YY/T 1465.5-2016Immunogenic evaluation method of medical devices - Part 5: Determination of a-Gal antigen clearance in medical devices utilizing animal tissues and their derivatives with M86 antibody (English PDF)

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 x 10^7 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 x 10^7 cells/mL. Store it at 4 °C for later use.

6.2.4 Preparation steps

Resuspend 1 x 10^6 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 alpha-Gal antigen removal process;

b) Material group after alpha-Gal antigen removal process;

c) Negative control group, materials that do not contain alpha-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 alpha-Gal antigen standard

Take alpha-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.

A.1 Test principle

The specific M86 antibody is combined with the alpha-Gal antigen in the test sample and stained by immunohistochemistry. By comparing the degree of color development before and after the alpha-Gal antigen removal process, the clearance rate of the alpha-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 alpha-Gal antigen, such as human acellular dermis or GGTA1 gene knockout pig skin.

b) Positive control group, which is pig skin sample without alpha-Gal antigen removal process.

c) Test sample group, which is pig skin sample with alpha-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

B.1 General

Sample processing should ensure full exposure of alpha-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. […]