Hello, I am trying to verify the efficacy of an assay method to quantify standard concentrations of Hyaluronic acid (HA). I follow the protocol which is mentioned below. It is a colorimetric method. The procedure is as follows:
1)1 ml of HA sample (known concentration) + 1ml of 0.1%SDS and vortex for 10 mins. (Treatment with SDS is to separate out the capsular HA in case of estimation from the fermentation broth. I am including this step even for estimation of standard HA just to find out if there is any loss happening in this step)
2)Centrifuge @10000rpm for 10mins
3)Add 0.4ml of supernatant to 1.6ml of absolute ethanol and keep it O/N @ 4 Deg C.
4)Centrifuge @10000 rpm for 10mins and discard the supernatant.
5)Dissolve the pellet in 1ml 0.9% NaCl.
6)Following the 5th step I go with the Carbazole method to colorimetrically estimate Hyaluronic acid (HA) @OD530.
I am skeptical on the efficacy of the above method of HA estimation as I am encountering a significant amount of loss of HA during the estimation. Based on the empirical results that I am getting, I think the loss is occurring somewhere in the 1st five steps of the aforementioned protocol. It would be great if I am told about what could be the reason for the lower estimation or where exactly is the loss occurring and also how to prevent the loss in estimation.
-Thanks & Regards
I fear you have to check each and every step objectively to know the loss in estimation. The role of SDS in the estimation has to be extrapolated and may concentration based HA presence needs to be standardized. The intake in the supernatants (step 2 and 4) parts as well as the 4 dc temperature backed HA concentration is also important. It seems the loss is substantial here since the calorimetry is sensitive enough to give the estimate unless the HA itself is in a non-assaying molecular associations.
we preparation of HA from Umbilical cord ....How we can check it ...which protocol .plz.
researcher
It is a kind of difficult to work with HA. We are using carbazole method and we are having high variation in the estimation. Though we did work further standartization of the method to decrease variability. Could this be due to the nature of the HA (tends to sitck together- less efficient homejenization?). We are producing HA from bacterial fermentation, I was wondering about the glucose leftover - or glucose sticked to the HA, is it possible ? Any answer could be pertiated thank you.
Hi.
We have carried out various experiments where it is shown that the underestimation of the AH concentration is in the precipitation stage with ethanol. Sometimes it is necessary to repeat the reduction with more ethanol to increase the recovery percentage of HA.
We have also seen that some components of the culture medium precipitate with the addition of ethanol and this produces many interferences with the carbazole method.
But during precipitation with ethanol, hyaluronic acid and some of the components of the culture medium precipitate. The way to remove impurities is to re-suspend the precipitate in distilled water or sodium chloride and re-precipitate with more ethanol at least 3 times. This procedure is very tiring and made us drop large amounts of reagents, so we decided to use a chromatographic method to quantify the HA.
Hello... maybe you could help me. I looking for a method for assay Hyaluronic acid cross linked in dermal Filler using GC- MS mass spectrum or HPLC- DAD . any idea? [email protected]. Barcelona- Spain. Tks a lot.
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