And kidney function was measured in serum, plasma, cerebrospinal fluid, or urine [21-26]. The aim of the present study was to establish a new Double-Antibody-Sandwich Enzyme-Linked immunosorbent assay(DAS-ELISA)-based measurement of HCC with the self-made monoclonal antibody and VHHs by applying the hybridoma technology and phage VHH display technology, to develop a?2014 Jiang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Jiang et al. Journal of Translational Medicine 2014, 12:205 http://www.translational-medicine.com/content/12/1/Page 2 ofHCC ELISA Test Kit with the highest sensitivity, low cost, and easy operation.MethodsReagents and instrumentsNucleic acid gel imaging system, nucleic acid electrophoresis apparatus and protein gel electrophoresis apparatus were purchased from Shanghai Tanon company. Microplate reader was purchased from Thermo Fisher Technology Ltd (Shanghai, China). Polyethylene glycol (PEG) was purchased from Merck Co, Mouse typerisotyping panel kit from Bio-RAD Co, and RPMI MEDIEM 1640 medium, penicillin-Streptomycin double antibody solution, newborn calf serum and HEPES from Life Technologies Gibco Co. Hypoxanthine-Aminopterin-Thymidine (HAT) supplemented medium, Hypoxanthine- Thymidine Boc-D-Lys-OH (HT) supplemented medium, Freund’s complete or incomplete adjutants were purchased from Sigma. Natural human cystatin C (N-HCC) was purchased from Enzo Life Sciences Ltd., Horseradish peroxidase-conjugated goat anti-mouse IgG from Santa Cruz Biotechnology Inc., or Tween-20 and Bovine serum albumin (BSA) from Amresco. BALB/c mice were from Shanghai Institutes for Biological Nutrition, according to the ethical permission approved by the committee of Animal Ethical Evaluation, Chinese Academy of Science. The natural camel single-domain heavy chain antibody library was kindly provided by Dr. Ario de Marco for Italian IFOMIEO center.Preparation of recombinant HCCThe total RNA was extracted from renal epithelial 293 (R)-1-(3-Chlorophenyl)ethan-1-ol T cells using the TransZol Up RNA kit. The cDNA was synthesized from RNA using the Superscript II reverse transcriptase with OligodT (18) primers, as the template for the PCR reaction. The primers specific for HCC were used to introduce the restriction sites BamH I and Xho I (The primers: 5′-GGATCCAGTCCCGGCAAGCCG-3′ and 5′-CCTCGAGCTAGGCGTCCTGACAGGT-3‘). PCR products (363 bp) corresponding to HCC fragments and then connected to pEASY-T1 simple T vectors [27-29]. The cloning T vectors which contain purpose gene and the prokaryotic expression vector pET-32a was digested with BamH I and Xho I twice and dephosphorylated and gel purified before the ligation incubation. The ligation was performed overnight at 16 by T4 DNA ligase. The recombinant plasmids were transformed into Rosetta and the transformants were selected on PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14445666 Luria-Bertani LB agar plates supplemented with PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/8833965 100 g/ml ampicillin. Single bacterial colony was picked from the transformned plate and verified by PCR, and the positive bacteria were induced to express the target protein. The positive single colonies inoculated (1:100) into.
