Toxicon 48 (2006) 1011–1017 A new in-vitro agglutination technique for potency estimation of antisnake venom serum (ASVS) R. Chander, M. Batra, D. Singh, Y. Kumar, S. Rawat, S. Kumar à Antisera Division, Central Research Institute, Kasauli (H.P) PIN-173204, India Received 21 July 2006; received in revised form 10 August 2006; accepted 11 August 2006 Available online 17 August 2006 Abstract Traditionally the potency of ASVS is assayed quantitatively by in-vivo neutralization test for lethality in mice. A sensitive and simple in-vitro agglutination assay for the quantitative determination of Antisnake Venom Serum (ASVS) potency is reported. The method is rapid, cheap, simple, economical and above all does not require the use of experimental animals for potency assay of in process, unpurified and purified sera batches. Among in-vitro procedures, agglutination assay was favored in comparison to flocculation as the later was found to give variable results and also time consuming (high Kf value). Before application, the method was standardized and validated for choice and concentration of particulate material (latex vs. bentonite), temperature and optimum antiserum concentration. It is well known fact that venoms lose toxicity on dilution however this study demonstrated that the bentonite adsorbed venoms of the entire four snake species viz., Cobra, Krait, Russell’s viper and Echis are stable even up to 30 days of storage. Among five lots each of unpurified serum, unprocessed plasma and purified sera tested, the results were found comparable with universally accepted in-vivo biological assay. The coefficient of correlation was found to be near 1.0 within 95% fiducial limits of acceptance and also significantly less variation was observed in the mean potency values and standard deviations. For all results p value was observed to be o0.01. Results indicate that in-vitro agglutination assay is suitable and can be used for potency estimation of in process as well as unpurified and purified ASVS batches. r 2006 Elsevier Ltd. All rights reserved. Keywords: Agglutination technique; Antiserum; Antisnake venom serum 1. Introduction In any language of the world, snake produces unimaginable fear and anxiety. This has been happening in our society since the dawn of civilization. Poisonous snakebite is common health hazard especially among the snake catchers, forest workers and agriculturists in India. In India alone, it is estimated that about 200,000 persons are bitten and 15,000 deaths occur due to poisonous snake- bites annually (Ananthapadmanabhan, 1991). The highest incidence is reported during rainy season particularly at night. The poisonous snakes respon- sible for most of the snakebite deaths in India are Cobra (Naja naja), Krait (Bungarus ceruleus), Russell’s viper (Daboia russelli) and Saw scaled viper (Echis carinatus)(Jena and Sarangi, 1993). Treatment of snakebite requires prompt and specific measures. Due to non-availability of rapid and dependable diagnostic test for identification of ARTICLE IN PRESS www.elsevier.com/locate/toxicon 0041-0101/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.08.007 à Corresponding author. Tel.: +91 1792 272060/272059x223; fax: +91 1792 272049. E-mail address: yasht26@yahoo.co.in (S. Kumar).