I am looking into osteoporosis in male C57BL/6 mice. Therefore I intent to measure P1NP and CTX in serum but I do not know which test of which brand would be the best to measure this. There are a lot of brands selling ELISA kits, however I do not know which one to order. Is there anyone with experience on this kind of measurements who could help me answer this question?
Hi Hanna,
You can obtain fasting serum samples from animals if they are not fed for at least 5-6 hours. This is due to the interference of the peptides to be analyzed (especially for CTX-I) with lipids/proteins that serum contains after food intake. Take into account that you have to be advised, since it is a regulated procedure.
It is preferable to analyse your samples immediately after collection. If not, you can keep them at 4C for up to 16h or make aliquots and store them at -20C, but do not freeze/thaw the samples more than twice.
Important note 1: It is of crucial importance that you add the samples to the wells as fast as you can (preferably within 10-15 minutes). Especially for the PINP determination, the first incubation time, when the competition for the capture of antibody is taking place, is relatively short (1h), the time difference between the first and the last added sample should be minimal, otherwise this will affect your results. Therefore, you can prepare your samples in eppendorfs, place them in a rack and use a multichannel pipette for placing the samples, standards and controls into the wells, just like when you add chromogen, stopping solution etc. This will limit the time significantly.
Important note 2: Use an excess of samples’ volume when you prepare them in order to have enough for each sample in duplicate, because of pipette accuracy errors, which often occur. For example, in PINP analysis, the samples must be diluted 1:10 with sample diluent and therefore you prepare 5μl serum + 45μL diluent (or 10+90 in duplicate). You can prepare 11+99μl so that you’ll be sure that you have enough volume.
Hope this could be of help. I wish you the best of luck with tour experiments.
I have used the IDS kits (RatLaps for CTx and mouse/rat P1NP) and they were very good and reliable. They're not cheap though. Have a look at their bone products page http://www.idsplc.com/int/products/?format=m
I agree with Rob, very reliable ELISA kits with minimum CVs. Best results can be obtained if the serum samples are fasting and not hemolyzed.