA) sulfation, it seems probable that these two series of oligosaccharides represent short NS domains with an increasing extension of the NA domain. Thus, the heparanase appears to have specificity for the relatively highly sulphated linkage region between the NS and NA domains (thus producing the base tri and pentasaccharides) and also for the unsulfated region represented by the extending GIcNAc-UA disaccharides (NA domain). Previous studies have indicated multiple substrates for heparanase activity. For example, earlier studies indicated that human heparanase cleaved a sequence within the highly modified NS domains of HS. Heparanase from CHO cells has been proposed to have substrate specificity for the mixed NA/NS domains. Studies on the heparanase from a rat parathyroid cell line identified relatively undersulfated structures at the cleavage site and also proposed that the cleavage occurred at the boundary of highly sulphated and undersulfated domains. At the same time studies on recombinant human heparanase with defined tetra and hexasaccharide structures indicated the requirement for a highly sulphated structure containing an unsulfated GIcA at the cleavage site. [0141] Although there appears to be only one candidate gene for heparanase, it is also plausible that there are two different enzymes with specificities for either NS or NA domains in HS. Heparanase activity is also likely to be responsible for the generation of the nonasaccharide (UA-HN-[UA-HN]2-UA-HNAC- UA) (Table 2, series 3) that represents a longer NS domain with heparanase cleavage occurring after the first HNAc in the adjacent NA domain. The final HS-derived oligosaccharide series (Table 2, series 4) represents similar structures to those seen in series 1 but with a HNAc as opposed to a UA at the reducing end. This suggests the action of an endohydrolase on the HNAc-UA linkage in HS. In this series of oligosaccharides the number of sulfates does not proportionately increase with oligosaccharide length, again indicating that the extension of the UA-HNAc disaccharide is into the NA domain, and thus the endoenzyme specificity is for the NA domain.
[0142] The absence of any oligosaccharides larger than a pentasaccharide containing only UA and HN suggests that the NA/NS domain of HS is more resistant to heparanase activity than the NA domains. Presumably, the larger NS regions are to be found in the high molecular weight structures eluting at V0 from the Bio-Gel P4 column. The structures observed, appear to arise primarily from the action of heparanase in NA domains of HS, these regions of low sulfation are separated by short (up to three) regions of sulfated UA-HN disaccharide repeats (NS domains).
The reducing terminus of each oligosaccharide is then trimmed back by the action of exoenzymes to the first IdoA2S residue where, as a result of the deficient enzyme, exodegradation is halted. A similar scheme involving an endo-N- acetylglucoasminidase activity is also proposed to account for the HS derived oligosaccharides in Table 2 series 4 (Figure 70).
[0143] The study of stored oligosaccharides from patients with a defective exodigestion of glycosaminoglycan chains (MPS patients) provides a unique, global insight into the process of endohydrolysis of glycosaminoglycans in the endosomal/lysosomal network. In urine from an MPS II patient we have observed a significant proportion of low molecular weight oligosaccharides (40% of uronic acid in hexadecasaccharide or smaller), indicating that the action of these endoglycosidases provides a significant contribution to the total degradation of GAG in the lysosome. Characterisation of the resulting oligosaccharides has provided us with new insight into the endoenzymes involved and their substrate specificities. In addition to the hyaluronidase and heparanase we have provided evidence for the action of an endo- glucuronidase activity on DS and an endo-N-acetylglucosaminidase activity on HS. We have also provided evidence that the majority of HS digestion occurs in the low sulfation (NA) regions. Further detailed characterisation of the stored substrates in MPS II and other MPS types will increase our understanding of these enzymes and their role in GAG turnover.
[0144] This invention comprises general and specific compositions for LSD diagnostics, including multiplex bead technology. Although species specific antibodies have been used as illustrative examples, other types of antibodies that bind LSD target proteins are not considered to deviate from the spirit and scope of the claimed invention.
REFERENCES CITED
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
U.S. PATENT DOCUMENTS
United States Patent 6,449,562 ("the '562 Patent") entitled "Multiplexed Analysis of Clinical Specimens Apparatus and Method," having Chandler et al. listed as inventors was issued on September 10, 2002. United States Patent 6,524,793 ("the '793 Patent") entitled "Multiplexed Analysis of Clinical Specimens Apparatus and Method," having Chandler et al. listed as inventors, was issued on February 25, 2003.
United States Patent Application Serial No. 09/956,857 ("the '857 Application") entitled "Multiple Reporter Read-out for Bioassays" was published on March 20, 2003.
PCT Application AU03/00731 entitled "identification of Oligosaccharides and their Use in the Diagnosis and Evaluation of Mucopolysaccharidoses and Other Related Disorders," having Hopwood et al., listed as inventors, filed on June 13, 2003 Other Publications
Carlsson, S.R., M. Fukuda, Structure of human lysosomal membrane glycoprotein 1, Assignment of disulfide bonds and visualization of its domain arrangement., J. Biol. Chem. 264:20526-20531 (1989).
Fransen, J.A., L.A. Ginsel, P.H. Cambier, J. Klumperman, R.P. Oude Elferink, J.M. Tager, Immunocytochemical demonstration of the lysosomal enzyme alpha- glucosidase in the brush border of human intestinal epithelial cells, Eur J Cell Biol 47:72-80 (1988).
Harlow, E., D. Lane, Antibodies, A laboratory manual, Cold Spring Harbor Laboratory (1988). Hua, CT. et al, Evaluation of the lysosome-associated membrane protein LAMP-2 as a marker for lysosomal storage disorders, CHn. Chem. 44(10): 2094-2102 (1988).
Isaac EL, Karageorgous LE, Brooks DA, Hopwood JJ and Meikle PJ. Experimental Cell Research 2000, 254: 204-209]. Isbrandt, D., G. ArIt, D.A. Brooks, JJ. Hopwood, K. von Figura, and C. Peters, Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome): Six unique arylsulfatase B gene alleles causing variable disease phenotypes, Am J Hum Genet 54(3): 454-63 (1994). Meikle et al., Prevalence of lysosomal storage disorders, JAMA 281 : 249-254 (1999).
Neufeld, E.F. and J. Muenzer, The mucopolysaccharidoses, The Metabolic & Molecular Basis of Inherited Disease, 7th Edition., pp. 2465-2494 (1995).
Umapathysivam, K., JJ. Hopwood, PJ. Meikle, Determination of acid alpha- glucosidase activity in blood spots as a diagnosis for Pompe Disease, CHn. Chem. 47(8): 1378-1383 (2001).
Qi and Grabowski [Qi TL and Grabowski GA J Biol Chem 1994, 269: 16746-16753.
Zola, H., D. Brooks, Techniques for the Production and Characterization of Monoclonal Hybridoma Antibodies, Monoclonal Hybridoma Antibodies: Techniques and Applications (1982). Kramer, K. L., and Yost, H. J. (2003) Annu Rev Genet 37, 461-484
Trowbridge, J. M., and Gallo, R. L. (2002) Glycobiology 12, 117R-125R
Lindahl, U., Kusche-Gullberg, M., and Kjellen, L. (1998) J Biol Chem 273, 24979- 24982
Lyon, M., and Gallagher, J. T. (1998) Matrix Biol 17, 485-493 Maccarana, M., Sakura, Y., Tawada, A., Yoshida, K., and Lindahl, U. (1996) J Biol Chem 271, 17804-17810
Prydz, K., and Dalen, K. T. (2000) JCe// Sci 113 Pt 2, 193-205 Kreil, G. (1995) Protein Sci 4, 1666-1669
Lokeshwar, V. B., Rubinowicz, D., Schroeder, G. L., Forgacs, E., Minna, J. D., Block, N. L., Nadji, M., and Lokeshwar, B. L. (2001) J Biol Chem 276,
11922-11932
Bame, K. J. (2001) Glycobiology 11, 91R-98R
Hulett, M. D., Freeman, C, Hamdorf, B. J., Baker, R. T., Harris, M. J., and Parish, C. R. (1999) Nat Med 5, 803-809 Vlodavsky, L, and Friedmann, Y. (2001) JCUn Invest 108, 341-347
Dempsey, L. A., Brunn, G. J., and Platt, J. L. (2000) Trends Biochem Sci 25, 349-351 Gingis-Velitski, S., Zetser, A., Kaplan, V., Ben-Zaken, 0., Cohen, E., Levy-Adam, F., Bashenko, Y., Flugelman, M. Y., Vlodavsky, L, and Ilan, N. (2004) J Biol Chem
Morimoto-Tomita, M., Uchimura, K., Werb, Z., Hemmerich, S., and Rosen, S. D. (2002) JBiol Chem 277, 49175-49185
Neufeld, E. F., and Meunzer, J. (2001) in The Molecular Basis of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and VaUe, D., eds), pp. 3421-3452, McGraw-Hill, New York
Unger, E. G., Durrant, J., Anson, D. S., and Hopwood, J. J. (1994) Biochem J304 ( Pt 1), 43-49
Bielicki, J., Hopwood, J. J., Wilson, P. J., and Anson, D. S. (1993) Biochem J289 ( Pt 1), 241-246
Blumenkrantz, N., and Asboe-Hansen, G. (1973) Anal Biochem 54, 484-489 Ramsay, S. L., Meikle, P. J., and Hopwood, J. J. (2003) MoI Genet Metab 78, 193-204 Fuller, M., Meikle, P. J., and Hopwood, J. J. (2004) Glycobiology 14, 443-450
Knudson, W., Gundlach, M. W., Schmid, T. M., and Conrad, H. E. (1984) Biochemistry 23, 368-375
Marchetti, D., Liu, S., Spohn, W. C, and Carson, D. D. (1997) J Biol Chem 272, 15891-15897 Pikas, D. S., Li, J. P., Vlodavsky, L, and Lindahl, U. (1998) JBiol Chem 273, 18770- 18777
Bame, K. J., and Robson, K. (1997) JBiol Chem 272, 2245-2251
Bame, K. J., Venkatesan, L, Stelling, H. D., and Tumova, S. (2000) Glycobiology 10, 715-726 Podyma-Inoue, K. A., Yokote, H., Sakaguchi, K., Ikuta, M., and Yanagishita, M. (2002) JBiol Chem 277, 32459-32465
Okada, Y., Yamada, S., Toyoshima, M., Dong, J., Nakajima, M., and Sugahara, K. (2002) JBiol Chem 277, 42488-42495
Toyoshima, M., and Nakajima, M. (1999) JBiol Chem 274, 24153-24160

Claims

CLAIMS:
1) A diagnostic composition comprising: a purified capture antibody capable of binding a target antigen; and a microsphere having the purified capture antibody conjugated to the microsphere; wherein the target antigen is: saposin (SeqID No. 1), LAMP-I (SeqID No.
2), α- iduronidase (SeqID No.
3), α-glucosidase (SeqID No.
4), β- glucosidase (SeqID No.
5), 2-sulphatase (SeqID No.
6), 4-sulphatase (SeqID No.
7), α-galactosidase (SeqID No.
8), sphingomyelinase
(SeqID No. 9), 3-sulphatase (SeqID No. 10), or sulphamidase; and wherein the microsphere comprises at least a first fluorophore and a second fluorophore.
2) The diagnostic composition of claim 1, further comprising a purified detection antibody, wherein the purified detection antibody is capable of binding the target antigen, and the purified detection antibody is conjugated to a detection label.
3) The diagnostic composition of claim 2, wherein the detection label is phycoerythrin or Biotin .
4) The diagnostic composition of claim 1, wherein the first fluorophore is spectrally distinct from the second fluorophore.
5) The diagnostic composition of claim 1, wherein the microsphere has a diameter of about 5 μm.
6) A diagnostic composition comprising: a first microsphere conjugated to a first purified capture antibody that selectively binds to an epitope of saposin (SeqID No. 1); a second microsphere conjugated to a second purified capture antibody that selectively binds to an epitope of LAMP-I (SeqID No. 2); a third microsphere conjugated to a third purified capture antibody that selectively binds to an epitope of α-iduronidase (SeqID No. 3); a fourth microsphere conjugated to a fourth purified capture antibody that selectively binds to an epitope of α-glucosidase (SeqID No. 4); a fifth microsphere conjugated to a first purified capture antibody that selectively binds to an epitope of β-glucosidase (SeqID No. 5); a sixth microsphere conjugated to a second purified capture antibody that selectively binds to an epitope of 2-sulphatase (SeqID No. 6); a seventh microsphere conjugated to a third purified capture antibody that selectively binds to an epitope of 4-sulphatase (SeqID No. 7); an eighth microsphere conjugated to a fourth purified capture antibody that selectively binds to an epitope of α-galactosidase (SeqID No. 8); a ninth microsphere conjugated to a third purified capture antibody that selectively binds to an epitope of sphingomyelinase (SeqID No. 9); and a tenth microsphere conjugated to a fourth purified capture antibody that selectively binds to an epitope of 3-sulphatase (SeqID No. 10); wherein the first through tenth microspheres comprise a specific ratio of fluorophores and are spectrally distinct from each other.
7) The diagnostic composition of claim 6, further comprising: a first purified detection antibody conjugated to a fluorescent detection label and that selectively binds to an epitope of saposin (SeqID No. 1); a second purified detection antibody conjugated to a fluorescent detection label and that selectively binds to an epitope of LAMP-I (SeqID No.
2); a third purified detection antibody conjugated to a fluorescent detection label and that selectively binds to an epitope of α-iduronidase (SeqID
No. 3); a fourth purified detection antibody conjugated to a fluorescent detection label and that selectively binds to an epitope of α-glucosidase (SeqID
No. 4); a fifth purified detection antibody conjugated to a fluorescent detection label and that selectively binds to an epitope of β-glucosidase (SeqID
No. 5); a sixth purified detection antibody conjugated to a fluorescent detection label and that selectively binds to an epitope of 2-sulphatase (SeqID No. 6); a seventh purified detection antibody conjugated to a fluorescent detection label and that selectively binds to an epitope of 4-sulphatase (SeqID
No. 7); an eighth purified detection antibody conjugated to a fluorescent detection label and that selectively binds to an epitope of α-galactosidase (SeqID
No. 8); a ninth purified detection antibody conjugated to a fluorescent detection label and that selectively binds to an epitope of sphingomyelinase (SeqID No. 9); and a tenth purified detection antibody conjugated to a fluorescent detection label and that selectively binds to an epitope of 3-sulphatase (SeqID No. 10). 8) A diagnostic composition comprising: a purified capture antibody capable of binding an epitope of saposin (SeqID No. 1); and a microsphere having the purified capture antibody conjugated to the microsphere; wherein the microsphere comprises at least a first fluorophore and a second fluorophore.
9) A diagnostic composition comprising: a purified capture antibody capable of binding an epitope of LAMP-I (SeqID No. 2); and a microsphere having the purified capture antibody conjugated to the microsphere; wherein the microsphere comprises at least a first fluorophore and a second fluorophore.
10) A diagnostic composition comprising: a purified capture antibody capable of binding an epitope of α-iduronidase (SeqID No. 3); and a microsphere having the purified capture antibody conjugated to the microsphere; wherein the microsphere comprises at least a first fluorophore and a second fluorophore.
H)A diagnostic composition comprising: a purified capture antibody capable of binding an epitope of α-glucosidase (SeqID No. 4); and a microsphere having the purified capture antibody conjugated to the microsphere; wherein the microsphere comprises at least a first fluorophore and a second fluorophore.
12) A diagnostic composition comprising: a purified capture antibody capable of binding an epitope of β-glucosidase (SeqID No. 5); and a microsphere having the purified capture antibody conjugated to the microsphere; wherein the microsphere comprises at least a first fluorophore and a second fluorophore.
13) A diagnostic composition comprising: a purified capture antibody capable of binding an epitope of 2-sulphatase (SeqID No. 6); and a microsphere having the purified capture antibody conjugated to the microsphere; wherein the microsphere comprises at least a first fluorophore and a second fluorophore.
14) A diagnostic composition comprising: a purified capture antibody capable of binding an epitope of 4-sulphatase (SeqID No. 7); and a microsphere having the purified capture antibody conjugated to the microsphere; wherein the microsphere comprises at least a first fluorophore and a second fluorophore.
15) A diagnostic composition comprising: a purified capture antibody capable of binding an epitope of α- galactosidase (SeqID No. 8); and a microsphere having the purified capture antibody conjugated to the microsphere; wherein the microsphere comprises at least a first fluorophore and a second fluorophore.
16) A diagnostic composition comprising: a purified capture antibody capable of binding an epitope of sphingomyelinase (SeqID No. 9); and a microsphere having the purified capture antibody conjugated to the microsphere; wherein the microsphere comprises at least a first fluorophore and a second fluorophore.
17) A diagnostic composition comprising: a purified capture antibody capable of binding an epitope of 3-sulphatase (SeqID No. 10); and a microsphere having the capture antibody conjugated to the microsphere; wherein the microsphere comprises at least a first fluorophore and a second fluorophore.
18) A diagnostic composition comprising: a purified capture antibody capable of binding an epitope of sulphamidase; and a microsphere having the capture antibody conjugated to the microsphere; wherein the microsphere comprises at least a first fluorophore and a second fluorophore.
PCT/AU2005/001821 2004-12-02 2005-12-02 Multiplex-bead complex for determination of lysosomal storage disorders WO2006058385A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US63261004P 2004-12-02 2004-12-02
US60/632,610 2004-12-02

Publications (1)

Publication Number Publication Date
WO2006058385A1 true WO2006058385A1 (en) 2006-06-08

Family

ID=36564687

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2005/001821 WO2006058385A1 (en) 2004-12-02 2005-12-02 Multiplex-bead complex for determination of lysosomal storage disorders

Country Status (2)

Country Link
US (1) US7615224B2 (en)
WO (1) WO2006058385A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8349319B2 (en) 2009-08-28 2013-01-08 Mount Sinai School Of Medicine Dose escalation enzyme replacement therapy for treating acid sphingomyelinase deficiency
WO2014197859A1 (en) 2013-06-07 2014-12-11 Genzyme Corporation Marker for acid sphingomyelinase disorders and uses thereof

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4833831B2 (en) * 2003-03-31 2011-12-07 ウイミンズ、アンド、チルドランズ、ホスピトル Multiple screening for lysosomal storage diseases (LDS)
US8497122B2 (en) 2008-04-11 2013-07-30 Washington University Biomarkers for Niemann-pick C disease and related disorders
WO2017011582A1 (en) 2015-07-13 2017-01-19 Gelb Michael H Methods for assaying palmitoyl protein thioesterase 1 and tripertidyl peptidase activity in dried blood spots for detection of neuronal ceroid lipofuscinoses in newborns
EP4126966A4 (en) * 2020-03-31 2024-08-07 Seattle Childrens Hospital D/B/A Seattle Childrens Res Institute Proteomic screening for lysosomal storage diseases

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997044668A1 (en) * 1996-05-17 1997-11-27 Women's And Children's Hospital Early detection of lysosomal storage disorders
WO2000055632A1 (en) * 1999-03-17 2000-09-21 Women's And Children's Hospital Diagnosis of lysosomal storage disorders using saposins and other markers
WO2002088322A2 (en) * 2001-05-02 2002-11-07 Incyte Genomics, Inc. Cell adhesion and extracellular matrix proteins
WO2003092601A2 (en) * 2002-04-30 2003-11-13 Seikagaku Corporation A method for detecting lysosomal storage diseases

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4833831B2 (en) * 2003-03-31 2011-12-07 ウイミンズ、アンド、チルドランズ、ホスピトル Multiple screening for lysosomal storage diseases (LDS)

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997044668A1 (en) * 1996-05-17 1997-11-27 Women's And Children's Hospital Early detection of lysosomal storage disorders
WO2000055632A1 (en) * 1999-03-17 2000-09-21 Women's And Children's Hospital Diagnosis of lysosomal storage disorders using saposins and other markers
WO2002088322A2 (en) * 2001-05-02 2002-11-07 Incyte Genomics, Inc. Cell adhesion and extracellular matrix proteins
WO2003092601A2 (en) * 2002-04-30 2003-11-13 Seikagaku Corporation A method for detecting lysosomal storage diseases

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3482767A1 (en) 2009-08-28 2019-05-15 Icahn School of Medicine at Mount Sinai Dose escalation enzyme replacement therapy for treating acid sphingomyelinase deficiency
US8658162B2 (en) 2009-08-28 2014-02-25 Icahn School Of Medicine At Mount Sinai Dose escalation enzyme replacement therapy for treating acid sphingomyelinase deficiency
US8709408B2 (en) 2009-08-28 2014-04-29 Icahn School Of Medicine At Mount Sinai Dose escalation enzyme replacement therapy for treating acid sphingomyelinase deficiency
EP3998078A1 (en) 2009-08-28 2022-05-18 Icahn School of Medicine at Mount Sinai Dose escalation enzyme replacement therapy for treating acid sphingomyelinase deficiency
US9114139B2 (en) 2009-08-28 2015-08-25 Icahn School Of Medicine At Mount Sinai Dose escalation enzyme replacement therapy for treating acid sphingomyelinase deficiency
US9655954B2 (en) 2009-08-28 2017-05-23 Icahn School Of Medicine At Mount Sinai Dose escalation enzyme replacement therapy for treating acid sphingomyelinase deficiency
US8349319B2 (en) 2009-08-28 2013-01-08 Mount Sinai School Of Medicine Dose escalation enzyme replacement therapy for treating acid sphingomyelinase deficiency
US10188705B2 (en) 2009-08-28 2019-01-29 Icahn School Of Medicine At Mount Sinai Dose escalation enzyme replacement therapy for treating acid sphingomyelinase deficiency
US10022428B2 (en) 2013-06-07 2018-07-17 Genzyme Corporation Marker for acid sphingomyelinase disorders and uses thereof
EP3584580A1 (en) 2013-06-07 2019-12-25 Genzyme Corporation Recombinant human acid sphingomyelinase for use in treating human asmd
US10888607B2 (en) 2013-06-07 2021-01-12 Genzyme Corporation Marker for acid sphingomyelinase disorders and uses thereof
WO2014197859A1 (en) 2013-06-07 2014-12-11 Genzyme Corporation Marker for acid sphingomyelinase disorders and uses thereof
US11998592B2 (en) 2013-06-07 2024-06-04 Genzyme Corporation Methods of treating acid sphingomyelinase deficiency

Also Published As

Publication number Publication date
US7615224B2 (en) 2009-11-10
US20070265432A1 (en) 2007-11-15

Similar Documents

Publication Publication Date Title
US8003337B2 (en) Method for detecting lysosomal storage diseases
US20120184050A1 (en) Multiplex screening for lysosomal storage disorders (lsds)
US7615224B2 (en) Multiplex-bead complex for determination of lysosomal storage disorders
AU2003233255B2 (en) Oligosaccharide biomarkers for mucopolysaccharidoses and other related disorders
US20090324617A1 (en) Cancer specific glycans and use thereof
CA2373119C (en) Polysaccharide structure and sequence determination
US20040132131A1 (en) Methods for comparitive analysis of carbohydrate polymers and carbohydrate polymers identified using same
CA2428150C (en) Methods for comparitive analysis of carbohydrate polymers
US11262348B2 (en) Cell-based assays for detection of antibodies or other factors that neutralize uptake of lysosomal enzymes
AU2004225901B2 (en) Multiplex screening for lysosomal storage disorders (LSDs)
AU2002225992B2 (en) Methods for comparitive analysis of carbohydrate polymers
WO2016022927A1 (en) Detection of oligosaccharides
AU2002225992A1 (en) Methods for comparitive analysis of carbohydrate polymers

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2007543656

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 05813353

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP