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Nagalase Activity Assay

Activity of α-N-acetylgalactosaminidase (Nagalase) in serum or plasma is determined through a proprietary method consisting of a two step immunocapture assay.

In a first step Nagalase is captured on a solid phase coated with a α-N-acetylgalactosaminidase specific antibody able to capture up to 10 ng/ml of Nagalase from serum or plasma. After removal of unbound material the activity of immobilized  Nagalase is measured by incubation with a specific α-N-acetylgalactosaminidase fluorogenic substrate.  The resulting Nagalase enzymatic activity is expressed as nmol/min per milliliter.

IMPORTANT: we are now accepting both plasma and serum samples for testing

 

Nagalase Testing Request Form 

Samples for Nagalase Activity Assay need to be collected as follows :

Testing on plasma:  Whole blood must be collected in 1x9ml (or 2x4,5ml) EDTA tubes. Tubes must be centrifuged by nurse/physician within one hour at 3000 rpm for 10 minutes. Plasma has to be aliquoted in new, sterile tubes and frozen. Immediately freeze at least 3ml of plasma and ship to our lab on dry ice. Sample must be kept frozen at all time.

Testing on serum:  Whole blood must be collected in serum tubes (like BD Vacutainer SST II Advanced tube, yellow cap). Tubes must be centrifuged (3000 rpm for 10 minutes) by nurse/physician AFTER one hour of cloting at room temperature. Serum has to be aliquoted in new, sterile tubes and frozen. Immediately freeze at least 3ml of serum and ship to our lab on dry ice. Sample must be kept frozen at all time. Centrifuged serum tube could be shipped on ambient temperature if its delivery is ensured within 24h after the blood sampling.

 

Activity of α-N-acetylgalactosaminidase (Nagalase) testing at RED Laboratories(adult population, over 18 years) 

N-acetylgalactosaminidase (Nagalase) is an enzyme that deglycosylates the Gc protein also known as vitamin D binding protein (VDBP), rendering it incapable of conversion to active GcMAF (Gc protein-derived Macrophage Activating Factor) and thereby preventing its regulation of macrophage activation.Activity of α-N-acetylgalactosaminidase (Nagalase) in serum or plasma is determined through a proprietary method consisting of a two step immunocapture assay.Nagalase is known to inactivate Gc-MAF and his natural precursor Gc-globulin. It is proposed that patients could be treated with substitution therapy with Gc-MAF and Nagalase has been projected as marker (indicator) of effectiveness for the Gc-MAF therapy.

Apparent nagalase activity in serum or plasma is measured kinetically at RED Laboratories through conversion of a fluorogenic substrate in function of time. The test is standardized against a large serum pool of carefully selected healthy persons (normal WBC count, no inflammation, CRP <1mg/L, no clinical history of immune disease of diabetes) the apparent nagalase activity measurement has allowed to establish a normal range of substrate conversion between 0,5 and 0,95  nMol/ml/min for adults.

Our extensive research in this field however revealed that the measurement of the apparent nagalase activity does only reflect the nagalase activity as potential inactivating enzyme activity in serum/plasma for GC-MAF, but it does not reflect the total nagalase activity in serum, because with the measurement through fluorogenic substrate conversion  one should taken into account the amount of natural substrates  present in the serum/plasma which compete for the substrate conversion in our assay. The known natural substrates for Nagalase are (i) Gc-MAF (at very low concentration ng/ml), (ii) Gc-globulin (at high concentration  on average 620 mg/L in serum) and (iii) bloodgroup A  substance (in extremely high concentrations  in total blood due to the amount of RBC, but only in low concentrations in serum or plasma). The amount of natural substrate for nagalase present in tissues is not known for the moment.This situation leads to the conclusion that the apparent Nagalase activity is not a good biomarker to measure the breakdown of Gc-MAF in the blood of patients and we should taken into account the presence of natural competitors of Nagalase  in order to establish the total nagalase content in serum/plasma.

The main natural competitor we can measure in blood is the Gc-gloubulin concentration which may vary between 150 and 1500 mg/L.  The Gc-globulin concentration may affect the Gc-MAF concentration in two ways: first as being the precursor of Gc-MaF affecting the buildup of Gc-MaF through the activating enzyme co-operation on B and T lymphocytes and secondly as a major competitor for Nagalase preventing the breakdown of Gc-MAF. In line with this, the results for Nagalase testing done at RED Laboratories will be reported as a group containing the values for (i) apparent nagalase activity, (ii) Gc-globulin concentration, (iii) corrected Nagalase activity (i.e. effective nagalase activity) in function of the major natural substrate competitor Gc-globulin and (iv) an alternative immunomodulator enzyme for which we have choose Dipeptidyl-peptidase or CD26 present on T and B lymphocytes and macrophages as well as in numerous other cell populations in the body.

Please find below the normal range values for these parameters:

Adults:

1. Apparent nagalase activity: 0,5-0,95 nMol/ml/min

2. Gc-globulin: 279-579 mg/L

3. Effective nagalase activity: 0,44-1,29 nMol/ml/min

4. Dipeptidyl-peptidase (DPP-4) activity: 18,80-33,79 nMol/ml/min

 

Activity of α-N-acetylgalactosaminidase (Nagalase) testing at RED Laboratories for children (below 11 years) and adolescents (10-19 years)

N-acetylgalactosaminidase (Nagalase) is an enzyme that deglycosylates the Gc protein also known as vitamin D binding protein (VDBP), rendering it incapable of conversion to active GcMAF (Gc protein-derived Macrophage Activating Factor) and thereby preventing its regulation of macrophage activation.Activity of α-N-acetylgalactosaminidase (Nagalase) in serum or plasma is determined through a proprietary method consisting of a two step immunocapture assay.Nagalase is known to inactivate Gc-MAF and his natural precursor Gc-globulin. It is proposed that patients could be treated with substitution therapy with Gc-MAF and Nagalase has been projected as marker (indicator) of effectiveness for the Gc-MAF therapy.

Apparent nagalase activity in serum or plasma is measured kinetically at RED Laboratories through conversion of a fluorogenic substrate in function of time. The test is standardized against a serum pool of carefully selected healthy persons (normal WBC count, no inflammation, CRP <1mg/L, no clinical history of immune disease of diabetes), the apparent nagalase activity measurement has allowed to establish a normal range of substrate conversion between 0,55 and 1,08 nMol/ml/min for children and between 0,52 and 1,01 nMol/ml/min for adolescents.

Our extensive research in this field however revealed that the measurement of the apparent nagalase activity does only reflect the nagalase activity as potential inactivating enzyme activity in serum/plasma for GC-MAF, but it does not reflect the total nagalase activity in serum, because with the measurement through fluorogenic substrate conversion  one should taken into account the amount of natural substrates  present in the serum/plasma which compete for the substrate conversion in our assay. The known natural substrates for Nagalase are (i) Gc-MAF (at very low concentration ng/ml), (ii) Gc-globulin (at high concentration in serum) and (iii) bloodgroup A  substance (in extremely high concentrations  in total blood due to the amount of RBC, but only in low concentrations in serum or plasma). The amount of natural substrate for nagalase present in tissues is not known for the moment.

This situation leads to the conclusion that the apparent Nagalase activity is not a good biomarker to measure the breakdown of Gc-MAF in the blood of patients and we should taken into account the presence of natural competitors of Nagalase  in order to establish the total nagalase content in serum/plasma. The main natural competitor we can measure in blood is the Gc-gloubulin concentration which may vary between 150 and 1500 mg/L.  The Gc-globulin concentration may affect the Gc-MAF concentration in two ways: first as being the precursor of Gc-MaF affecting the buildup of Gc-MaF through the activating enzyme co-operation on B and T lymphocytes and secondly as a major competitor for Nagalase preventing the breakdown of Gc-MAF.

In line with this, the results for Nagalase testing done at RED Laboratories will be reported as a group containing the values for (i) apparent nagalase activity, (ii) Gc-globulin concentration, (iii) corrected Nagalase activity (i.e; effective nagalase activity) in function of the major natural substrate competitor Gc-globulin and (iv) an alternative immune modulator enzyme for which we have choosen Dipeptidyl-peptidase (DPP-4) or CD26 present on T and B lymphocytes and macrophages as well as in numerous other cell populations in the body and which is a strong immune modulator that increases upon immune stimulation. Please find below the normal range values for these parameters (established using 108 children and 79 adolescents):

Children (below 11 years old):

1. Apparent nagalase activity: 0,55-1,08 nMol/ml/min

2. Gc-globulin: 360,7-661,2 mg/L

3. Effective nagalase activity: 0,49-1,18 nMol/ml/min

4. Dipeptidyl-peptidase (DPP-4): 33,5-57,1 nMol/ml/min

Adolescents  (10-19 years old):

1. Apparent nagalase activity: 0,52-1,01 nMol/ml/min

2. Gc-globulin: 387,1-553,1 mg/L

3. Effective nagalase activity: 0,47-1,09 nMol/ml/min

4. Dipeptidyl-peptidase (DPP-4): 22,5-54,4 nMol/ml/min