Journal: PLoS ONE
Article Title: Anti-Lubricin Monoclonal Antibodies Created Using Lubricin-Knockout Mice Immunodetect Lubricin in Several Species and in Patients with Healthy and Diseased Joints
Figure Lengend Snippet: mAb-7h12 can measure lubricin in human plasma, serum, and synovial fluid by competition ELISA. (A) Photograph showing the result of a competition ELISA performed in triplicate. Biotin-labeled mAb-7h12 (0.2 μg/ml) was pre-incubated with purified lubricin (the concentrations of purified lubricin are indicated on top of each column) and added to lubricin-coated wells. After several washes, the mAb bound to the lubricin-coated wells, was detected colorimetrically using horseradish peroxidase (HRP) conjugated to streptavidin. Note when antibody is not pre-incubated with lubricin (0 μg/ml), all antibody binds to the pre-coated wells and the HRP-streptavidin detection of bound antibody turns these wells dark blue. In contrast, pre-incubating the antibody with increasing amounts of purified lubricin reduces antibody binding to the wells and there is less color formation by HRP-streptavidin. (B) Standard curve derived from the O.D. 415 nm values for the competition ELISA shown in panel A . The x-axis indicates the concentration of lubricin (μg/ml) that was pre-incubated with the antibody; the y-axis indicates the average difference in O.D. reading compared to the 0 μg/ml lubricin control. (C) Photograph showing the result of a competition ELISA performed in duplicate (individual rows). Biotin-labeled mAb-7h12 (0.2 μg/ml) was pre-incubated with 1:50 dilutions of human plasma or serum, or 1:50,000 dilutions of human synovial fluid. The plasma samples are from patients with CACP (CA697–1, CA698–1, CA698–2) and their unaffected family members (CA697–2, CA698–4, CA698–5). The serum samples are from healthy controls (OT701–1; OT702–1). The synovial fluid samples are from patients with OA, RA, or CACP (CA). Note that plasma and synovial fluid from patients with CACP do not reduce antibody binding to lubricin-coated wells as indicated by the dark blue color, whereas plasma, serum, or synovial fluid from unaffected individuals does reduce antibody binding to the lubricin-coated wells. Based upon the measured O.D. values for these samples (data not shown), no detectable lubricin is present in serum and synovial fluid from patients with CACP, whereas ~ 200 μg/ml is present in the RA and OA synovial fluid samples and ~ 0.2 μg/ml is present in the plasma and serum samples from unaffected relatives and controls. (D) mAbs 9g3, 5cll, 6a8 and 7h12 can be used interchangeably with recombinant human lubricin in a competition ELISA format.
Article Snippet: Detection of the avidin/biotin/peroxidase-conjugated secondary antibody was performed using DAB FastTabs substrate (Sigma-Aldrich) for 5 minutes, followed by counterstaining with hematoxylin or hematoxylin and eosin.
Techniques: Enzyme-linked Immunosorbent Assay, Labeling, Incubation, Purification, Binding Assay, Derivative Assay, Concentration Assay, Recombinant