ED-ALP105

Intended use
Immunohistochemical staining kit for help in the diagnosis of Alport Syndrome on kidney and skin biopsies.
Patients with Alport syndrome have mutations in the gene for the alfa 5 chain of type IV collagen. This results in a lack of the alfa 5 (IV) chain from glomerular basement membrane (GBM) and the skin basement membrane as well as a lack of the alfa 3 (IV) chain from GBM.
For research and in-vitro diagnostic use only.

Interpretation of staining
For proper interpretation a negative (without primary antibody) and a positive control (MAB1) should be run alongside specimens. The positive control serves to indicate that specimen processing and staining were carried out correctly and should show intense staining along basement membranes, in the glomerulus mainly to the mesangial matrix and the subendothelial aspects of the glomerular basement membrane (GBM). The negative control serves to assess non-specific staining which should be taken into consideration when interpreting results.
MAB 3 and MAB 5 normally stains the entire thickness of the glomerular basement membrane as well as the distal tubular BM. In Alport families with X-linked disease the MAB 3 and MAB 5 staining are absent in males and have a discontinuous distribution in females. The findings can be seen in 60-70% of the families. In autosomal recessive patients staining of MAB5 is seen on Bowmans capsule and collecting ducts BM while MAB3 is negative.
MAB3 is not normally found in the skin while MAB 5 stains the epidermal basement membrane. This staining is usually lacking in X-linked male patients while females exhibit a segmental distribution of the antigen. In autosomal recessive patients staining of MAB 5 is normal on the epidermal basement membrane.

Principle of the Wieslab® Alport syndrome assay
Staining of frozen sections by monoclonal antibodies:
- 3 µm thick cryostat sections are cut, air dried, fixed in acetone.
- The section to be stained with MAB 5 (anti-alfa 5 chain antibody) is now denatured by glycine/urea solution.
- Add the monoclonal antibodies MAB 1, MAB 3 and MAB 5.
- Incubate with FITC-labelled secondary anti-mouse antibodies.
- Add mounting media containing p-phenylene diamine to delay fluorescence quenching.

An alternative method, based on DAKO LSAB kit K0680 can also be used.
In this method formalin-fixed tissue can be used

Kit components and storage of reagents
Monoclonal antibody to alfa 1 chain (MAB 1)
Monoclonal antibody to alfa 3 chain (MAB 3)
Monoclonal antibody to alfa 5 chain (MAB 5)
Glycine/urea solution (Store in a freezer, since urea slowly decomposes).
Store the monoclonal antibodies at 2-6° C.