br Abbreviations Apo A Apolipoprotein A Apo A Apolipoprotein
Abbreviations: Apo-A1, Apolipoprotein A-1; Apo-A2, Apolipoprotein A2; HDL, high density lipoprotein; AUC, Area under the curve; EDTA, ethylenediaminete-traacetic acid; r, correlation coeﬃcient; RIPA, recombinant immunoblot assay; ROC curves, Receiver operating characteristics curves; TBS, tris-buﬀered saline; μg, micro gram; μl, microliter; μM, micromolar; mM, millimolar; ng, nano gram; OR, odds ratio; p, p. value; PVDF, polyvinylidenedifluoride; SD, standard deviation; SPSS, statistical package for the social sciences
E-mail address: [email protected] (D.E.-S. Ellakwa).
20% of the total HDL-C protein content. Additionally, Apo-A2 has been related to various types of cancer in diﬀerent clinical studies (Bandarian et al., 2016). In the present study, blood and urine levels of Apo-A1 and Apo-A2, as were assessed by western blot analysis to measure expres-sion levels of the proteins and genes. The expression of Apo-A1 and Apo-A2 levels with tumor stage and grade was also assessed.
2. Subjects and methods
2.1. Study population
Subjects of the present study were selected from Kasr El-Ainy Hospital, Urology Department Cairo University, Egypt and diagnosed as 701977-08-4 cancer by histopathological examination of biopsy cystoscopy samples. Laboratory work was conducted in Unit of Biochemistry and Molecular Biology, Faculty of Medicine, Cairo University. The present study was directed as stated by those professional ethics of declaration of Helsinki. Every one of subjects of the present study has signed written informed consents which were aﬃrmed by the Ethics Committee of Cairo University.
Groups of the study: Group I: Involved 50 patients with urinary bladder carcinoma diagnosed by pathological examination of cysto-scopy biopsy tissue samples. Group II: Involved 50 patients with cystitis (benign condition). Group III: Involved 50 healthy normal subjects. Inclusion criteria include: age 48–65 years, bladder biopsy showing histological evidence of bladder cancer, patients not receiving any medications, surgery or radiological interventions, no associated chronic diseases or their complications or any other type of tumors. Exclusion criteria include: The presences of any chronic systemic illness were excluded from this study such as patients with chronic kidney, liver, cardiac diseases, hypertension, diabetes mellitus, autoimmune disease, pregnant females, severe obesity or patients receiving any medications.
2.2. Sample collection and processing
Whole blood samples on ethylenediamine tetraacetate (EDTA) were collected and divided into two parts; 3 mL for separation of the mononuclear cell layer by Ficoll Paque (Munich, Germany) and 3 mL for serum separation. Blood and serum samples were stored at −80 °C till the time of laboratory assays.
2. Voided morning urine samples (50–100 ml) were collected from all patient groups before they receive any medications. Urine samples were collected before cystoscopy, surgery or any radiological inter-ventions. Samples were centrifuged at 4000g for 20 min. Urine super-natant was stored at −80 °C until used for protein assay.
2.3. Western blot technique was conducted for assessment of Apo-A1 and Apo-A2 protein levels usingV3 Western Workflow™ Complete System, Bio-Rad® Hercules, CA, USA.
Briefly, urine samples and blood samples containing 5 mg proteins were homogenized in recombinant immunoblot assay (RIPA) buﬀer, then centrifugation at 12,000 rpm for 20 min. The protein concentra-tion for each homogenized sample was determined using Bradford assay. Equal amounts of protein (20–30 μg of total protein) were se-parated by SDS-PAGE and then transferred to a polyvinylidenedi fluoride (PVDF) membrane. The membrane was blocked in tris-buﬀered saline (TBS) buﬀer containing 5% skim milk and 0.1% Tween 20 at room temperature for 1 h and incubated withApo-A1 and Apo-A2 pri-mary antibodies supplied by Novex overnight at pH 7.6 at 4 °C with gentle shaking. After washing, peroxidase-labeled secondary antibodies were added, and the membranes were incubated at 37 °C for 1 h. Band intensity was analyzed by ChemiDocTM imaging system with Image LabTM software version 5.1 (Bio-Rad Laboratories Inc., Hercules, CA, USA). Total proteins normalization was used to normalize each target protein levels. Apo-A1 monoclonal antibody was obtained from Novex, Catalogue number 701239. Apo- Apo-A2 monoclonal antibody was Gene Reports 16 (2019) 100463
obtained from Novex Catalogue number 701236. Dilution of Apo-A1 antibodies used is 1: 3000, dilution ofApo-A2is 1: 3000, and final concentration is 2 μg/mL for all antibodies. Rat anti-mouse secondary antibody Horse Radish Peroxidase conjugate was obtained from ThermoFisher Scientific (USA, Rockford) Catalogue number 18-4015-
82. Detection of proteins blots in PVDF membranes was conducted by
Chemiluminescent Detection (Immun-Star™ WesternC™ Chemiluminescence Kit, Catalogue No. 1705061, BIO-RAD, USA). Protein standards for western Blot were obtained from Bio-Rad Precision plus western C protein standards Catalogue No. 161-0385. http://www.biorad.com/en-eg/applications-technologies/detection-methods#3Chemiluminescence detection was conducted using ChemiDocMP imaging system with Image Lab software version 5.1 (Bio-Rad, USA).