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Feasibility of use of single dose immunization regime in Antibody Induction Method for testing potency of tetanus component in DTP group of vaccines and comparison of validated ELISA with TNT for antibody titration

¹Department of Microbiology, Panjab University, Chandigarh, INDIA-160014.

²Department of Parasitology, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh,

   ^{INDIA-160012.}

³Central Research Institute, Kasauli, Himachal Pradesh, INDIA-173204.

⁴Serum Institute of India, Pune, INDIA.

*Corresponding Author:

Prof. Rakesh Sehgal, (MD, DNB, MAMC).

²Department of Parasitology, Postgratuate Institute of Medical Education and Research (PGIMER), Chandigarh,

   ^{INDIA-160012.}

Telephone:  +91-172-2755168

E-Mail: sehgalrksh@gmail.com

Abstract

Conventionally, for estimation of potency of tetanus toxoid by Antibody Induction Method (AIM), two doses (1/10^th of single human dose/dose) of tetanus toxoid given on 0^th and 28^th day, injected subcutaneously into guinea pigs has been recommended. But this assay has a serious drawback of 'booster effects' as the animal is immunized on two occasions besides usage of large number of animals. Hence, feasibility of using single dilution given at single occasion was assessed. For this a three dilution regime was used wherein three dilutions of the standard and test tetanus toxoid (TT) were injected subcutaneously into guinea pigs. Post-immunization sera were raised at 4^th and 6^th week and titrated by in-vivo Toxin Neutralization Test (TNT) done in mice and in-vitro indirect enzyme-linked immunosorbent assay (ELISA). For TNT, L+/10 doses of toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus antitoxin detection. It was found that if the animal was bled at 4^th week post-immunization, anti-tetanus antibody titers as shown by ELISA were 4-5 times higher than the titers estimated by TNT while at 6^th week ELISA and TN titers were comparable (r²=0.999). When the time of bleeding, post-immunization, was increased from 4^th to 6^th week, a 5 fold increase in neutralizing antibody titer was seen. Also, standardized ELISA was found to be of high validity and can be used as an alternate for evaluation of anti-tetanus neutralizing antibodies, thus permitting the in-vitro evaluation of the tetanus antitoxin. Moreover, besides saving mice, ELISA saves five days required for TNT and the pressure on the laboratory is significantly reduced.

Keywords: Double-Dose Single-Dilution; Single-Dose Single-Dilution; Tetanus Toxoid; ELISA; Toxin Neutralization Test; Validation; Time of bleeding.

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