title: A solid-phase assay for the quantitation of total protein eluted from balafilcon, lotrafilcon, and etafilcon contact lenses.
abstract: OBJECTIVE
  To compare two variations of a membrane-based protein assay utilizing Amido black (AB) detection with a commercially available 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA) assay for use in the quantitation of individual tear proteins, pooled human tear proteins, and protein extracted from ex vivo lotrafilcon A, balafilcon A, and etafilcon A contact lens materials.
  
  
  METHODS
  Ex vivo contact lens extracts, pooled human tears, and individual tear proteins (human serum albumin (HSA), bovine lactoferrin, human secretory immunoglobulin A (sIgA), human lysozyme) were subjected to three solid-phase assays: AB on polyvinylidene difluoride (AB on PVDF) and AB on nitrocellulose (AB on NC) and the CBQCA assay. Micro-bicinchonic acid (micro-BCA) assay was also employed with lens extracts to determine total protein concentration. Individual and pooled tear proteins were referenced to a micro version of the quantitative ninhydrin protein assay.
  
  
  RESULTS
  The CBQCA demonstrated the greatest overall sensitivity and lowest intra- and inter-assay variability. AB on NC demonstrated the most accurate ability to quantify total protein in pooled human tear samples, although it also displayed the greatest protein-to-protein variation using individual tear proteins. The CBQCA assay displayed the greatest cross-reactivity with unworn balafilcon and lotrafilcon lens extracts, whereas AB on NC demonstrated the least. AB on NC measured similar amounts of total protein in extracted ex vivo lenses as the CBQCA assay if background interference was subtracted from CBQCA values. AB on PVDF measured the lowest amount of deposited protein from ex vivo lenses.
  
  
  CONCLUSIONS
  Both the AB on NC and CBQCA assays can be used to measure protein in extracts of lotrafilcon, balafilcon, and etafilcon lens materials.
authors.affiliation: Centre for Contact Lens Research, School of Optometry, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: Mary-Ann
authors.initials: MA
authors.lastname: Glasier
authors.forename: Lakshman N
authors.initials: LN
authors.lastname: Subbaraman
authors.forename: Michelle
authors.initials: M
authors.lastname: Senchyna
authors.forename: Lyndon
authors.initials: L
authors.lastname: Jones
mesh_terms: D000580:Amido Black; D001565:Benzoates; D003263:Contact Lenses, Hydrophilic; D003429:Cross Reactions; D003720:Densitometry; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000032:analysis*; D006801:Humans; D020100:Hydrogels; D008689:Methacrylates; D011485:Protein Binding; D011804:Quinolines; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012828:Silicones; D013050:Spectrometry, Fluorescence; D005466:Fluorocarbons
chemical_list: D000580:Amido Black; D001565:Benzoates; D005136:Eye Proteins; D020100:Hydrogels; D008689:Methacrylates; D011804:Quinolines; D012828:Silicones; C523195:balafilcon A; C114176:etafilcon; C481129:lotrafilcon A; D005466:Fluorocarbons; C042434:tear proteins; C069617:3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde