Appendix: 1 Chemicals, Reagents and Buffers 1. BUFFERS FOR WBC EXTRACTION WBC lysis buffer (for 1 liter) Ammonium chloride 8.3 gm (150 mm/l) Sodium bicarbonate 0.84 gm (10 mm/l) 1 X reagent EDTA 29 mg (0.1 mm/l) Adjust the ph at 7.4 Keep it at 4 o C 0.9% NaCl 0.9 gm NaCl in 1 liter of D.D. water 2. BUFFERS FOR PROTEIN ESTIMANATION: Solution A: 50ml of 2% sodium carbonate in 0.10N sodium hydroxide 0.50ml of 1% copper sulfate in deionized water 0.50ml of 2% sodium potassium tartarate in deionized water Mixed solution A fresh every day that assays are done. Solution B: 1N Folin reagent (Diluted 2N phenol reagent v: v with deionized water)
3. REAGENTS REQUIRED FOR MPS TYPE I (Hurler Syndrome): Chemicals: Sodium Acetate (3H 2 O), Glycine, Sodium hydroxide, Glacial acetic acid, 2- Hexadeanoylamino 4-nitrophenylphosphorylcholine (MW: 575.7), 2-Nhexadecanoylamino 4-nitrophenol (392.5), Ethanol. Reagents: 1. Acetate Buffer (0.5M, ph: 5.6): Dissolved 13.6g of sodium acetate in water and adjusted the ph to 5.6 and make up the final volume to 200ml. 2. Glycine Buffer: (0.1M, ph: 10.5) Dissolved 0.751g of glycine in water and adjusted the ph, by titrating with 0.5 N NaOH, to 10.5. Make up the final volume to 100ml. 3. Glycine Buffer: Ethanol Mixture (1:5 v/v): 4. 2-Hexadeanoylamino-4-nitrophenylphosphorylcholine: Dissolved the 16.5 mg of 2- Hexadeanoylamino-4-nitrophenylphosphorylcholine in 1ml of water. 5. Stock Standard Solution (2.0 mm): Dissolved 7.85 mg of 2-N-hexadecanoylamino 4- nitrophenol in 10 ml of ethanol: glycine buffer mixture. 6. Working Standards: Prepared a series of working standards (WS1-WS5) i.e. 20, 40, 80, 160 and 200µm, from the stock standard in glycine buffer: ethanol mixture.
4. REAGENTS REQUIRED FOR MPS TYPE II (Hunter Syndrome) Reagent 1: 0.1M sodium acetate buffer ph 5.0 containing 10mmol/L lead acetate and 0.02% sodium azide was used. Reagent details: Sodium acetate, anhydrous: (MW 82.03), Glacial acetic acid : ( MW 60.05), Lead acetate trihydrate: (MW 379.3), Sodium azide: (MW 65.01) Preparation: Dissolved 1.361g sodium acetate and 379 mg lead acetate in 80ml of distilled water. Adjusted to ph 5.0 with acetic acid before making up to 100 ml. Reagent: 1.25 mmol/l 4-methylumbelliferyl-α-iduronate 2-sulphate in acetate buffer. Preparation: Dissolved the substrate in acetate buffer at a concentration of 0.6mg/ml. Reagent 2: Mcllvain s buffer ph 4.5 (0.4mol/L Na phosphate/0.2 mol/l citrate) Reagent details: Disodium hydrogen phosphate, anhydrous :( MW 210.14) Citric acid monohydrate :( MW 210.14) Preparation: Dissolved 5.68g disodium orthophosphate Na 2 HPO 4 in 100mls distilled water. Dissolved 10.52g citric acid in 50 ml distilled water. Adjusted the ph of the sodium phosphate solution to 4.5 with the citric acid. Reagent: LEBT (Lysosomal enzyme purified from bovine testis) Preparation: Reconstituted with 1ml distilled water.
5. CHEMICALS REQUIRED FOR GAGs ESTIMATION: 1. 1,9-Dimethylmethylene blue (DMB) chloride 2. Bovine Serum Albumin: Heparin 3. Tris 4. HCL 5. Formic Acid 6. Formic Acid (22.25M) 7. Sodium Hydroxide 8. Sodium Dodecyl sulfate (SDS) 9. Ethanol Reagents: 1. Formate Buffer (0.1M,pH 3.5): 1.125ml of formic acid was added to 400 ml of water and ph was adjusted to 3.5 with sodium hydroxide (2M) and adjusted the final volume to 500ml. 2. GAG s Precipitation DMB (0.2mM) reagent: Dissolved 6.96mg DMB dye in 2ml of 95% ethanol and adjusted the final volume to 100ml with 0.1M formate buffer ph 3.5. Stored in a brown color bottle at room temperature.
3. Tris buffer 0.2M, ph 7.5: 12.114 g of Tris was dissolved in ~400 ml of water and adjusted ph to 7.5 with 2 N HCI and adjusted the final volume to 500 ml with water. 4. Bovine serum albumin solution (200 mg %): 50 mg of BSA was dissolved in 25 ml of water. 5. Protein based dye reagent (0.05 mm DMB, 0.1 M Tris, 25 mg% BSA ph: 7.5) A. Bovine serum albumin (BSA) solution (125 mg %): 125 mg BSA was added in to ~50 ml distilled water; allowed hydrating and dissolving. Adjusted the final volume to 100 ml with water. B. DMB dye solution A (0.1 mm DMB in 0.2 M Tris buffer, ph 7.5): 8.7 mg of DMB dye was dissolved in 5 ml of 95% ethanol and adjusted the final volume to 250 ml with 0.2 M Tris HCI buffer, ph 7.5. 100 ml of BSA (A) was mixed with 250 ml of DMB dye solution (B) and adjusted the final volume to 500 ml with water. Stored in a brown color bottle at room temperature. Stabled for 2 months at ambient temperature. 6. Stock reference standards (5 mg/ml): Dissolved 10 mg of heparin in saline and maked up to 2 ml.
7. Ref. standard for use (5-500 µg/ml): Stock standards were diluted in normal saline to achieve the desired concentration of 5-500 µg/ml. 8. Sodium dodecyl sulfate (25 mg %) solution: 25 mg of sodium dodecyl sulfate was dissolved in 100 ml of water. 6. MATERIAL REQUIRED FOR CHITOTRIOSIDASE ASSAY: Microplate: One microplate with 96 wells was used coated with anti-human chitotriosidase polyclonal antibody as a capture antibody. 10 X Wash Buffer: 100 ml of 10X wash buffer containing 2% Tween-20 Dilution Buffer: 40 ml of 1X buffer for reconstitution of Human Chitotriosidase standard and sample dilution. Human Chitotriosidase Standard: 18ng of lyophilized recombinant human chitotriosidase. HRP conjugated Detection Antibody: 12 ml of HRP (horseradish peroxidase) conjugated anti human chitotriosidase polyclonal antibody. Substrate Reagent: 20 ml of the chromogenic substrate, tetra meththylbenzidine (TMB). Stop Solution: 20 ml of 1N H 2 SO 4.
Preparation of Reagents 1. The working solution of Wash Buffer was prepared by adding 100 ml of the 10X Wash Buffer to 900 ml of deionized (distilled) water and stored at 4 o C for one month or 20 o C for long-term storage. 2. Human Chitotriosidase Standard was reconstituted with 0.5 ml of Dilution Buffer. The concentration of the reconstituted Human Chitotriosidase was 3.6 ng/ml, which was referred as a Master Standard of human chitotriosidase. Prepared Standard solutions as follows: The Master Standard was used to produce a dilution series. Mixed each tube thoroughly before the next transfer. The 3,600 pg/ml standard (Std.1) served as the high standard. The Dilution Buffer served as the zero standard (Blank.)
7. MATERIAL USED FOR CCL18 ASSAY: 1. Capture Antibody: 180µg/mL of mouse anti-human PARC was reconstituted with 1.0ml of PBS. After reconstitution, the antibody was stored at 2-8ºC for up to 60 days or aliquated and stored at -20 ºC to -70 ºC for up to 6 months. Diluted for a working concentration of 1.0 µg/ml in PBS. 2. Detection Antibody: 36 µg/ml of biotinylated goat anti human PARC reconstituted with 1.0ml of reagent diluents. Reconstituted were stored at 2-8ºC for up to 60 days or aliquated and stored at -20 ºC to -70 ºC for up to 6 months. For making a working concentration of 200ng/mL, diluted in reagent diluents. 3. Standard: Standard was reconstituted with 0.5mL of reagent diluents for making 110ng/mL of recombinant human PARC. Reconstituted standard was store at 2-8ºC or aliquoted and stored at -70 ºC for up to 2 months. 4. Streptavidin-HRP: 1.0ml of streptavidin conjugated to horseradish peroxidase was stored at 2-8 ºC for up to 6 months. For making working concentration the substrate, diluted with reagent diluent. Buffer Used: 1. PBS: 137mM NaCl,2.7mM KCl, 8.1mM Na 2 HPO 4, 1.5mM KH 2 PO 4, ph7.2-7.4,0.2 0.2µm filtered 2. Wash Buffer: 0.05% Tween 20 in PBS ph7.2-7.4. 3. Reagent Diluent: 1% BSA in PBS ph7.2-7.4 0.2µm filtered. 4. Substrate Solution: 1:1 mixture of color reagent A(H 2 O 2 ) and color reagent B (Tetramethylbenzidine) 5. Stop Solution: 2N H 2 SO 4.
8. MATERIALS USED FOR HCII-T ASSAY: 1. Capture Antibody (THCII-EIA-C) containing 0.5 ml of polyclonal affinity purified anti-thrombin antibody for coating plates was used. 2. Detecting Antibody (THCII-EIA-D) containing 0.5 ml of peroxidase conjugated polyclonal anti-hcii antibody for detection of captured THCII complex was used. 3. Coating Buffer containing 50 mm Carbonate, 1.59g of Na 2 CO 3 and 2.93g of NaHCO 3 dissolved in 1 litre of distilled water was used. ph was adjusted to 9.6. Buffer was stored at 2-8 o C for 1 month. 4. PBS (base for wash buffer and blocking buffer) containing 8.0g NaCl, 1.15g Na 2 HPO 4, 0.2g KH 2 PO 4 and 0.2g KCl dissolved in 1 litre of distilled water was used. ph was adjusted to 7.4 and buffer was stored for 1 month at 2-8 o C. 5. Wash Buffer {PBS-Tween (0.1%, v/v)} containing 1.0 ml of Tween-20 dissolved in 1 litre of PBS buffer. ph was adjusted to 7.4 and stored at 2-8 o C for 1 week. 6. Blocking Buffer {PBS-BSA (2%, w/v)} containing 5.0g of Bovine Serum Albumin dissolved in 200 ml of PBS. ph was adjusted to 7.4 and volume was maintained up to 250 ml with PBS and stored at -20 o C. 7. Sample Diluent { HBS-BSA-T20, 5.95g HEPES (free acid)} containing 1.46 g NaCl, 2.5 g Bovine Serum Albumin dissolved in 200 ml H2O. 0.25 ml of Tween-20 was added; ph was adjusted to 7.2 with NaOH, then make up to a final volume of 250 ml with H2O and stored at -20 o C. 8. Substrate Buffer (Citrate-Phosphate buffer ph 5.0) containing 2.6g citric acid and 6.9g Na 2 HPO 4 up to a final volume of 500 ml with purified H 2 O. Buffer was stored at 2-8 o C for 1 month.
9. OPD Substrate (o-phenylenediamine.2hcl) containing Toxic1 (5 mg tablets): 5 mg OPD was dissolved in 12 ml substrate buffer then 12 µl 30% H 2 O 2 was added. 10. Stopping Solution containing 2.5 M H 2 SO 4 11. Materials for making TAT reference standards: - Purified human Heparin Cofactor II - HCII deficient plasma, lyophilized - Human thrombin 12. Other: 96-wells Microplates