A turbidimetric assay for the measurement of clotting times of procoagulant venoms in plasma

Margaret O'Leary, Geoffrey Isbister

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Introduction

Assessment of the procoagulant effect of snake venoms is important for understanding their effects. The aim of this study was to develop a simple automated method to measure clotting times to assess procoagulant venoms.

Methods

A turbidimetric assay was developed which monitors changes in optical density when plasma and venom are mixed. Plasma was added simultaneously to venom solutions in a 96 well microtitre plate. After mixing, the optical density at 340 nm was monitored in a microplate reader every 30 s over 30 min. The clotting time was defined as the lag time until the absorbance sharply increased. The turbidimetric method was compared to manual measurement of the clotting time defined as the time when a strand of fibrin can be drawn out of the mixture. The two methods were done simultaneously, with the same venom and plasma, and compared by plotting the manual versus turbidimetric clotting times. Within-day and between-day runs were done and the coefficient of variation (CV) was calculated.

Results

Plots comparing manual clotting times to the lag time in the turbidimetric assay showed good correlation between the two methods for brown snake (Pseudonaja textilis) venom, including 24 determinations in triplicate over six days for seven different venom concentrations. Good correlation was also found for four other venoms: tiger snake (Notechis scutatus), Carpet viper (Echis carinatus), Russell's viper (Daboia russelii) and Malaysian pit piper (Calloselasma rhodostoma). Between-day CV was in the range 10–20% for both methods, while within-day CV < 10%.

Discussion

The turbidimetric assay appears to be a simple and convenient automated method for the measurement of clotting times to assess the effects of procoagulant venoms.

Original languageEnglish
Pages (from-to)27-31
Number of pages5
JournalJournal of Pharmacological and Toxicological Methods
Volume61
Issue number1
DOIs
Publication statusPublished - Jan 2010

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Venoms
Assays
Plasmas
Density (optical)
Snake Venoms
Russell's Viper
Piper
Tigers
Snakes
Fibrin

Cite this

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title = "A turbidimetric assay for the measurement of clotting times of procoagulant venoms in plasma",
abstract = "IntroductionAssessment of the procoagulant effect of snake venoms is important for understanding their effects. The aim of this study was to develop a simple automated method to measure clotting times to assess procoagulant venoms.MethodsA turbidimetric assay was developed which monitors changes in optical density when plasma and venom are mixed. Plasma was added simultaneously to venom solutions in a 96 well microtitre plate. After mixing, the optical density at 340 nm was monitored in a microplate reader every 30 s over 30 min. The clotting time was defined as the lag time until the absorbance sharply increased. The turbidimetric method was compared to manual measurement of the clotting time defined as the time when a strand of fibrin can be drawn out of the mixture. The two methods were done simultaneously, with the same venom and plasma, and compared by plotting the manual versus turbidimetric clotting times. Within-day and between-day runs were done and the coefficient of variation (CV) was calculated.ResultsPlots comparing manual clotting times to the lag time in the turbidimetric assay showed good correlation between the two methods for brown snake (Pseudonaja textilis) venom, including 24 determinations in triplicate over six days for seven different venom concentrations. Good correlation was also found for four other venoms: tiger snake (Notechis scutatus), Carpet viper (Echis carinatus), Russell's viper (Daboia russelii) and Malaysian pit piper (Calloselasma rhodostoma). Between-day CV was in the range 10–20{\%} for both methods, while within-day CV < 10{\%}.DiscussionThe turbidimetric assay appears to be a simple and convenient automated method for the measurement of clotting times to assess the effects of procoagulant venoms.",
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A turbidimetric assay for the measurement of clotting times of procoagulant venoms in plasma. / O'Leary, Margaret; Isbister, Geoffrey.

In: Journal of Pharmacological and Toxicological Methods, Vol. 61, No. 1, 01.2010, p. 27-31.

Research output: Contribution to journalArticleResearchpeer-review

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