EN 455-3-2006 pdf download.Medical gloves for single use Part 3 : Requirements and testing for biological evaluation.
Mark the cuff of one glove of each pair at a point (200 ± 10) mm from the tip of the middle finger and weigh the glove (m1) to the nearest 0,1 g. For each pair, insert the second glove inside the marked one so that they fit together as shown in Figure A. 1 a).
NOTE The method for introducing one glove into the other is not of critical Importance provided the gloves are maniputated as little as possible. One way it can be achieved is by sliding a rod in the thumb and the little finger of the inner glove to h&p to introduce them into the corresponding fingers of the second glove. Use the rods to Insert the three other fingers,
A.6.2.2 Pour into the inner glove a sufficient quantity of dye solution (A.3.2.3) to fill all five fingers. Introduce 25 ml extraction buffer (A.3.2.2) at (25 ± 5) °C between inner and outer gloves. For larger gloves this volume may be increased to a maximum of 50 ml. Remove most air bubbles and seal the gloves with the clamp (A.4.14) at the 20 cm mark so as to produce a watertight seal as shown in Figure A.1 b).
A.6.2.3 Fix the gloves to the shaker (A4.15) and shake for (120 ± 5) mm at (25 ± 5) °C.
A.6.2.4 Remove the clamp and separate the gloves carefully. Take care not to contaminate the extract with the dye solution. If the extract is coloured blue, it shall be discarded and the extraction repeated with fresh gloves.
A.6.2.5 Carefully transfer the extract into a centrifuge tube (A.4.3) and clarify the extract either by centrifugation at not less than 2000 g for 15 mm or filtration through a single use filter unit (A.4.4), or a combination of both as appropriate. Either store the resulting clear solution refrigerated at 2 °C to 8 °C and carry out the determination within 48 h or alternatively freeze aliquots of the solution at .18 °C or lower for a period not exceeding 2 months before analysis.
A.6.2.6 Cut the section of the cuff above the 20 cm mark from the extracted outer glove, wipe the liquid off the surface with tissue, allow to dry at room temperature and weigh it to the nearest 0,1 g (m2). Calculate the mass (m) of the extracted part of the glove as follows:
ni = n1 m2
A.6.3 Protein standard
A.6.3.1 Stock protein solution
Prepare a solution of ovalbumin (A.3.8) with a nominal concentration of I mg/mI by dissolving 25 mg ovalbumin in 25 ml extraction buffer (A.3.2.2). Filter the solution through a 0,22 .im fdter (A.4.4) and determine the true concentration of ovalbumin by using an LJV spectrophotometer to measure the absorbance at 280 nm using a quartz cuvette (A.4.7). If the absorbance is divided by 0,715) it will give the exact concentration in mg/mI. The solution is stable for 2 days under refrigeration or for 2 months frozen at -18 °C. Thawing requires heating to 45°C for 15 min.
A.6.3.2 Protein standard solutions
Prepare serial dilutions of the protein stock solution (A.6.3.1) using the extraction buffer (A.3.2.2). to make
solutions with nominal concentrations of about 100 ig/ml. 50 .tgIml. 20 igIml, 10 pg/mI, 5 pg/mI and 2 pg/mI.
Use extraction buffer as a blank. The solutions are stable for 2 days refrigerated or for 2 months frozen at –
18 °C. Thawing requires heating 1045 °C for 15 mm.
A.6.4 Precipitation and concentration of protein
A.6.4.1 Carry out the procedure in duplicate at (25 ± 5) °C.
Assuming a molecular weight of 43000 D and a molar extinction of 30745 at 280 nm and pH 7.4, the extinction of 1 mg/mi ovalbumin in 0.1 M TES buffer pH 7,4 Is 0,715 using a light path of 1 cm [3].
A.6.4.2 Accurately transfer 1 ml each of the blank, protein standard solutions (A.6.3.2) and the four glove
extracts (A.6.2.5) into micro tubes (A.4.6). Add 0,1 ml of DOC (A.35), mix by vortexing and aflow to stand for
10 mm. Add 0,1 ml of TCA (A.3.6) and 0,1 ml PTA (A.3.7), mix by vortexing and allow to stand for a further
30 mm.
A.6.4.3 Centrifuge at 6000 g for 15 mm. Decant the supematant liquid and drain for 5 mm by inverting each centrifuge tube on an absorbent paper.
A..6.4.4 Add 0,2 ml of 0,1 M sodium hydroxide solution (A.34) to each tube including the blank. Mix on a vortex mixer to re-dissolve the precipitated proteins. Ensure that the proteins are completely re-dissolved to a clear solution. Depending on the glove this sometimes needs an overnight standing reingerated at (5 ± 3) °C. If any precipitate remains, add a further, measured quantity of the sodium hydroxide solution by 0.2 ml increments up to a total of I ml and use a 0,2 ml aliquot for subsequent steps. It can be useful to dilute the extract of such samples prior to precipitation.EN 455-3-2006 pdf download.