Melan A Specific (MSVA-900M)

The Melan A (melanocyte antigen) protein, also termed “melanoma antigen recognized by T cells 1” (MART-1) is coded by a gene on chromosome 9p24.1. The 18 kDa protein has a single transmembrane domain and consists of 118 amino acids. The function of the protein is unknown. A small fragment of the protein (about nine amino acids) is bound by MHC class I complexes and presented to cytotoxic T cells. The MART-1/melan A antigen is specific for the melanocyte lineage, found in normal skin, the retina, and melanocytes, but not in other normal tissues. Melan A is thus a highly useful marker for benign and malignant melanocytic tumors.

It is of note, that MSVA-900M is fully specific for Melan A. Several other Melan-A antibodies, not only recognize the Melan A protein but – as a result of cross-reactivity – also an unknown structure which is possibly related to corticosteroids. The MSVA antibody MSVA-901M+ (Melan A+) has such properties and can therefore be used as a marker for benign and malignant melanocytic tumors as well as steroid producing tissues.

Using the antibody MSVA-900M (Melan A Specific), a strong staining can be observed in melanocytes in skin and non-keratinizing squamous epithelia from various sites. Staining is absent in all other normal tissues, including the adrenal gland.

These findings are largely comparable to the RNA data summarized in the Human Protein Atlas (Tissue expression Melan A).
It is of note, that the different antibodies developed within the  protein atlas project contain antibodies of the Melan A+ type showing also strong immunostaining of adrenocortical cells as a result of an antibody .

Suggested positive tissue control: Skin: Virtually all melanocytes should show a strong Melan A immunostaining including a weak to moderate staining in melanocytic dendrites.

Suggested negative tissue control: Kidney: Melan A staining must be absent in all cells.

Normal tissue gallery

Melan A is expressed in the vast majority of primary malignant melanomas. Melan A is also expressed in all types of cutaneous naevi and in other tumors of melanocytic differentiation, such as clear cell sarcoma, melanotic neurofibroma, melanotic schwannoma as well as PEComas (perivascular epitheloid cell tumor) including angiomyolipoma, lymphangioleiomyoma(-tosis), and pulmonary sugar tumor. Melan A expression is sometimes reduced and/or only patchy in desmoplastic melanoma and in metastatic melanomas. 

The TCGA findings on Melan A RNA expression in different tumor categories have been summarized in the Human Protein Atlas.

Cancer tissue gallery

No data available at the moment

IHC users have different preferences on how the stains should look like. Some prefer high staining intensity of the target stain and even accept some background. Others favor absolute specificity and lighter target stains. Factors that invariably lead to more intense staining include higher concentration of the antibody and visualization tools, longer incubation time, higher temperature during incubation, higher temperature and longer duration of the heat induced epitope retrieval (slide pretreatment). The impact of the pH during slide pretreatment has variable effects and depends on the antibody and the target protein. Accordingly, multiple different protocols can generate identical staining results.

All images and data shown here and in our image gallery are obtained by the manual protocol described below. Other protocols resulting in equivalent staining are described as well.

Manual protocol

Freshly cut sections should be used (less than 10 days between cutting and staining). Heat-induced antigen retrieval for 5 minutes in an autoclave at 121°C in pH 7,8 Target Retrieval Solution buffer. Apply MSVA-900M at a dilution of 1:150 at 37°C for 60 minutes. Visualization of bound antibody by the EnVision Kit (Dako, Agilent) according to the manufacturer’s directions.

Agilent / Dako – Autostainer Link 48

Pretreatment in PT-Link for 30 minutes at 95°C (pH high); FLEX peroxidase blocking for 5 minutes (room temperature), MSVA-900M 1:150 for 20 minutes (room temperature), FLEX+ mouse/rabbit (LINKER) for 15 minutes (room temperature), horseradish peroxidase (HRP) for 20 minutes (room temperature), FLEX DAB+Sub-Chromo for 10 minutes (room temperature), FLEX hematoxylin for 5 minutes (room temperature).

These images reflect stainings by the protocol described above. It is of note that a comparable staining result can also be obtained by different protocols. In general, a longer pretreatment, a longer incubation time of the primary antibody, a higher antibody concentration, and a longer incubation time of FLEX+LINKER result in stronger staining, potentially at the cost of more background staining. Modifications of the protocol with a strengthening effect on staining intensity in combination with changes of other parameters that result in lower staining intensity can result in a comparable result as shown above.

Leica – BOND RX

Dewax at 72°C for 30 seconds; Pretreatment in Bond Epitope Retrieval Solution (ER2 – EDTA pH9) for 20 minutes at 100°C; Peroxidase blocking for 5 minutes (room temperature), MSVA-900M 1:150 for 15 minutes (room temperature), Post primary (rabbit anti mouse) for 8 minutes (room temperature), Polymer (goat anti rabbit) for 8 minutes (room temperature), mixed DAB refine for 10 minutes (room temperature), hematoxylin for 5 minutes (room temperature).

These images reflect stainings by the protocol described above. It is of note that a comparable staining result can also be obtained by different protocols. In general, a longer pretreatment, a longer incubation time of the primary antibody, a higher antibody concentration, a higher temperature during incubation, and a longer incubation time of Post primary and or the Polymer result in stronger staining, potentially at the cost of more background staining. Modifications of the protocol with a strengthening effect on staining intensity in combination with changes of other parameters that result in lower staining intensity can result in a comparable result as shown above.

Roche – Ventana Discovery ULTRA

Pretreatment for 64 minutes at 100°C (pH 8,4); CM peroxidase blocking for 12 minutes (room temperature), MSVA-900M 1:100 for 20 minutes at 36°C, secondary antibody (anti-mouse HQ) for 12 minutes at 36°C, anti-HQ HRP for 12 minutes at room temperature, DAB at room temperature, hematoxylin II at room temperature for 8 minutes, bluing reagent at room temperature for 4 minutes.

These images depict staining results obtained by the protocol described above. It is of note, that the Ventana machines generally require higher antibody concentrations than other commonly used autostainers because the antibodies are automatically diluted during the procedure. Various other protocols can result in an identical result as shown above. A longer pretreatment, a longer incubation time of the primary antibody, a higher antibody concentration, a higher temperature during incubation, and a longer incubation time of secondary antibody and or the anti-HQ HRP result in stronger staining, potentially at the cost of more background staining.

Potential pitfalls

Several Melan A antibodies (but not MSVA-900M) share a cross reactivity for structure related to steroid hormone producing cells. Therefore, some users of “Melan A antibodies” use these for diagnosing adrenal cortical, sex-cord and pituitary tumors. This antibody is specific for melanocytes and does not stain steroid producing cells. Our antibody MSVA-901M+ (Melan A CR) has this cross-reactivity and stains steroid hormone producing cells.

• The exact function of Melan A is unknown.
• The utility of Melan A as a cancer vaccine target is under investigation.

Utility of MSVA-900M Melan A is documented by strong positive staining in cell types that are well documented to react with Melan A such as melanocytes and absence of staining in all tissues known to not express Melan A such as adrenal cortex as well as tissues notorious for non-specific IHC background such as kidney, colonic mucosa, and epidermis. 

Normal tissue gallery