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YY/T 1465.5-2016 (YY/T1465.5-2016, YYT 1465.5-2016, YYT1465.5-2016)
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YY/T 1465.5-2016: PDF in English (YYT 1465.5-2016)

YY/T 1465.5-2016
YY
PHARMACEUTICAL INDUSTRY STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
ICS 11.120.20
C 30
Immunogenic evaluation method of medical devices - Part 5:
Determination of α-Gal antigen clearance in medical devices
utilizing animal tissues and their derivatives with M86
antibody
ISSUED ON: JULY 29, 2016
IMPLEMENTED ON: JUNE 01, 2017
Issued by: China Food and Drug Administration.
Table of Contents
Foreword ... 3
Introduction ... 4
1 General provisions ... 5
2 Normative references ... 5
3 Terms and definitions ... 6
4 Test principle ... 6
5 Reagents and instruments ... 6
6 Test steps ... 6
7 Data analysis ... 9
8 Test report ... 10
Appendix A (Informative) Example of determination of α-Gal antigen clearance rate in
enzymatically hydrolyzed pig skin products by immunohistochemistry ... 11
Appendix B (Informative) Example of sample processing ... 14
References ... 16
Immunogenic evaluation method of medical devices - Part 5:
Determination of α-Gal antigen clearance in medical devices
utilizing animal tissues and their derivatives with M86
antibody
1 General provisions
This Part of YY/T 1465 provides a method for determining the clearance rate of α-Gal
antigen in medical devices of animal origin, which is suitable for the evaluation of the
effectiveness of the α-Gal antigen clearance process.
The method in this Part is applicable to materials that can fully expose the α-Gal antigen
through grinding. If the α-Gal antigen cannot be fully exposed through grinding, other
confirmed suitable methods may be considered.
Note: Appendix A gives examples of immunohistochemistry evaluation methods.
2 Normative references
The following documents are essential to the application of this document. For the dated
documents, only the versions with the dates indicated are applicable to this document;
for the undated documents, only the latest version (including all the amendments) is
applicable to this standard.
GB/T 16886.2 Biological evaluation of medical devices - Part 2: Animal welfare
requirements (GB/T 16886.2-2011, ISO 10993-2:2006, IDT)
GB/T 16886.20 Biological evaluation of medical devices - Part 20: Principles and
methods for immunotoxicology testing of medical devices (GB/T 16886.20-2015,
ISO/TS 10993-20:2006, IDT)
YY/T 0771.1 Medical devices utilizing animal tissues and their derivatives - Part 1:
Application of risk management (YY/T 0771.1-2009, ISO 22442-1:2007, IDT)
Chinese Pharmacopoeia 2010 Edition
avoiding excessive destruction. The recommended particle size distribution range of
the processed sample is (0 μm ~ 90 μm). Examples of different types of sample
processing are given in Appendix B.
Note 1: Animal-derived biological materials are mainly composed of structural proteins, such
as collagen fibers. Experimenters should use appropriate sample preparation methods, to ensure
that the α-Gal antigen in the test sample is fully exposed.
Note 2: Three parallel samples are prepared for each sample; the average value is taken as the
final measurement result.
Note 3: According to the request of the client, 3 samples of different batch numbers or the same
batch number can be taken, to evaluate the process stability of different batches or the same
batch.
6.2 Preparation of test system control materials
6.2.1 General
Considering that the methods in this standard are affected by many factors, appropriate
control substances should be used, to verify the rationality of the test system before
testing. It is recommended to use mouse myeloma (SP2/0) cells or rabbit red blood cells
(RRBC). If other reference substances are selected, their effectiveness should be
confirmed.
6.2.2 SP2/0 cells
SP2/0 (ATCC CRL-1581TM) is obtained from the fusion of spleen B cells of BALB/c
mice immunized with sheep red blood cells and mouse myeloma cells. It does not
secrete immunoglobulins. Use RPMI1640 complete medium to culture, until the growth
state is good; adjust the cell concentration to 1 × 107 cells/mL for later use.
6.2.3 RRBC
6.2.3.1 It is recommended to use untested healthy New Zealand rabbits, SPF grade. If
other strains of rabbits are selected, their suitability should be explained. Animals
should be kept for at least 5 days before blood collection, to adapt to the laboratory
environment. All animal testing should be conducted in laboratories approved by
national accreditation agencies and in compliance with all applicable regulations on
laboratory animal welfare, meanwhile it should also comply with the requirements of
GB/T 16886.2.
6.2.3.2 Take 20 mL of fresh rabbit whole blood. Add it to an Erlenmeyer flask
containing glass beads. Shake for 10 minutes, to remove fibrin. Add about 10 times the
amount of 0.9% sodium chloride solution. Shake well. Centrifuge at 2000 r/min for 5
minutes. Remove the supernatant. Wash the precipitated red blood cells 3 times, using
0.9% sodium chloride solution, according to the above method, until the supernatant is
no longer red. The obtained red blood cells are mixed with 0.9% sodium chloride
solution to 1 × 107 cells/mL. Store it at 4 °C for later use.
6.2.4 Preparation steps
Resuspend 1 × 106 SP2/0 cells or RRBC in 40 μL of 1% BSA. Mix well. Dilute 20 μL
of cells to create a total of 4 concentration gradients (including a blank group without
cells). Add 1% BSA solution to the cell stock solution and diluent, respectively, 80
μL/tube. Then the volume ratio of the highest concentration group containing the cell
stock solution is 20%. The volume ratio of the remaining groups decreases in a gradient
with the doubling dilution. Three replicate holes for each concentration gradient are
available for use.
6.3 Test grouping
Test grouping includes:
a) Material group without α-Gal antigen removal process;
b) Material group after α-Gal antigen removal process;
c) Negative control group, materials that do not contain α-Gal antigen;
d) Test solvent control group.
6.4 Determination of optimal M86 antibody dilution
6.4.1 General
There may be certain differences in the potency of different batches of M86 antibodies;
the optimal dilution of each batch of M86 antibodies should be determined. Divide the
commercially available M86 into appropriate volumes. Store it for later use.
6.4.2 Preparation of α-Gal antigen standard
Take α-Gal-BSA. Dissolve it in the coating solution at pH 9.6. Add it to the microplate,
100 μL/hole. The recommended final concentration is 1 μg/mL ~ 10 μg/mL. Coat
overnight at 4 °C. Use 1% BSA sealed for later use.
6.4.3 Optimal dilution of M86 antibody
Add 100 μL of diluted M86 antibody solution to each microplate, for a total of 8
dilutions. The recommended initial dilution is 1:25. The dilution is between 1:25 and
1:3200. Mix well. Incubate with shaking at room temperature for 1.5 hours. Use
washing solution to wash the plate three times. Add HRP-labeled anti-mouse secondary
antibody. Incubate with shaking at 37 °C for 1 hour. Then use washing solution to wash
the plate three times. After color development, use a microplate reader to read the
absorbance value. Draw a reaction curve between the absorbance value and the
corresponding M86 dilution. Take twice the concentration value of the dilution, that
just enters the plateau area of the reaction curve, as the optimal dilution. Dilute the
stored M86 antibody before each use.
6.5 M86 antibody co-incubation
Add 160 μL of the M86 antibody solution, at the dilution ratio determined in 6.4, to
each control substance and each test tube, at the same initial concentration grouped
according to 6.3, that is, the total volume of each reaction tube is 200 μL. Mix well.
Shake and incubate at 4 °C overnight. Centrifuge the tube at 10000 g for 5 minutes.
Take the supernatant for later use.
6.6 Indirect inhibition ELISA method to detect the remaining M86 antibodies in
the supernatant solution
Add excess α-Gal antigen solution, 100 μL/hole, to the corresponding holes of the 96-
hole plate. Incubate at 4 °C overnight. Use washing solution to wash the plate three
times. Take 100 μL of the supernatant prepared according to 6.5. Add it to the 96-hole
plate coated with the corresponding antigen. After shaking and incubating at 37 °C for
2 hours, use the washing solution to wash the plate three times. Add HRP-labeled anti-
mouse secondary antibody. Incubate with shaking at 37 °C for 1 hour. Then use the
washing solution to wash the plate three times. After color development, read the
absorbance value with a microplate reader.
7 Data analysis
7.1 First, perform 3 repeated measurements on the test system's control substance and
test sample. Take the average absorbance value of the 3 measurements. Calculate the
M86 binding inhibition rate (remaining M86 binding rate), according to formula (1).
Where:
I - M86 binding inhibition rate;
a - OD value of the material group that has undergone the α-Gal antigen removal
process, the material group that has not been α-Gal antigen removed, the negative
control or the test system control substance;
b - OD value of solvent control.
7.2 Use the binding inhibition rate of the test system's control substance M86 (repeated
measurements 3 times) and the corresponding dilution, to obtain the binding inhibition
rate curve equation. R2 should not be less than 0.96.
Appendix A
(Informative)
Example of determination of α-Gal antigen clearance rate in enzymatically
hydrolyzed pig skin products by immunohistochemistry
A.1 Test principle
The specific M86 antibody is combined with the α-Gal antigen in the test sample and
stained by immunohistochemistry. By comparing the degree of color development
before and after the α-Gal antigen removal process, the clearance rate of the α-Gal
antigen is calculated.
A.2 Reagents and instruments
A.2.1 Reagents
A.2.1.1 Neutral formalin.
A.2.1.2 5%BSA.
A.2.1.3 Xylene.
A.2.1.4 Mouse M86 antibody.
A.2.1.5 PBS.
A.2.1.6 Tissue repair solution.
A.2.1.7 Ethanol.
A.2.2 Instruments
A.2.2.1 Oven.
A.2.2.2 Tissue sectioning equipment.
A.2.2.3 Optical microscope.
A.3 Experimental procedures
A.3.1 Sample preparation
Take the samples to be tested that have been preserved and fixed in formalin, embedded
in conventional paraffin, sliced, baked in a 60 °C oven overnight for later use.
A.3.2 Test grouping
Carry out the test according to the following groupings (5 samples per group, 3 slices
prepared in parallel for each sample):
a) For the negative control group, it is recommended to use tissues that do not contain
α-Gal antigen, such as human acellular dermis or GGTA1 gene knockout pig skin.
b) Positive control group, which is pig skin sample without α-Gal antigen removal
process.
c) Test sample group, which is pig skin sample with α-Gal antigen removed by
enzymatic hydrolysis.
A.3.3 Test procedures
A.3.3.1 Slices are deparaffinized in conventional xylene and subject to gradient ethanol
treatment.
A.3.3.2 Rinse with distilled water and soak in PBS, 5 minutes/time, 2 times in total.
A.3.3.3 Microwave repair for 10 minutes. First heat the repair solution to 95 °C ~
100 °C. Then put in the slices.
A.3.3.4 Incubate with 3% hydrogen peroxide for 10 minutes at room temperature, to
block the activity of endogenous peroxidase.
A.3.3.5 Rinse with PBS, 5 min/time, 3 times in total.
A.3.3.6 Block with 5% BSA solution for 30 minutes.
A.3.3.7 Add the appropriate dilution of M86 antibody determined in 6.4; incubate at
4 °C overnight.
A.3.3.8 Rinse the slices with PBS, 5 minutes/time, a total of 3 times.
A.3.3.9 Add the secondary antibody working solution according to the instructions.
Incubate at room temperature or 37 °C for 15 min ~ 20 min. Rinse the slices with PBS,
5 min/time, a total of 3 times.
A.3.3.10 DAB color development takes 3 min ~ 15 min. Rinse thoroughly with running
water.
A.3.3.11 If necessary, the nuclei can be counterstained with hematoxylin (0.5% ~ 1%),
dehydrated, transparent, sealed.
A.3.4 Test results
Appendix B
(Informative)
Example of sample processing
B.1 General
Sample processing should ensure full exposure of α-Gal antigen and avoid excessive
destruction. It is recommended to use tissue homogenizer1 ) for sample processing.
Other equivalent processing methods can also be used; however, users should confirm
the selected test parameters.
B.2 Sample processing
B.2.1 Bone samples
For bone implant samples, take 100 mg of the sample into a 1.5 mL grinding bottle.
Add eight 3 mm and one 5 mm ceramic magnetic beads. Rotate at 6800 r/min, 30 s/time.
Cool with liquid nitrogen and grind 2 or more times (interval time: 30 s), until the
sample is completely crushed (particle size less than 100 μm). Place it on ice for later
use. Take an appropriate amount of test sample. Weigh it accurately and record it. Put
it into a 0.5 mL centrifuge tube. Add 0.1 mL of 1% BSA solution. Take the homogenate
for doubling gradient dilution, at least 4 gradients. Take 40 μL each. Place it in a 0.5
mL centrifuge tube for later use.
B.2.2 Dry sample
Take an appropriate amount of test sample. Weigh it accurately and record it. Use sterile
ophthalmic scissors to cut the sample into appropriate size. Put it into a 1.5 mL grinding
bottle. Add 300 μL of 1% BSA solution. Add eight 3 mm and one 5 mm ceramic
magnetic beads. The rotation speed is 6800 r/min, 30 s/time. Cool it with liquid nitrogen
and grind it 2 or more times (interval time is 30 s), until the sample is completely
crushed (the particle size is less than 100 μm) and a uniform tissue homogenate is
formed. Take the homogenate and carry out doubling gradient dilution, for at least 4
gradients. Take 40 μL of each and place it in a 0.5 mL centrifuge tube for later use.
B.2.3 Wet sample
Take an appropriate amount of test sample. Place it in a 1.5 mL sterile centrifuge tube.
Vacuum dry or assist drying in a 37 °C drying oven. Then take the test sample (≤ 300
µg). Weigh it accurately and record it. Use sterile ophthalmic scissors to cut the sample
1 BertinPrecellys24 is an example of a suitable commercially available product. This information is given for the
convenience of users of this standard and does not imply endorsement of the product. Other equivalent products may
be used if they have the same effect.
......
 
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.