purified human lubricin  (Corning Life Sciences)

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

doi: 10.1371/journal.pone.0116237

Figure Lengend Snippet: (A) Five μg of human lubricin purified from synovial fluid (PHL), and 1 μl of synovial fluid recovered from patients with osteoarthritis (OA), CACP, or from bovine, porcine, goat, dog, rat and guinea pig were electrophoresed under reducing conditions on 4–20% SDS-PAGE gradient gels, transferred to PVDF, and immunodetected using the biotin labeled mAbs. Note that the mAbs detect protein in the synovial fluid from patients with OA and from other species, but not from the patient with CACP, who is genetically deficient for lubricin. (B) 0.7 μg of Purified human lubricin (PHL), porcine gastric mucin (PM) and bovine submaxillary mucin (BSM) were electrophoresed under non-reducing conditions on 4–12% SDS-PAGE gradient gels, transferred to nitrocellulose and immunodetected using the mAbs. For mAb-8e3, 1.4 μg of PHL was loaded. Note that anti-lubricin mAbs did not cross react with BSM or PM.

Article Snippet: Individual wells in polystyrene 96-well plates (Corning Incorporated, Corning, NY) were coated using 100 μl of purified human lubricin [ ] in PBS (10 μg/ml) at 4°C overnight.

Techniques: Purification, SDS Page, Labeling

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

doi: 10.1371/journal.pone.0116237

Figure Lengend Snippet: Serum-free conditioned media from 293T cells expressing T-Fc (i.e., KEPAPTTT) recombinant protein or variants of this octapeptide motif were resolved on reducing SDS-PAGE gel and immunodetected with the mAbs. (A) Recombinant T-Fc immunodetected using antibodies indicated in each panel. Removal of O-linked glycans by enzymatic digestion with neuraminidase and O-glycosidase caused loss or attenuated recognition by all mAbs, except anti-HA antibody. Interestingly, all mAbs showed significantly increased immunoreactivity to the recombinant protein treated with neuraminidase alone, suggesting sialic acid modifications that usually occur at sugar chain termini interfere with epitope-antibody interactions. (B) Recombinant HA-KEPAPTTT-Fc (T-Fc) and HA-IgG-Fc (Fc) proteins were treated by neuraminidase and/or O-glycosidase and immunodetected using anti-human IgG-Fc. Note sugar modification on the KEPAPTTT peptide, but not on the Fc fragment, is responsible for changes in polypeptide mobility upon deglycosylation. (C) Recombinant octapeptides fused downstream of HA and upstream of IgG-Fc immunodetected using antibodies indicated in each panel. Anti-HA antibody showed that all mutant recombinant proteins were expressed. Recombinant protein HA-KEPAPTTT-Fc (1) was immunodetected by all monoclonal antibodies. These antibodies did not detect the variant forms of the recombinant protein: HA-KEPAPTAT-Fc (2); HA-KEPAATTT-Fc (3); HA-KEPAPTT-Fc (4); HA-PAPTTT-FC (5). Note that the anti-mouse IgG, used as secondary antibody for mAb-9g3, showed strong cross-reactivity with the human IgG-Fc present in all recombinant proteins. HRP-conjugated Streptavidin was used as a secondary antibody to detect biotin-labeled mAbs 7h12, 5c11 and 6a8, and showed no background by itself (data not shown). (D) Schematic depicting the peptide and O-linked glycosylation motifs that occur commonly in the mucin-like domain of human lubricin. Sialylated and non-sialylated O-linked oligosaccharides can be added to the Threonine (T) residues; two potential oligosaccharides (sialylated and non-sialylated) are indicated and their relative affinities to the mAbs are indicated by the arrow weights.

Article Snippet: Individual wells in polystyrene 96-well plates (Corning Incorporated, Corning, NY) were coated using 100 μl of purified human lubricin [ ] in PBS (10 μg/ml) at 4°C overnight.

Techniques: Expressing, Recombinant, SDS Page, Modification, Mutagenesis, Variant Assay, Labeling

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

doi: 10.1371/journal.pone.0116237

Figure Lengend Snippet: (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: Individual wells in polystyrene 96-well plates (Corning Incorporated, Corning, NY) were coated using 100 μl of purified human lubricin [ ] in PBS (10 μg/ml) at 4°C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Labeling, Incubation, Purification, Binding Assay, Derivative Assay, Concentration Assay, Recombinant

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

doi: 10.1371/journal.pone.0116237

Figure Lengend Snippet: Biotin-labeled mAb-7h12 was mixed with plasma samples from a patient with CACP (CA697–1) or his unaffected father (CA697–2), serum samples from a patient with rheumatoid arthritis (RA) or an age- and sex-matched healthy control (Control), synovial fluid samples from a patient with osteoarthritis (OA), rheumatoid arthritis (RA), or CACP (CA), and with human (hLub-IP) and bovine (bLub-IP) lubricin that had been purified from synovial fluid. Antibody was recovered using streptavidin beads and proteins co-precipitated with mAb-7h12 were eluted, subjected to SDS-PAGE, transferred to PVDF, and immunodetected with mAb-7h12 (upper panel) or polyclonal Ab-J108N (lower panel). Note mAb-7h12 immunoprecipitated lubricin from plasma and serum that is also recognized by polyclonal antibody J108N. In contrast, lubricin immunoprecipitated from OA and RA synovial fluid is not recognized by polyclonal antibody J108N. J108N also does not recognize immunoprecipitated purified human lubricin (hLub-IP). The is no non-specific binding of lubricin to streptavidin beads as demonstrated by the lack of immunodetectable protein when mAb-7h12 is not used in the co-IP (no Ab control) of plasma from the unaffected CACP parent or synovial fluid from a patient with RA. As positive controls for immunodetection purified human and bovine lubricin (hLub and bLub) were directly subjected to SDS-PAGE for immunodetection.

Article Snippet: Individual wells in polystyrene 96-well plates (Corning Incorporated, Corning, NY) were coated using 100 μl of purified human lubricin [ ] in PBS (10 μg/ml) at 4°C overnight.

Techniques: Labeling, Purification, SDS Page, Immunoprecipitation, Binding Assay, Co-Immunoprecipitation Assay, Immunodetection