Development and characterisation of a monoclonal antibody family against aquaporin 1 (AQP1) and aquaporin 4 (AQP4)

Abstract

Recent studies have discovered the existence of water-channel molecules, the so called aquaporins (AQP) presumably involved in active, ATP dependent water transport between the intracellular and extracellular compartments. Both genetic and protein sequences and structures of the AQPs are known and crystallographic analyses of some members of the AQP family have been performed. Specific antibodies are required to examine their histological locations and analyse their roles in physiological and pathological pathways of water transportation and osmotic regulation. Until recently some polyclonal antibodies have been developed against certain members of the AQP family. However, to date highly specific monoclonal antibodies against aquaporins do not exist. We have developed a monoclonal antibody family against the aquaporin 1 (AQP1) and aquaporin 4 (AQP4) molecules. Well-conserved epitop sequences of AQP1 and AQP4 proteins were selected by computer analysis and their synthetic peptide fragments were used as the antigens of immunisation and the following screening. Antibodies were characterised by immunoserological methods (ELISA, dot-blot and immunoblot), flow cytometry and immunohistochemistry of formaldehyde-fixed and paraffin-embedded tissue samples. RT-PCR tests controlled the specificity of the immune reactions. Our monoclonal antibodies recognised AQP1 and AQP4 in all the techniques mentioned above and apparently they are useful both in various quantitative and qualitative measurements of the expressions of AQP1 and AQP4 in several species (human, rat, mouse, invertebrates, even plants). According to preliminary immunohistochemical studies our monoclonal anti-AQP1 and anti-AQP4 antibodies are appropriate tools of patho-morphological examinations on routine formol-paraffin tissue samples.

Aquaporin-1 in blood vessels of rat circumventricular organs

“…Outside the rat brain, AQP1 is widely expressed in the plasma membranes of continuous capillary endothelia and of non-vascular endothelia throughout the body: in cardiac, skeletal and smooth muscle capillary beds, in mammary glands, in bronchial, tracheal and nasopharyngeal capillary beds and in descending vasa recta in the kidney (Nielsen et al 1993(Nielsen et al , 1995Verkman 2006). In contrast to continuous capillary endothelia, AQP1 appears to be absent from the plasma membranes of fenestrated capillary endothelia throughout the rat, viz., in the small intestine and colon and in the fenestrated ascending vasa recta and glomerular and peritubular capillaries of the kidney (Nielsen et al 1993(Nielsen et al , 1995Gannon and Carati 2003), although Nagy et al (2002) report the presence of AQP1 in rat glomerular capillaries, which conflicts with the findings of Nielsen et al (1993Nielsen et al ( , 1995.…”

“…Most, if not all, microvessel endothelia in these areas are AQP1-immunoreactive. With the exception of the contradictory findings in renal glomeruli (Nielsen et al 1993(Nielsen et al , 1995Nagy et al 2002), these capillaries are so far the only fenestrated capillaries in the rat to be definitively shown to be AQP1-positive; all other fenestrated capillaries in the rat outside the brain appear to be AQP1-negative, although many continuous capillaries outside the brain are AQP1-positive (Nielsen et al 1993).…”

“…The mouse monoclonal anti-AQP1 antibody (Abcam) was prepared against a synthetic peptide (GQVEEYDLDD DINSRVEMKPK) corresponding, except for one amino acid, to the 21 C-terminal amino acids at positions 249-269 from rat AQP1 and was thoroughly characterised by Nagy et al (2002) by using enzyme-linked immunosorbent assay, dot-blot, Western blot, immunohistochemistry and reverse transcription with the polymerase chain reaction (RT-PCR).…”

Characterization of changes in global gene expression in the brain of neuron-specific enolase/human Tau23 transgenic mice in response to overexpression of Tau protein

“…Aquaporins (AQPs) are a family of transmembrane proteins that act as water selective channels (26). In the brain, AQP1 is found in the apical pole of the choroidal plexus of epithelial cells and ependimocytes (27,28). A possible relationship between the AQP increase and spongiform degeneration has been suggested by AQP1 increase in Creuzfelt-Jakob disease (CJD) patients and in Bovine spongiform encephalopathy (BSE) found in infected mice (29).…”

Aquaporin–graphene interface: relevance to point-of-care device for renal cell carcinoma and desalination

“…[25] would be bound through a special linker molecules to the surface of a single layer graphene embedded in a microfluidic channel [26]. A scheme of single layer graphene attached to a peptide epitope of AQP1 and interacting with AQP1 is shown in Fig.…”