We think that you can use our assays for ADAMTS-5 full length but we do not have any data. We know that it is not possible to use native Aggrecan. The giant glycosylations have negative effects on the binding of the capture antibodies to aggrecan-ARGSVIL fragments.

Our research data showed very clear that anti-ARGSVIL antibody binds the digested native aggrecan only in Western-blot and not in ELISA.

Yes, you can use such a protein. Please note that the protein should be unglycosylated. We provide a recombinant diluted aggrecan-IGD with this kit or you can use the higher concentrated aggrecan-IGD with cat. no. 30 411 004.

Yes, we determine the activity of our truncated ADAMTS5 (without amino acids Δ625-930) with this kit. If you are interested we can send a recommendation for this experiment.

ProteaDetect^_® kits can also be used after the expired date. This date depends on our antibody (peroxidase conjugate). After expired date we expect a decrease absorption of the samples. To be sure that the absorption values can be obtained for the smaller dilution in the right range of 2.5-3.5, we recommend:

1.   Please make a small test using the peptide standard. For this, use two wells in the first row (it means the row with
      the smaller dilution). If the absorption values are less than 2, we recommend:

2.   Instead of 1:100 dilution of POD, a dilution of 1:80 or 1:50 (the kit has a twice amount of POD as needed for one
     plate). Here, ensure that incubation time of TMB with POD is under observation (It can happens that 30 min, as
     the manual recommends, is too much. In this case time must be decreased in order to obtain the absorption values
     among 2.5-3.5 (and not MAX).

It should cause no problem after 2-3 freezing thaws of the components in the kit.

Please note:
A.     If many samples are handled at the same time, we recommend to use multi-channel pipette and to be very
        careful to avoid air bubbles, especially when TMB and sulphuric acid are added.
B.     The samples should have the same temperature when starting the experiment, in order to get a real comparison
        between samples and reproducible results.
C.     After shaking out fluids from the plates (e.g. after washing) be sure that bubbles are removed from the plates
        (with a tip).

If your customer needs to stock the ELISA components for a long period of time, we recommend the following:
A.     By room temperature: wash buffer and sulphuric acid
B.     By 4○C: microtiter plate, antibody-peroxidase conjugate, TMB
C.     By -20 ○C: all the other buffers: ELISA buffer, EDTA dilution buffer, reaction buffer, ± inhibitor solution
D.     By -80○C: all proteins and peptides: ADAMTS4-standard, Aggrecan-IGD, ARGSVIL-Peptide standard,
        ± Inhibitor solution

The expiry date of the kit is calculated (for ½ year) to ensure that:
A. TMB solution, which is not stable for too long period of time, will not become too brown, because this
    increases the background
B. The activity of the antibody-peroxidase conjugate, which decreases in time, will not become too small.

Besides sulphuric acid (stop solution) that has a concentration of 0.25 M and is only corrosive, all compounds of our kits contain no hazardous additives.

In the wild type aggrecan aa 332 is G not A, but in the recombinant protein that we offer the G is mutated to A.

These assays can be used to measure the activity of every aggrecanase that release ARGSVIL-Peptid after IGD cleavage, but only for serum-, EDTA- and heparin free biological samples

ProteaDetect^® kits have been tested with synovial fluid samples from calf, rabbit and human. We recommend sample dilutions to a final concentration of 10-20% in the reaction mix (avoiding the inhibition of the aggrecanase reaction by undiluted synovial fluid). The samples must be serum-, EDTA-, heparin-free and the kits cannot be used for tissues, because there are components that will inhibit the assay.

The activity of ADAMTS-proteins dependents on structure, length (full-length or truncated forms) and domains. In the kit, the concentration of ADAMTS4 is 0.3 µM, while the provided ADAMTS5 has 2 µM. Therefore is ADAMTS5 (until 0.2 µM) diluted 1:10 when making the activity measurements (ELISA). Also in this case higher activity values are reached. This is anyway a description for our proteases TS4 (from kit) and TS5.
For more information, see the article from Fushimi K, Troeberg L, Nakamura H, Lim NH, Nagase H., J Biol Chem. 2008 Mar 14;283(11):6706-16. Epub 2007 Dec 22. Functional differences of the catalytic and non-catalytic domains in human ADAMTS-4 and ADAMTS-5 in aggrecanolytic activity.

The sensitive aggrecanase assay works in the same way as the aggrecanase assay. The difference is that the aggrecan-IGD has been mutated. This difference leads to a higher sensitivity for the sensitive aggrecanase assay with 0.003 to 0.1 nM ADAMTS4 compared to normal aggecanase assay with 0.024 to 1.5 nM ADAMTS4.

60 µl ADAMTS4 of 0.3 µM

When carrying out two controls, one for standard curve of ARGSVIL and the other for standard curve of proteolytic reaction with purified aggrecanase (both duplicated), then 50 determination (25 duplicates) can be carried out.

For each standard 16 wells are needed (a total of 32). The microtiter plate contain 96 wells, it means 96 wells minus 32 wells for standards = 54 wells.

The assay consists of two modules, the aggrecanase module and the ELISA module. The aggrecanase module provides chemicals for 50 proteolytic reactions (components: aggrecan IGD, ADAMTS4, inhibitor solution, reaction buffer, EDTA-dilution buffer), while the ELISA module contains reagents for 96 determinations including reagents for 2 standard curves of ARGSVIL-peptide and 3 standard curves of proteolytic reactions (components plate, ELISA buffer, ARGSVIL-peptide standard, antibody peroxidase conjugate, wash buffer, detection solution TMB, sulphuric acid). ADAMTS4 can then be used for at least 50 reactions.

Aggrecan-IGD is the connecting peptide between G1 and G2 (T₃₃₁ - G₄₅₈). It comprises the following amino acids:
T A E D F V D I P E N F F G V G G E E D I T V Q T V T W P D M E L P L P R N
I T E G E   A R G S V I L  T V K P I F E V S P S P L E P E E P F T F A P E I G A T A F A E V E N E T G E A T R P W G F P T P G L G P A T A F T S E D L V V Q V T A V P G Q P H L P G G (His-tag).

Proteolytic cleavage of aggrecan-IGD by aggrecanases releases an aggrecan peptide with the N-terminal sequence ARGSVIL. ARGSVIL-peptide is bound to the coated antibody. The antigen for the second antibody (peroxidase conjugate) is the sequence that follows ARGSVIL’s sequence:
T V K P I F E V S P S P L E P E E P F T F A P E I G A T A F A E V E N E T G E A T R P W G F P T P G L G P A T A F T S E D L V V Q V T A V P G Q P H L P G G (His-tag).

For the detection of truncated ADAMTS4 using ProteaDetect^® aggrecanase activity assay, is a minimum concentration of 0.012 nM ADAMTS4 and 2 pM ADAMTS4 if using ProteaDetect^® Sensitive aggrecanase activity assay.

ProteaDetect^® Aggrecanase activity assay kit cannot be used for blood samples due to the fact that HSA (human serum albumin) binds to the aggrecanase catalytic site and inhibits its activity. In fact it is not possible to determine the concentration of any aggrecanase using ProteaDetect^® kit, but only the activity.

Both assays can only be used to measure the activity of all three recombinant truncated forms of ADAMTS that we offer: ADAMTS1, ADAMTS4 and ADAMST5 and of any other aggrecans that release the ARGSVIL-Peptid after IGD cleavage.