Multiplex-bead complex for determination of lysosomal storage disorders
Multiplexing bead technology is used for simultaneous screening of multiple LSD 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-1, 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.
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 are: saposin (SeqID No. 1), LAMP-1 (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; 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-1 (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 contain a specific ratio of fluorophores and are spectrally distinct from each other; and
wherein β-galactosidase or galactocerebrosidase, heparan-N-sulphatase, α-N-acetylglucosaminidase, and galactose-6-sulphatase are not present.
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-1 (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);
a tenth purified detection antibody conjugated to a fluorescent detection label and that selectively binds to an epitope of 3-sulphatase (SeqID No. 10) and
wherein β-galactosidase or galactocerebrosidase, heparan-N-sulphatase, α-N-acetylglucosaminidase, and galactose-6-sulphatase are not present.