CHARACTERIZATION OF BACTERIAL STRAINS FOR USE IN A PHAGOCYTIC IMMUNOASSAY

A THESIS SUBMITTED IN PARTIAL FULFILLMENT FOR THE DEGREE OF MASTER OF SCIENCE IN BIOTECHNOLOGY

SUBMITTED BY

MADHAVI SHINDE (M.Sc.BT-10020)

UNDER THE GUIDANCE OF

DR. MANOJ KUMAR

HEAD, IMMUNOBIOLOGY AND MOLECULAR RESEARCH LAB

Serum Institute of India Ltd., Pune 2011-2012

DEPARTMENT OF BIOTECHNOLOGY AND BIOINFORMATICS PADMASHREE DR. D.Y. PATIL UNIVERSITY JULY 2012

ABSTRACT
Streptococcus pneumoniae is the major causative agent of pneumonia worldwide. Thus there are constant efforts to develop a vaccine that will protect adults and children against infection with major serotypes of S. pneumoniae. Effectiveness of these vaccines can be evaluated by means of several enzyme based and cell based assays. Opsonophagocytic Assay (OPA) and Enzyme Linked Immunosorbent Assays (ELISA) are examples of assays that are usually used for vaccine potency evaluation. OPA is usually used as second reference assay after ELISA because ELISA gives total antibodies that are produced in response to the vaccine whereas OPA gives the estimate of functional antibodies. Evaluation of pneumococcal vaccines requires estimating antibody responses against 7-11 serotypes and performing OPA for such high number of serotypes becomes tedious. Thus there is a need for developing an assay that can estimate antibody response to multiple serotypes at a time. Multiplexed OPA (MOPA) is an assay that can detect antibody response against 4 serotypes at a time. MOPA forms main experimental procedures of the project. Aim of the project was Characterization of Bacterial Strains for Use in a Phagocytic Immunoassay. The main objectives of project were to prepare pneumococcal stocks, characterize pneumococcal strains and then use the characterized strains in an immunoassay i.e. Multiplexed OPA. The pneumococcal stocks were prepared using Todd Hewitt Yeast Extract- Glycerol Broth. The pneumococcal strains were characterized by determining their antibiotic resistance and titration values. Finally the characterized strains were used in MOPA to check whether the bacteria work under the assay conditions or not. The results of the experiments performed were satisfactory and according to the criteria established by MOPA experiments in various laboratories across the world. Antibiotic sensitivity experiments carried out determined that the serotypes under study were resistant to one of the four antibiotic under study (Streptomycin, Spectinomycin, Optochin and Trimethoprim) but resistant to other three. This characteristic of bacterial strain was the major factor that helped in multiplexing the OPA. Titration values were used to decide the optimal dilution of bacteria to be used in the assay to get ~80-120 bacterial colonies per spot. MOPA was performed with sera samples taken from individuals pre and post vaccination with a pneumococcal vaccine. The assay results showed that there was significant increase in antibody titers for a particular serotype post vaccination. Use of MOPA as a reference assay for determining the antibody titers reduced the amount of time and sera volume required as compared to that of simple

OPA. In addition to this, use of TTC dye in overlay agar gives coloured colonies which make it easier to count the bacterial colonies and calculate titers using an automated counter and software. Thus using MOPA for determining effectiveness of pneumococcal vaccines can reduce time as well as overall expenditure required.