Immunoevaluation and Characterization of Tetanus Toxoid

Natural polymer like potato starch is a mixture of amylose and amylopectin, . Extraction of Potato starch was performed by rasping, centrifugation, refining and drying method. In our research method, we employed potato starch as a biodegradable polymer; It has a great impact on pharmaceutical applications due to its bioavailability, non toxic, high change density and biodegradability. In our research work, we have selected Potato starch polymer as a model of immunomodulatory effect of vaccine of tetanus antigen. According to WHO, Tetanus is a systemic infectious disease caused by genus Cl Tetanii. It has been estimated that the tetanus fever endemicity among large populations and global emergence of multidrug resistant strains to impose greater urgency on the evaluation of existing and new vaccines to prevent mortality of neonates and in pregnancy. Recently available recombinant vaccine was seems to be side effect and cost effective. The starch polymer in the form of microspheres was preferred in order to replace the alum to elicit sustained immune response because alum induces local granuloma and hypersensitivity reaction to some individual. We have employed microencapsulation technique by using 0.5% ml glutaraldehyde as a crosslinking agent. The particle size was analyzed as 40.23μm. Invitro studies was analyzed by SEM, stabilities studies Immunogenicity studies was carried out by incubating the sample, centrifuged and tested for an antigen and the Compatibility study was performed by Infrared Spectroscopy, the antigen integrity was studied by SDS PAGE and ELISA. Immunoglobulin titer values was found out ( IgG, IgA,IgM, IgE) to show the increase level of antibody response. Key-words: Natural polymer, Tetanus antigen, Immunological evaluation, Antibody Corresponding Author: L.Nirmala, SRM College of Pharmacy Department of Pharmaceutical Biotechnology SRM University Katangkulathur Kanchipuram District email: [email protected] INTRODUCTION: Tetanus is more common in the developing countries, where the climate is warm and in rural areas, poor hygienic practices in India. It was a common cause of death particularly in the newborn and mother in pregnancy. The world spread use of vaccines over the last few decades has resulted in a reduction in the incidence of many infectious diseases in developed countries. Nevertheless, there are still significant challenges for vaccine developments including the need to make improvement in existing vaccine by making them safer, more immunogenic and to extend vaccine coverage in certain populations particularly in the developed world. One of the most important issues in vaccinology is the need for new adjuvant in vaccine delivery system Most of the vaccines currently in development are based on purified subunits, recombinant molecule, synthetic peptides or nucleic acids which are often poorly immunogenic, expensive and produce adverse effect It is clear that new generation of vaccines will require better adjuvant delivery systems to induce optimal immune response. The system under discussion employ either biodegradable polymer or system requiring removal after use and can release the drug either by membrane or matrixcontrolled diffusion. Recent trends in potential carrier delivery have seen microencapsulation of pharmaceutical substances in biodegradable polymers as an emerging technology. Currently,Biodegradable polymer in the form of microsphere has shown the ideal perquisite for microsphere carrier in vaccine delivery system The enhanced immunogenicity of particulates antigens is unsurprising, since pathogens are particulates of similar dimensions and the immune system has evolved to deal with these Particulates delivery system It presents multiple copies of antigens to the immune system and promote Immunoevaluation and Characterization of Tetanus Toxoid NJIRM 2011; Vol. 2(1).Jan-March eISSN: 0975-9840 pISSN: 2230 9969 P ag e4 2 trapping and retention of antigen in local lymph node moreover particles are taken up by macrophages and dendrite cells leading to enhanced antigen penetration and the release of cytokines to promote the induction of an immune response Sustaining response for a longer duration including local mucosal immune response, generating antibody with increased avidity and neutralization capacity eliciting cytotoxic T lymphocyte (CTLs) to enhance immune response. Potato Starch can be modified through physical, chemical or enzymatic processes and used as food additives because of their considering safety and low cost. Very low levels are used and these are approved by the FDA (CFR 172.892) MATERIAL AND METHODS: sample of Typhoid antigen obtained from Serum Institute of India Guindy ( Chennai), Organic solvent of AR. grade, Tween-80 (hi-media), Glutaraldehyde (spectrochemicals). Starch (chemical formula (C6H10O5) n,) is a mixture of amylose and amylopectin. The former consists of long, unbranched chains of D-glucose residues connected by (α1→4) linkages. Potato was washed efficiently to remove dirt, fungi, rotten spots. Rasping was performed to release the tuber cells and Starch. The potato juice is generally rich in sugar and protein. So when opening the cells the juice is instantly exposed to air and reacts with the oxygen, forming coloured components, which may adhere to the starch. This can be reduced by adding sufficient amount of sulpurdioxide gas to maintain the juice and pulp light yellow. The sample was centrifuged at 5,000rpm and supernatant sample was collected, dried, and stored. Tetanus vaccine encapsulated Potato starch microparticles were prepared by microencapsulation techniques (Emulsion crosslinking) Based upon the result obtained by changing various parameters such as effect of polymer concentration and effect of crosslinking agent concentrations various batches were prepared, to standardize an ideal batch. Starch solution (1% to 10% / ml) was prepared in a 250 ml beaker by continuous stirring for one hour at 5000 rpm. An emulsion was prepared by mixing the vegetable oil and toluene with two drops of Tween80. To the gel 1ml of Tetanus antigen and 1 ml of 0.5% Glutaraldehyde was added and stirring is continued for one hour. Add 2 ml of gel containing tetanus antigen in to the emulsion with continuous stirring. At the end the precipitates were centrifuged, washed with various organic solvents’ A white powder were obtained (microspheres) many batches were formulated, pooled and stored in a refrigerator at 4C. Characterization of micro particles: The morphology and size distribution of dried microspheres was evaluated by Scanning Electron Microscopy(SEM) and the size of the microparticles was determined by optical microscope using calibrated scale. The magnitude of loading Tetanus antigen in microparticles was performed by mixing with phosphate buffer saline (pH7.4). Under shaking at room temperature and kept for 3 hrs. The suspension was centrifuged at 4000 rpm for 15 min to remove free antigen. This process was analyzed by Lowry’ method. The stability of the formulation Tetanus antigen encapsulated starch microparticles and unloaded starch microparticles were determined over period of weeks. Both antigens loaded and unloaded micro particles were kept at 4C. At predetermined time intervals the samples were taken at 0, 1, 2, 3, 4, 5, 6, and 7 days. The morphology was determined by light microscope and the size was determined by using stage-eyepiece micrometer. The swelling ratio of the starch microparticles was determined as the percentage of particle size change after incubation in the phosphate buffer (pH 7.4). Weight amount of microparticles with similar diameter were chosen. Diameter of the beads was measured before and after incubating in phosphate Immunoevaluation and Characterization of Tetanus Toxoid NJIRM 2011; Vol. 2(1).Jan-March eISSN: 0975-9840 pISSN: 2230 9969 P ag e4 3 buffer (pH 7.4) for 12 hrs under optical microscope. The swelling percent was calculated as follow: Compatibility study was performed by Infrared Spectroscopy and the antigen integrity was studied by SDS PAGE and ELISA In vitro release profile: The 200 mg of the Tetanus vaccines micro particles were taken in a 250ml conical flask. To this 50ml of phosphate buffer (pH7.4) was added. The flask was kept in the shake cum incubator. The shaker was adjusted to 80 horizontal strokes per minute at 37 C. From this 1ml of solution was taken in test tube and fresh phosphate buffer was added immediately in the flask. This was repeated at various pre determined time intervals 2hrs, 4hrs, 6hrs, 8hrs, 10hrs, 12hrs, 14hrs, 16hrs, 18hrs, 20hrs. The collected samples were centrifuged and supernatant solution analyzed by Bronsted Lowry method. Immunoglobulin titer: Bleeding of immunized Wister rats done by Retroorbital plexus by using a capillary tube. The samples were pooled and serum was separated. The serum sampled was analyzed by Enzyme linked Immunosorbent assay. The potency of Tetanus vaccine encapsulated starch microparticles may be tested by measuring the specific antibody level was done by Immunoprecipitation assay. The immunoglobulin titer is found by Immune precipitation assay with Nephelometric end point detection. RESULTS : The polymer was prepared in different analysis method at different concentration (1%,2%,3%,4%,5%,6%,7%,8% and 9%). At 8% of Potato starch suitable for the microparticles formation for the antigen was release as Shown in Table 1. 0.5% of cross inking concentration shows the smooth particles formation, which is shown in Table 2. Antigen capacity before and after loading shows the size reduction as represented in Table 3 and The characteristic of the microparticle size shows the loading capacity and efficiency and swelling property shown in Table 4 Table: 1 The effect of the polymer concentration Polymer Polymer conc. Stabilizing agent Conc. of stabilizing agent (%) Size & shape