Microplate-based antithrombin activity bioassay for Shuxuetong Injection through aptamer-thrombin capturing coupled with chromogenic substrate hydrolysis

Abstract

Shuxuetong Injection (SXT) is one kind of herbal injections derived from leech (Whitmania pigra) and earthworm (Pheretima aspergillum). It exhibits powerful therapeutic effects on “blood stasis and stagnation (yu xue yu zhi)”, particularly in patients with stroke and myocardial infarction. However, adverse reactions caused by quality problems occur occasionally. We described an aptamer-based bioassay for antithrombin activity of SXT. The antithrombin activity was obtained by calculating the inhibition rate of thrombin catalytic activity toward chromogenic substrates in the presence of SXT. The choice of aptamers was investigated, and one 29-mer aptamer (denoted as Apt29) was chosen. Taking advantage of aptamer-thrombin complex on microplate to specifically capture thrombin-binding molecules from SXT, this assay effectively eliminates the interference from other complex components with the interactions among thrombin, antithrombin molecules and substrates, which existed in the conventional substrate method, and achieves accuracy and sensitivity of antithrombin activity detection. The average intra-day and inter-day precisions, expressed as relative standard deviation (RSD), were 3.3% and less than 10%, respectively. Eight batches of SXT were detected. The antithrombin activity was ranged from 22.1% to 37.8% and the RSD was 22.5%, which showed the batch-to-batch variability. The accuracy of the method was validated by comparing the antithrombin activity of SXT detected by it with that measured by the fibrinogen clotting assay and the fluorometric peptide substrate assay. The approach proved to be applicable to test the antithrombin activity of SXT. This assay offers benefits in fast, simple and high throughput analysis and will be helpful for bioactivity determination and quality control of herbal injections.