Quantitation of rat lacrimal secretion: a novel sandwich ELISA with high sensitivity.
Modulation of lacrimal acinar cell tear secretion may involve multiple factors acting both in subtle and pronounced ways. Functional screens of recombinant protein products arising from gene array technologies, or protein fractions derived from lacrimal conditioned media or extracellular matrix, will require a highly sensitive assay capable of monitoring tear protein secretion by small replicate cultures. To improve significantly on current methods, a rat- and mouse-specific sandwich ELISA was developed. For this purpose, chickens and rabbits were immunized with serum-free secretion media from carbachol and VIP-stimulated rat lacrimal acinar cell cultures. Immune sera were characterized by ELISA, Western blotting and immunohistochemistry, and subsequently optimized for use in a sandwich ELISA. Both antisera detected a wide range of different rat tear proteins, and immunostained only the secretory granule-rich juxtalumenal region in sections of rat lacrimal gland. Chicken, but not rabbit, antiserum cross-reacted with rabbit and human tears. In sandwich ELISA, capture with purified chicken immunoglobulin fraction and detection with rabbit antiserum detected as little as 1 ng ml-1 tear protein in 10,000-fold diluted rat secretion medium--a level of sensitivity 8000 times greater than the rat tear peroxidase assay. Such specificity and sensitivity greatly reduce the quantity of media needed for assay, and makes feasible functional screens for scarce factors that may influence lacrimal secretory processes, and in turn possibly play a role in human lacrimal insufficiency syndromes.
Sanghi
S
S
Department of Cell Biology, University of Virginia, Charlottesville 22908, USA.
Kumar
R
R
Walton
S
S
Laurie
G W
GW
D000818:Animals; D015153:Blotting, Western; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005136:Eye Proteins / Q000032:analysis*; D006801:Humans; D007106:Immune Sera; D007150:Immunohistochemistry; D007765:Lacrimal Apparatus / Q000378:metabolism*; D051379:Mice; D011817:Rabbits; D051381:Rats; D012680:Sensitivity and Specificity; D012859:Sjogren's Syndrome; D013666:Tears / Q000378:metabolism*
D005136:Eye Proteins; D007106:Immune Sera