Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: Serum proteins separated by two dimensional gel electrophoresis in early and advanced NAFLD, with and without HCC . These are representative blots comparing immune depleted and desalted serum from a patient with NAFLD and no fibrosis, a patient with NAFLD cirrhosis, and a patient with NAFLD cirrhosis and cancer. Serum profiles separated by 2D gel electrophoresis from 5 patients in each group, run in duplicate, were compared using Progenesis software. 5 spots were characterised by MS after excision. Spots 1,2 and 3 were isoforms of apolipoprotein A1, spot 4 was apolipoprotein A4, and spot 5 was identified as CD5 antigen like, or CD5L.

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Software

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: CD5L discriminates poorly between cirrhotic patients with and without HCC . Serum levels of CD5L were measured by ELISA assay in cirrhotic patients with (218 ± 221; n = 45) and without (194 ± 166; n = 49) HCC, as shown in 2A. Characteristics of the individuals are shown in Table 1. There was no significant difference between the two groups. ROC analyses is shown in 2B. The area under the curve is 0.495 (95% confidence intervals 0.376 and 0.614). A level > 400 ng/ml has a sensitivity of only 20%, but a specificity of 88%. Levels > 500 ng/ml have a specificity of 96%, > 600 ng/ml of 98% and > 700 ng/ml of 100%. For comparison, the mean CD5L serum level from patients without cirrhosis (detailed in figure 3) is also shown.

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: CD5L discriminates between steatohepatitis patients with and without cirrhosis . Serum levels of CD5L were measured in a total of 113 patients with either ALD or NAFLD. Fibrosis was scored histologically on liver biopsy as per the Brunt scoring system. The numbers of individuals in each group are shown within the boxes of chart 3A. The difference in CD5L levels between the different stages of fibrosis is statistically significant by univariate analysis (p = 0.004) controlled for both age and sex. This difference The ROC analyses for the identification of those individuals with stage 4 fibrosis (cirrhosis) is depicted in 3B. The area under the curve is 0.719 (95% confidence intervals 0.623 and 0.816), p < 0.0001. A level of CD5L 50 ng/ml has a sensitivity of 78% and a specificity of 46%, while a level of 100 ng/ml has a sensitivity of 63% and a specificity of 72%. A level greater than 200 ng/ml has a specificity of 95% (sensitivity 41%), and greater than 300 ng/ml of 97% (sensitivity 27%).

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques:

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: CD5L mRNA expression is not altered in association with either fat, inflammation or fibrosis scores in pre-cirrhotic NAFLD liver tissues . mRNA CD5L expression was quantified by real-time PCR, relative to GAPDH and a normal liver sample, in 21 pre-cirrhotic NAFLD biopsy tissues, 13 normal liver samples taken at the time of liver resection, one cirrhotic liver and one HCC. As shown in 4A-C, there was no difference in any pre-cirrhotic NAFLD biopsy tissues in association with the degree of fat, inflammation or fibrosis. There was a significant increase in the NAFLD tissues as a group (n = 21), compared with normal liver tissues (n = 13) as represented in 4D (6.945 ± 0.722 versus1.68 ± 0.269; p = 0.000, ***). The elevated CD5L mRNA expression in one cirrhotic and HCC tissue pair, obtained at the time of laparoscopic radiofrequency ablation, is also presented in 4C and is in keeping with the elevated serum CD5L levels identified in the larger cohort of patients studied.

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: BMC Cancer

Article Title: A proteomic strategy to identify novel serum biomarkers for liver cirrhosis and hepatocellular cancer in individuals with fatty liver disease

doi: 10.1186/1471-2407-9-271

Figure Lengend Snippet: The identification of the differentiating protein spots on 2D gels.

Article Snippet: A direct ELISA for novel candidate CD5L was optimised using different concentrations of serum (raw, 1;10, 1:100, 1:500 and 1:1000), primary anti-CD5L antibody (R&D systems) and recombinant CD5L protein (R&D systems).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Bimolecule detection for Extracellular Vesicle Screening

doi: 10.1101/2020.07.23.217018

Figure Lengend Snippet: ( A ) The EMARS products for serum EVs from EML4-ALK transgenic mouse. To average experimental results over each group, an aliquot of the serum (10 μL each) from 10 animals in each group (WT), large lung tumor-bearing (TL), and small lung tumor-bearing mice (TS) was mixed in equal proportions, and then applied to EV purification and EMARS. The EMARS products were concentrated and purified by immunoprecipitation with the anti-fluorescein antibody Sepharose. The resulting samples were subjected to SDS-PAGE analysis with fluorescence detection. “IP” indicates the immunoprecipitated samples, and “Lys” indicates the lysate samples before immunoprecipitation. The right panel indicates the same gel as the left panel, but exposed for a longer time. ( B ) Confirmation of candidate partner molecules (CD5L and PZP) with mouse CHL1 in EVs. The EMARS products of WT, TL, and TS were respectively applied to immunoprecipitation (anti-fluorescence antibody Sepharose) and western blot analysis with anti-CD5L (left panel) antibody. After the stripping as described in the “ Materials and methods ”, the membranes were re-stained with anti-PZP antibody (right panel). Arrows indicate the detected band of CD5L and PZP proteins (including predicted dimer). Asterisk indicates unknown bands (predicted as non-specific or partial fragments). ( C, D ) Confirmation of candidate partner molecules (SLC4A1 and THBS1) with mouse CHL1 in EVs. The western blot analysis was performed with anti-SLC4A1 antibody ( C ). After stripping, the membranes were re-stained with anti-THBS1 antibody ( D ). Arrows indicate the detected band of SLC4A1 and THBS1 proteins (including predicted dimers). Asterisks indicate unknown bands (predicted as non-specific or partial fragments). ( E ) Expression of SLC4A1 (left column) and CHL1 proteins (right column) in tumor tissues from two male and two female EML4-ALK transgenic mice. The fragments of lung cancer tissues were mashed and washed gently with PBS, and then lysed with SDS-PAGE sample buffer directly. The resulting samples were subjected to Western blot analysis with anti-SLC4A1 antibody and anti-CHL1 antibody. Arrows indicate the detected band of monomer SLC4A1 and CHL1 proteins.

Article Snippet: After blocking with 5% skim milk solution, transferred membrane was reacted with first antibodies as follows: anti-mouse and human CHL1 antibody (AF2147 and MAB2126; R&D systems, MN; 1 μg/ml), HRP-conjugated anti-mouse and human CHL1 antibody (described above), anti-α2 integrin antibody (ab133557; Abcam; 1 μg/ml), anti-β1 integrin antibody (610467; BD transduction laboratories, NJ; 0.25 μg/ml), anti-FGFR3 antibody (c-15; Santa cruz, TX; 0.2 μg/ml 5% BSA-PBS), anti-TSG101 antibody (EXOAB-TSG101-1; System Biosciences, CA; 1:500), anti-CD63 antibody (EXOAB-CD63A-1; System Biosciences, CA; 1:500), anti-CD5L antibody (AF2834; R&D systems, MN; 0.4 μg/ml), anti-Pregnancy Zone Protein (PZP) antibody (PAG324Ra01; CLOUD-CLONE, TX; 0.5 μg/ml), anti-SLC4A1 antibody (18566-1-AP; PROTEINTECH, IL; 0.6 μg/ml), anti-Thrombospondin 1 (THBS1) antibody (PAA611Hu01; CLOUD-CLONE, TX; 0.5 μg/ml), and anti-caspase 14 antibody (MAB8215; R&D systems, MN; 0.5 μg/ml).

Techniques: Transgenic Assay, Purification, Immunoprecipitation, SDS Page, Fluorescence, Western Blot, Stripping Membranes, Staining, Expressing

Journal: Thyroid : official journal of the American Thyroid Association

Article Title: CD5L Constitutes a Novel Biomarker for Integrated Hepatic Thyroid Hormone Action.

doi: 10.1089/thy.2019.0635

Figure Lengend Snippet: Figure 4 Relative CD5L abundance in serum of patients with acute hypo- or

Article Snippet: CD5L was detected by rabbit antibodies against the N-terminus of human CD5L (AHP1169, Bio-Rad Laboratories GmbH, Feldkirchen, Germany) in combination with goat anti-rabbit-immunoglobulin-HRP antibody (P0448, Agilent Dako, Santa Clara, United States).

Techniques: