Purification of proteins which is recognized by monoclonal antibodies produced from human osteosarcoma cells (MG-63) for peptide sequencing by mass spectrometry

• Grant number: 15/23096-9
• Support Opportunities: Scholarships abroad - Research
• Start date: February 01, 2016
• End date: March 01, 2016
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Principal Investigator: Luciene Machado dos Reis
• Grantee: Luciene Machado dos Reis
• Host Investigator: Renato Costa Monteiro Filho
• Host Institution: Hospital das Clínicas da Faculdade de Medicina da USP (HCFMUSP). Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Institution abroad: Institut National de la Santé et de la Recherche Médicale (Inserm), France

Abstract

We produced monoclonal antibodies using cell membrane proteins from MG-63 cells (human osteosarcoma). After several steps of analysis, we obtained 19 monoclonal antibodies. These were tested in cells from bone explant culture and normal hematopoietic cells, in order to verify their specificity and cross-reactivity with hematopoietic cells. Some of the clones were sub-cloned and since then, we are working with the sub-clones PSP 4-5 (recognizes an antigen of 100 kDa), PSP 42-22 (recognizes an antigen of 26 kDa) and PSP 85-9 (recognizes an antigen of 20 kDa). These antibodies were tested by immunohistochemistry in normal human tissue (bone, heart muscle, adipose tissue, cartilage and skin fibroblasts) and were not specific for any tissue / cells, i.e., probably recognize common proteins in tissues which are originate from mesenchymal cells. We tested these antibodies in explant culture cells from normal bone tissue and metabolic bone diseases (osteoporosis, adynamic bone disease, fibrous osteitis), showing some interesting differences in their pattern of expression according to the condition. Then, we studied the expression of these antibodies in bone tumors (osteosarcomas, osteoblastomas, chondrosarcoma and leiomyosarcoma) and detected significant differences between osteosarcoma and osteoblastomas. Last year, we isolated and sequenced the antigen recognized by the PSP 42-22 antibody (Serologically defined colon cancer antigen 3 - SDCCAG3, ranging from 40 to 47 kDa and four distinct isoforms) and the PSP 85-9 (Yip1 interacting factor B homolog - YIF1B, ranging 31 to 34 kDa with six different isoforms) using a cDNA library. However, the size of the identified proteins is smaller than expected, suggesting that there were an alternative splicing. To confirm the identity of these antigens is necessary to sequencing them by mass spectrometry. This technique requires the use of very pure proteins. The objective of this study is to obtain the pure antigens by immunoprecipitation of MG-63 total lysate using monoclonal antibodies PSP 42-22 and PSP 85-9 conjugated to microspheres of Protein G immobilized on agarose. As soon the antigens are identified we will compare with the proteins already identified.