Anterior Gradient 2 / AGR2 (HMV325)

Anterior gradient protein 2 homolog (AGR-2), also known as secreted cement gland protein XAG-2 homolog, is a 20 kDa protein disulfide isomerase (PDI) which is coded by the AGR2 gene on chromosome 7p21. AGR2 is a resident endoplasmic reticulum protein which plays an important role in oxidative protein folding in the endoplasmic reticulum. It is expressed in many different tissues/cell types and exerts functions that are relevant for embryonal development, mucus maturation, tissue regeneration, and wound healing. AGR2 is critical for maintaining epithelial barrier function in the intestine. AGR2 knockout mice show a loss of intestinal mucus and develop ileitis and colitis. AGR2 upregulation (and downregulation) has been associated with tumor initiation, progression, and metastasis in several cancer types.

Images describing the AGR2 staining pattern in normal tissues obtained by the antibody HMV325 are shown in our “Normal Tissue Gallery”.

A strong, predominantly cytoplasmic AGR2 staining occurs in a broad range of different epithelial cell types. These findings are largely consistent with the RNA data described in the Human Protein Atlas (Tissue expression AGR2). 

Positive control = Colon: A strong predominantly cytoplasmic AGR2 staining should be seen in all epithelial cells.

Negative control = Colon: AGR2 staining should be absent in all non-epithelial cell types.

Normal tissue gallery

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.

All images and data shown here and in our image galleries 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 HMV325 at a dilution of 1:200 at 37°C for 60 minutes. Visualization of bound antibody by the EnVision Kit (Dako, Agilent) according to the manufacturer’s directions.

• The diagnostic, prognostic, and predictive role of AGR2 expression in tumors and in preneoplastic disease needs to be clarified.

There are two ways how the specificity of antibodies can be documented for immunohistochemistry on formalin fixed tissues. These are: 1. Comparison with a second independent method for target expression measurement across a large number of different tissue types (orthogonal strategy), and 2. Comparison with one or several independent antibodies for the same target and showing that all positive staining results are also seen with other antibodies for the same target (independent antibody strategy). 

Orthogonal validation: For the antibody HMV325 specificity for detection of AGR2 is suggested by the strong concordance of the immunostaining data with data from three independent RNA screening studies, including the Human Protein Atlas (HPA) RNA-seq tissue dataset, the FANTOM5 project, and the Genotype-Tissue Expression (GTEx) project, which are all summarized in the Human Protein Atlas (Tissue expression AGR2). AGR2 positivity by HMV325 is detectable in all tissues with documented AGR2 RNA expression (Salivary glands, stomach, duodenum small intestine, appendix, colorectum, gallbladder, urinary bladder, lung, pituitary gland, epididymis, prostate, seminal vesicle, breast, cervix, fallopian tube). However, an AGR2 positivity by HMV325 also occurs in several cell types without documented RNA expression. These include: squamous epithelial cells of the tonsil, hair follicles, and of corpuscles of Hassall’s of the thymus, endometrium, a subset of tubuli and collecting ducts of the kidney, intercalated ducts and – in some cases – acinar cells of the pancreas

Comparison of antibodies: True expression of AGR2 in all cell types for which AGR2 positivity was seen by HMV325 is corroborated by confirmation of all AGR2 stainings obtained by HMV325 by another independent commercially available antibody (termed “validation antibody”). Confirmed AGR2 positive cell types also included squamous epithelial cells of the tonsil, hair follicles, and of corpuscles of Hassall’s of the thymus, endometrium, a subset of tubuli and collecting ducts of the kidney as well as intercalated ducts and acinar cells of the pancreas.

Normal tissue gallery