Fluorochrome-conjugated MoAbs for Alport's syndrome

FITC-Anti Collagen IV α5(IV) Chain, Human (Mono) + Texas Red-Anti Collagen IV α2(IV) Chain, Human (Mono)

Reference: Kagawa et al. (1997) Epitope-defined monoclonal antibodies against type-IV collagen for diagnosis of Alport's syndrome. Nephrol. Dial. Transplant. 12 : 1238-1241

This product can be used for staining of human renal and skin biopsy sections very easily and rapidly because two monoclonal antibodies are conjugated with two different fluorochromes. FITC-conjugated-anti α5(IV) demonstrates presence or absence of the α5(IV) chain, and Texas-Red-anti α2(IV) reveals the renal basement membrane structure.

Alport's syndrome, an inherited disease, shows absence or reduction of the α5(IV) chain in the glomerular, tubular and Bowman's capsular basement membranes. Normal human kidney has the α1(IV) to α6(IV) chains in the renal basement membranes.

●Double exposure of normal human kidney section stained with the product.

FITC fluorescence (α5 chain) is observed in the GBM, part of the TBM and Bowman's capsular BM. BMs where two fluorochromes are present look orange to yellow.

●Double exposure of Alport human kidney section stained with the product.

Because no FITC fluorescence (α5 chain) is observed in the kidney of a patient with X-linked Alport's syndrome, only Texas Red fluorescence (α2 chain) is confirmed.

● CFT-45325 is prepared to stain human cryostat sections of biopsy specimens from kidney and skin by direct immunofluorescence.

Wash cryostat sections with phosphate buffered saline (PBS), wipe PBS around them with a paper wiper, and incubate them with 20-50 micro liter of the staining solution for 30-60 min at room temperature. After washing them with PBS, mount them and observe them with a fluorescence microscope.

Do not dilute the staining solution.
Do not use stale cryostat sections.
Do not fix sections with any fixatives.

● In case that a section is not stained or very poorly stained with FITC-anti-α5(IV) but the background staining of Texas Red-anti-α2(IV) is normally stained

1) The disease is diagnosed as Alport's syndrome. Or,

2) There is still a possibility that a yet-unknown amino acid substitution is present in the region of the epitope sequence.

For accurate diagnosis, it is important to consider clinical symptoms, and to make additional staining with other monoclonal antibodies against type IV collagen.

1) Kagawa M, Kishiro Y, Naito I, Nemoto T, Nakanishi H, Ninomiya Y, Sado Y (1997) Epitope-defined monoclonal antibodies against type-IV collagen for diagnosis of Alport's syndrome. Nephrol. Dial. Transplant. 12: 1238-1241.

2) Yoshioka K, Hino S, Takemura T, Maki S, Wieslander J, Taketoshi Y, Makino H, Kagawa M, Sado y, Kashtan CE (1994) Type IV collagen α5 chain: Normal distribution and abnormalities in X-linked Alport syndrome revealed by monoclonal antibody. Am. J. Pathol. 144: 986-996.

3) Ninomiya Y, Kagawa M, Iyama K, Naito I, Kishiro Y, Seyer JM, Sugimoto M, Oohashi T, Sado Y (1995) Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies. J. Cell Biol. 130: 1219-1229.

4) Naito I, Kawai S, Nomura S, Sado Y, Osawa G, Japanese Alport Network (1996) Relationship between COL4A5 gene mutation and distribution of type IV collagen in male X-linked Alport syndrome. Kidney Int. 50: 304-311.