Dear Jana Horakova

I think, you can use only, Toluidine blue O, no need for zinc chloride double salt. You can purchase from Sigma Aldrich

https://www.sigmaaldrich.com/catalog/product/sial/t3260?lang=ko®ion=KR

You can find the details for qunatify the heparin using the following refernce:

In detail, 0.04 wt.% TBO solution was prepared by dissolving the TBO powder in aqueous 0.01 M HCl/0.2 wt.% NaCl, and then the samples were incubated in 5 ml TBO solution under static conditions at 37 C for 4 h and rinsed three times with UP water. Subsequently, the Hep–TBO complex that formed on sample surface was eluted and dissolved in 5 ml 80% ethanol/0.1 M NaOH mixture (4/1 v/v) solution. Ultimately 150 ll of the supernatant was transferred to a 96-well plate and the absorbance was measured at 530 nm by a microplate reader (lQuant, Bio-tek Instruments Inc.), and the Hep density on the sample surface was evaluated using a calibration standard curve. There were six parallel samples in each group, and SS-DM was set as blank control.

For standard curve preparation, 2 ml 0.04 wt.% TBO was first added to 2 ml of a known concentration Hep solution and incubated at 37 C with gentle shaking for 4 h, the Hep–TBO complex spontaneously formed and precipitated in the mixture. Then the mixture was centrifuged at 3500 rpm for 10 min, the supernatant was removed and the precipitate was carefully rinsed twice with aqueous 0.01 M HCl/0.2 wt.% NaCl. Finally, 5 ml 80% ethanol/ 0.1 M NaOH mixture (4/1 v/v) solution was added to dissolve the precipitate and the absorbance was measured at 530 nm.

Dear Jana,

There is another method that is considered more accurate than toulidine blue assay for heparin quantification.You may try factor Xa anti-thrombogenic (FXa) assay.

https://www.biovision.com/factor-xa-activity-fluorometric-assay-kit.html

Factor Xa assay (FXa; Biovision Inc., Milpitas, CA, USA) depends on the ability of heparin to change the conformational change of antithrombin leading to inactivation of several coagulation factors including FXa. This utilizes the ability of Factor Xa to cleave a synthetic substrate thereby releasing a fluorophore, AMC, which can be quantified by spectrophotometer. Briefly, samples were incubated with 100 µl of antithrombin (25 mU) in 50 mM Tris buffer for 10 min under constant agitation to allow the antithrombin to react with heparin. Heparin standards were used as controls (0–100 ng dissolved in 50 mM Tris, 7.5 mM Na2 EDTA, 175 mM NaCl buffer). FXa substrate was added and incubated for 5 min to allow heparin-antithrombin complex to inactivate FXa. Supernatants were then transferred into a 96-well plate, absorbance readings were recorded at 350/460 nm every minute for the 15 min duration of the reaction.

Please refer to the supplementary method and results in the attached file!

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Best Regards!

Thank you both very much!!!!

Regarding the anti Xa assay - antithrombin is not a part of the kit? Is it possible to use other buffers instead of Tris (PBS, physiological solution)?