Product no: 511.21 Batch no: TK0676 Primer Lot no: 523-x DynaMix PlusTM no: T0640 Expiry date: 2008-01 Size: 20 tests

Instructions For Use

Dynal AllSet+™ Minor Antigen (mHA) Typing Kit

For Research Use Only

INSTRUCTIONS FOR USE Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit

CONTENTS Page INTENDED USE…………………………………………………………………………..….…………… 3

SUMMARY AND EXPLANATION……...... ………………...………………………………………… 3

PRINCIPLES OF THE PROCEDURE ……………………………...... ……………..……………… 3

REAGENTS...... …...... …………...….…………………………… 4 Provided in Typing kit ...………………………………..……….……………………………… 4 Storage ………………………………………………………………..……..…………..……… 4 WARNINGS AND PRECAUTIONS ………………………………………………….…………………… 5 EQUIPMENT REQUIRED BUT NOT PROVIDED BY DYNAL ……….………………………………… 5 PROCEDURE ...... ……………………………….……………………………………….….…………… Sample Collection and Preparation...... …………………………………… 6 PCR Amplification Set-up ...... …...... ………...……………………… 6 PCR Amplification ...... ……………………...... ……………………… 6 PCR Detection using agarose …………………………………… 7 8 INTERNAL CONTROLS ...... ……...... …...……………………………….………… Internal Positive Control …………………………………………………………………… 9 Integrated Negative Control well ……………………………………………………… 9 9 INTERPRETATION ….……………………………………………………………………………………… 10 TROUBLESHOOTING GUIDE ...…...... ………...... …...……………………………. 11 BIBLIOGRAPHY ...... ………...... …………...…...... …...... …....…………………………… 12 WARRANTY ……………………………………………………………………………………………….. 13 TRADEMARKS USED IN THIS DOCUMENT/PRODUCT ……………..…..…………………………… 13 PATENTS USED IN THIS DOCUMENT/PRODUCT ….………………….………………………………… 13 CONTACT DETAILS ...... ………………………. 14

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INSTRUCTIONS FOR USE Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit

For Research Use Only

INTENDED USE DNA typing of Minor Histocompatibility antigen alleles.

SUMMARY AND EXPLANATION

Minor Histocompatibility (H) antigens may crucially influence the outcome of HLA-identical allogeneic stem cell transplantation 1. Typing of minor H antigens enables the identification of recipient/donor pairs eligible for adoptive immunotherapy and of pairs with increased risk for GvHD. The minor H antigen genotyping SSP Kit has been designed to address these issues in the minor H antigen component of the 14th International Histocompatibility and Immunogenetics Workshop (www.lumc.nl/ihiw14).

The kit is a PCR-based method designed to detect the alleles of 11 different minor H antigens 2. The minor H antigen typing protocol has been constructed to match the common HLA typing protocols. It can therefore easily be incorporated in routine tissue typing procedures. The method has been tested on DNA from EBV-LCL that were typed using the relevant minor H antigen specific clones.

PRINCIPLES OF THE PROCEDURE

Almost all DNA-based typing technologies utilize the PCR technique 3 to amplify the target to be investigated. In most DNA-based methods of tissue typing, the PCR technique is used as a pre-typing amplification step to increase the amount of target DNA. The typing process then requires a post- amplification step to discriminate between the different alleles. Unlike other PCR based methods, the SSP Methodology 4,5,6,7 employed by Dynal Biotech Ltd – AllSet+™ SSP, discriminates between the different alleles during the PCR process. This shortens the post-amplification processing time to a simple gel electrophoresis detection step.

The Dynal SSP method is a PCR based technique, which uses Sequence Specific Primers (SSP), for DNA based typing.

The Dynal SSP products consist of panels of primer mixes where each primer mix contains one or more specific primer pairs, i.e. the allele- and/or group-specific primers, as well as a control primer pair matching non-allelic sequences in the samples. The control primer pair functions as an internal PCR control to verify the efficiency of the PCR amplifications.

Dynal SSP is based on the principle that a perfectly matched primer will be more efficiently used in the PCR reaction than a primer with one or several mismatches at its 3’ –end. The specificity of the typing system is part of the PCR amplification step, and post-amplification processing of the samples is reduced to a minimum.

The assignment of alleles merely consists of determining whether amplification has occurred or not, i.e. visualisation and detection of the amplification by agarose gel electrophoresis. The PCR-SSP technique provides a high degree of resolution because each primer pair identifies two linked, cis-located polymorphic sites. The method provides high sensitivity, specificity and reproducibility.

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INSTRUCTIONS FOR USE Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit

REAGENTS

Reagents provided in the Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit

Item Description Each well in the PCR tray contains a dried SSP primer 1. PCR Trays with pre-aliquoted and solution consisting of allele- and/or group-specific dried SSP Primer Mixes primers as well as a control primer pair matching non- allelic sequences. The control primer pair amplifies a fragment of a conserved gene present in all samples. There is an integrated negative control in well # 21.

2. PCR Caps Caps for sealing the PCR Trays.

3. Master Mix 3 vials each containing 1.8ml Ready to use 1 x concentration. The Master Mix has been optimised for use with AmpliTaq® DNA Polymerase (Roche Molecular Systems, Inc.) supplied at 5i.u./μl Formulation information available from [email protected]

Combined Instructions For Use and Batch Specific 4. Combined Product Instructions Product Information (BSPI) booklet. For Use and Batch Specific Product

Information booklet 5. Interpretation worksheet A worksheet to determine which allele or group of alleles the respective primer mixes identify.

Storage

1. PCR Trays and Master Mix must be stored at 2-8˚C. 2. Do not freeze reagents. 3. If correctly stored, these reagents and primer mixes are stable until the expiration date indicated on this Batch Specific Instructions For Use Booklet.

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INSTRUCTIONS FOR USE Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit

WARNINGS AND PRECAUTIONS

1. For Research Use Only. 2. All work with Dynal AllSet+™ Products should be performed using Good Laboratory Practice and according to local guidelines e.g. EFI standards, CPA guidelines. 3. Biohazard warning Handle all samples as if capable of transmitting disease. All work should be performed wearing appropriate gloves and protection. 4. Biohazard warning Screw cap tubes should be used for specimen preparation to prevent specimen splashing and potential contamination. 5. Biohazard warning: All specimens should be handled as if infectious using safe laboratory procedures such as those outlined in Biosafety in Microbiological and Biomedical Laboratories 8 and in the NCCLS Document M29-T 9. Thoroughly clean and disinfect all work surfaces with 1% bleach (NaClO). Autoclave any equipment or materials that have come in contact with clinical specimens before discarding. 6. Biohazard warning: The ethidium bromide used for staining of DNA is a potential carcinogen. Always wear nitrile gloves when handling stained gels and ethidium bromide. 7. Caution: Wear UV-Blocking eye protection and do not view UV light source directly with naked eye when viewing or photographing gels. 8. Caution: Pipettes used for post-PCR manipulations should not be used for pre-PCR manipulations. 9. Caution: Contamination. Avoid microbial contamination of reagents when removing aliquots from reagent bottles. Always wear gloves to avoid any risk of sample contamination. The use of sterile disposable pipettes and filter pipette tips is recommended. Do Not use reagents with evidence of turbidity or microbial contamination. 10. Caution Disposal: Dispose of unused reagents and waste in accordance with country, federal, state and local regulations. 11. Material Safety Data Sheet (MSDS) is available to download at www.dynalbiotech.com or on request from your local Dynal Biotech Office. (See contact details listed on last page of this insert.)

EQUIPMENT REQUIRED BUT NOT PROVIDED BY DYNAL BIOTECH:

1. Manual pipetting devices (e.g Eppendorf, Gilson) 2. Disposable filter pipette tips for above 3. Vortex mixer 4. Microcentrifuge 5. PCR tray microtube storage rack 6. Perkin-Elmer GeneAmp® PCR system 9600 or 9700 or any licensed thermalcycler of equivalent specification 7. Hot-plate / microwave for heating agarose solutions 8. Electrophoresis apparatus (e.g ABgene Electro-Fast® Stretch 108 Complete, AB-0708) 9. UV transilluminator 10. Gel Photographic/Image documentation system 11. Recombinant Taq Polymerase (Dynal recommends AmpliTaq®) 12. Electrophoresis grade agarose 13. Ethidium bromide

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INSTRUCTIONS FOR USE Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit

PROCEDURE

NOTE: This procedure should be performed in two areas of the laboratory (Pre-PCR Amplification and Post-PCR Amplification) as indicated in the following instructions.

SAMPLE COLLECTION AND PREPARATION

Caution: Handle all specimens as if they are capable of transmitting infectious agents.

a) THE USE OF HEPARINISED BLOOD IS NOT RECOMMENDED 10. b) DNA can be extracted from human nucleated cells by any preferred method. c) To obtain optimal PCR-SSP amplification and typing results, use high purity DNA (OD 260:280 ratio 1.6-1.8). d) The final concentration of the DNA prior to PCR-SSP should be approximately 50ng/µl. e) Extracted DNA should be diluted in dH20. f) DNA may be stored at -20˚C or colder for extended periods of time (> 1 year) with no adverse affects on results. DNA stored at +4˚ for long periods of time will degrade, giving rise to more artefacts, e.g. non-specific amplifications.

PCR AMPLIFICATION SET-UP (Performed in the Pre-PCR area)

To perform a single typing, make the following PCR mix.

• 208 μl Master Mix • 49.4 μl Sample DNA (at 50 ng/µl) • 2.08 μl AmpliTaq® DNA polymerase (at 5i.u./μl)

• Mix well • Dispense 10 µl of the above PCR mix into each well of the Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing tray (Except Well No. 21 which contains the internal negative control – See page 9 for details) • Put on the PCR caps and close tightly to ensure that the tubes are fully sealed. A capping tool can be used. PCR compatible adhesive sealing sheets may also be used. The samples are now ready for PCR amplification • Store remaining kit reagents at 2-8˚C

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INSTRUCTIONS FOR USE Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit

TRAY LAY-OUT

The correct orientation of the tray is secured by the primer mix in well number #1,also identified by the use of a blue dye.

e.g mHA-X 1 2 3 4 5 6 7 8

9 10 11 12 13 14 15 16

17 18 19 20 21

ANY OTHER NUMBERS MOLDED INTO THE PLASTIC TRAY E.G.1,2,3 SHOULD BE IGNORED

PCR AMPLIFICATION SET-UP (Performed in the Post-PCR area)

1. Place the AllSet+™ Minor Histocompatibility Antigen tray into the thermalcycler Program your thermalcycler using the following parameters:

Steps Temperature Time (sec.) Action (˚c) A denaturation step 96 120 Denaturation

Denaturation 96 15 10 cycles Annealing 65 60 Extension 96 10 Denaturation 20 cycles 61 50 Annealing 72 30 Extension

For specific thermalcycler information, refer to the manufacturer’s handbook.

2. Start the program. 3. When the program is complete, remove the samples from the thermalcycler.

NOTE: Do not bring amplified DNA into the Pre-PCR amplification area.

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INSTRUCTIONS FOR USE Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit

PCR DETECTION USING AGAROSE GEL ELECTROPHORESIS

Note: It is recommended that the same buffer is used for both making the gel and as the running buffer i.e. if you use 0.5 x TBE in the gel, also use 0.5 TBE as the running buffer.

1. Prepare a 2% (w/v) agarose (ABgene MultiABgarose, AG-0100c) gel in 0.5 x TBE buffer.

a) Dissolve the agarose (2g/100ml 0.5 x TBE buffer) by boiling in a microwave oven until thoroughly dissolved. • Cool to 60˚C. • Add ethidium bromide to a final concentration of 0.5μg/ml gel.

b) Cast a 3-4mm thick gel with 3mm wide wells. • Allow to set for at least 30 minutes.

2. Transfer the agarose gel to a submarine gel electrophoresis unit. The gel should be covered by 0.5 x TBE to a depth of approximately 1-2mm above the gel surface.

• Carefully remove the comb. • Load the entire PCR product in sequence directly and carefully onto the gel.

3. Run the gels in 0.5 x TBE buffer. Run minigels (a gel less than 10cm between electrodes) for 10 minutes at 15V/cm and larger gels for 20 minutes at 10V/cm.

To calculate the voltage, measure the distance (in centimetres) between the ELECTRODES in the electrophoresis bath (the length of the gel is NOT IMPORTANT).

4. Examine the gel under UV illumination and document by photography.

a) The same 0.5 TBE buffer can be used several times, but performance may be enhanced by the use of fresh buffer.

WARNING: Ethidium Bromide is a powerful mutagen. Wear nitrile gloves and eye protection when handling gels or solutions containing Ethidium Bromide.

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INSTRUCTIONS FOR USE Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit

Internal Positive PCR Control

The control primer pair 11 is included to verify the efficiency of the PCR reaction. Its presence confirms that the lack of a specific amplification in any given reaction is truly due to the absence of that polymorphism in the DNA sample, and not due to a failure of the PCR amplification.

Due to the different concentrations and annealing temperatures of the specific primer pairs as compared to the internal control primer pair: - The intensity of the control band often decreases in the presence of a specific amplification and - The internal control primers are more sensitive to non-optimal PCR conditions and can be used to monitor the PCR efficiency.

Failed amplification of the internal control fragments in lanes without specific amplifications should be considered as a sign of a non-optimal system. Please see the trouble shooting section for possible reasons and correct actions.

The following internal positive control, which amplifies a segment of the HLA-DRA gene is used in this kit.

1009bp Forward primer 5' GGCCGAGTTCTATCTGAATCCTGAC Reverse primer 5' GGTCCATACCCCAGTGCTTGAGAAG

Integrated Negative Control Well

This kit contains an integrated negative control in well #21

The Negative Control well is intended to check for contamination that may be present in DNA diluent, buffers or Taq Polymerase. The well contains the internal control primer pair to detect contamination with PCR amplified fragments derived from Dynal SSP products amplified via the internal control primers of the kit in use. Contamination with human genomic DNA will also be detected.

Instructions for Use - negative control well

Mix 2µl of the diluent normally used to dilute your sample DNA (minus DNA) with 8µl of the Dynal Mastermix/DynaMix solution and 0,1µl of Taq polymerase and then add to the dried pre-aliquoted negative control well (well #21). Where there is no contamination, no detectable PCR fragment will be seen when run under the same PCR conditions as Dynal AllSet+™ sets.

In the case of contamination, the negative control well will give rise to an internal positive control band.

This product may not detect contamination from other PCR based typing kits.

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INSTRUCTIONS FOR USE Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit

Interpretation

Alleles are assigned by the presence of a specific PCR product. A specific amplification in one lane indicates that the DNA sample contains the allele defined by the primer pair in that PCR reaction. The interpretation table supplied (and printed below) can be used to assign the minor H alleles present in the sample tested.

The approximate sizes of the specific PCR products can be used in order to distinguish between a true positive amplification and: - primer oligomer artifacts (primer dimers) - carry over from adjacent wells - non-specific amplifications

Tube No. Minor b.p. Polymorphism

1 190 H HA-1 2 190 R 3 271 V HA-2 4 271 M 5 128 T HA-3 6 317 M 7 186 R HA-8 8 186 P 9 213 H HB-1 10 210 Y 11 136 Y ACC-1 12 136 C 13 197 D ACC-2 14 197 G 15 185 R HwA-9 16 185 G 17 207 R HwA-10 18 207 stop 19 UGT2B17 353 Ex1a 20 HY 136 HY 21 Neg Ctrl 1009 Page 10 of 14

INSTRUCTIONS FOR USE Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit

TROUBLE SHOOTING GUIDE

PROBLEM REASON ACTION No amplification (neither amplification of the Check the volume and concentration of DNA added were internal control fragments, Insufficient amount of DNA correct. nor specific amplification) The DNA contains PCR inhibitors e.g. proteins, ethanol (from Measure the DNA purity. Try alternative DNA extraction “ precipitation steps). method. “ The DNA has been extracted from heparinised blood Use non-heparinised blood. “ The DNA is dissolved in a buffer containing EDTA (e.g. TE) Repeat the DNA extraction and dissolve DNA in sterile water. Make sure PCR caps are tightly closed – use a capping tool, or Random failure of PCR tubes that are not tightly closed will lead to evaporation use PCR compatible adhesive sealing sheets as an alternative amplification and subsequent failure of amplification (available from Dynal Biotech – Product Code 990.99). Check the correct number of wells have been loaded and that

Gel-loading mistakes each well contains approximately the same volume of PCR “ mixture. Calibrate all pipettes routinely according to the suppliers Use of non-calibrated pipettes “ recommendations. “ Pipetting errors Perform pipetting with more care. Sample DNA, Taq polymerase or PCR pre-mixtures have not Mix briefly by vortexing before use. “ been mixed properly before use Weak internal control Measure the DNA quality. If necessary repeat the DNA Impure DNA fragments extraction with freshly prepared solutions. “ Low amount of DNA Measure the DNA concentration and adjust to 50ng/μl. Check the Dynal SSP PCR parameters have been programmed

Too high annealing temperature correctly. Thermalcyclers should be professionally calibrated at “ least once every 12 months.

The quality of the Taq Polymerase is poor Use AmpliTaq® from Perkin-Elmer/Roche. “ Non-specific amplification Impure DNA Measure the DNA quality. Repeat the DNA extraction with (ladders or smears) freshly prepared solutions. Increasingly weak Prepare fresh ethidium bromide solution to achieve better amplification signals over The ethidium bromide agarose gel staining solution is old staining of the gel. time “ One of the UV lamps is broken Check UV light equipment. “ Incorrect Kit Storage Follow storage instructions on product packaging. Check the Dynal SSP PCR parameters have been programmed False negative Too high annealing temperature correctly. Thermalcyclers should be professionally calibrated at amplifications least once every 12 months. “ Low amount of DNA Check the volume and concentration of DNA used. Measure the DNA quality. Repeat the DNA extraction with Impure DNA “ freshly prepared solutions. “ The amount of DNA Taq Polymerase used is too low Check volume carefully “ The quality of the Taq Polymerase is poor Use AmpliTaq® by Perkin Elmer/Roche. Use gloves, pipette tips containing filter plugs, use separate False positive areas for pre and post-PCR. Employ careful and accurate May be caused by Contamination amplifications sample handling in all steps. Check for contamination using the Dynal Contamination Control - Product Code 990.05. Measure the DNA quality. If necessary, repeat the DNA Impure DNA “ extraction with freshly prepared solutions. Check the Dynal SSP PCR parameters have been programmed Too low annealing temperature. correctly and have your Thermalcyclers professionally calibrated “ at least once every 12 months. Strange amplification Check the batch number of the product used is the same as the Incorrect interpretation table is used patterns batch number on the interpretation table being used. Check all amplifications are the correct size. An artifact (carry-

The amplification pattern contains a ‘false positive’ over, primer-dimer) may have been mis-interpreted as a specific “ amplification. Repeat the typing, if reproducible, please contact Dynal Biotech

New allele, not covered by the interpretation table for information on amplification patterns of recently described “ alleles. Indistinct, blurred bands The electrophoresis buffer might be warm Use the TBE buffer recommended. Run at a lower voltage. “ The comb used has too thick slots Use thinner combs. Amplicon fails to load Different strength running buffer to gel buffer used Use same buffer for gel and running buffer, 0.5 x TBE properly recommended, Pipette properly. “ Running buffer at wrong concentration 0.5 x TBE is recommended. “ Air bubbles in pipette Pipette carefully.

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INSTRUCTIONS FOR USE Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit

BIBLIOGRAPHY

1) Goulmy E. Human minor histocompatibility antigens. Curr Opin Immunol 1996: 8: 75-81

2) Spierings E. et al. Manuscript in preparation

3) Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350-4.

4) Wu, D. Y., Ugozzoli, L., Pal, B. K. & Wallace, R. B. (1989). Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia. Proc Natl Acad Sci U S A 86(8), 2757-60.

5) Newton, C. R., Graham, A., Heptinstall, L. E., Powell, S. J., Summers, C., Kalsheker, N., Smith, J. C. & Markham, A. F. (1989). Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 17(7), 2503-16.

6) Olerup, O. & Zetterquist, H. (1992). HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation [see comments]. Tissue Antigens 39(5), 225- 35.

7) Bunce, M., O'Neill, C. M., Barnardo, M. C. N. M., Krausa, P., Browning, M. J., Morris, P. J. & Welsh, K. I. (1995b). Phototyping: Comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilising sequence-specific primers (PCR- SSP). Tissue Antigens 46(5), 355-367.

8) Richmond, Y. and McKinney, W. (eds.) 1993. Biosafety in microbiological and biomedical laboratories. HHS Publication Number (CDC) 93-8395.

9) National Committee for Clinical Laboratory Standards. Protection of Laboratory Workers from Infectious Disease Transmitted by Blood, Body Fluids and Tissue. Tentative Guideline. NCCLS Document M29-T Villanova, PA:NCCLS, 1989.

10) Satsangi J, Jewell DP, Welsh K, Bunce M, Bell JI.Effect of heparin on polymerase chain reaction. Lancet. 1994 Jun 11;343(8911):1509-10.

11) Bein G, Glaser R, Kirchner H. Rapid HLA-DRB1 genotyping by nested PCR amplification. Tissue Antigens. 1992 Feb;39(2):68-73.

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INSTRUCTIONS FOR USE Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit

WARRANTY

The products are warranted to the original purchaser only to conform to the quantity and contents stated on the vial and outer labels for the duration of the stated shelf life. Dynal Biotech's obligation and the purchaser's exclusive remedy under this warranty is limited either to replacement, at Dynal Biotech's expense, of any products which shall be defective in manufacture, and which shall be returned to Dynal Biotech, transportation prepaid, or at Dynal Biotech's option, refund of the purchase price.

Claims for merchandise damaged in transit must be submitted to the carrier.

This warranty shall not apply to any products that shall have been altered outside Dynal Biotech, nor shall it apply to any products that have been subjected to misuse or mishandling. ALL OTHER WARRANTIES, EXPRESSED, IMPLIED OR STATUTORY, ARE HEREBY SPECIFICALLY EXCLUDED, INCLUDING BUT NOT LIMITED TO WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Dynal Biotech's maximum liability is limited in all events to the price of the products sold by Dynal Biotech. IN NO EVENT SHALL DYNAL BIOTECH BE LIABLE FOR ANY SPECIAL, INCIDENTAL OR CONSEQUENTIAL DAMAGES. Some states do not allow limits on warranties, or on remedies for breach in certain transactions. In such states, the limits set forth above may not apply.

This product may not be repacked, reformulated or resold in any form without the written consent of Dynal Biotech Ltd., U.K.

TRADEMARKS USED IN THIS DOCUMENT/ PRODUCT

Dynal® is a registered trademark of DYNAL BIOTECH A.S.A, Oslo, Norway AllSet™ and AllSet+™ are trademarks of DYNAL BIOTECH A.S.A, Oslo, Norway AmpliTaq® is a registered trademark of Roche Molecular Systems, Inc. USA. Electro-Fast® is a registered trademark of Advanced Biotechnologies Ltd (ABgene®) GeneAmp® is a trademark of Roche Molecular Systems, Inc. USA DynaMix Plus™ is a trademark of DYNAL BIOTECH Ltd., U.K.

PATENTS USED IN THIS DOCUMENT/PRODUCT

* The Dynal SSP products are sold under license from ZENECA Ltd. ARMS™ in the subject of European Patent No. 0332435 and corresponding world wide patent property. ARMS™ is a trademark of ZENECA Ltd.

** This product is optimized for use in the Polymerase Chain Reaction (“PCR”) Process which is covered by patents owned by Roche Molecular Systems Inc. and F. Hoffmann-La Roche Ltd (“Roche”). No license under these patents to use the PCR Process is conveyed expressly or by implication to the purchaser by the purchase of this product. Further information on purchasing licenses to practice the PCR Process may be obtained by contacting, in the United States, the Director of Licensing at Roche Molecular Systems,, Inc., 1145 Atlantic Avenue, Alameda, California, 94501, and outside the United States, the PCR Licensing Manager, F. Hoffman- La Roche Ltd, Grenzacherstr. 124, DH-4070 Basel, Switzerland.

This product is sold under license from LEIDEN UNIVERSITY MEDICAL CENTER and THE UNIVERSITY OF VIRGINIA PATENT FOUNDATION (patent series WO 97/05168 relating to the H-Y antigen; WO 97/05169 relating to the HA-2 antigen; and WO 99/05173 relating to the HA-1 antigen), and from LEIDEN UNIVERSITY MEDICAL CENTER (patent series WO 99/05313 relating to methods for typing of the HA-1 antigen).

Manufactured by: Dynal Biotech Ltd., U.K. Developed by: LEIDEN UNIVERSITY MEDICAL CENTER, Department of Immunohematology and Blood Transfusion.

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INSTRUCTIONS FOR USE Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit

CONTACT DETAILS

For country-specific contact information visit our web site at

www.invitrogen.com

11 Bassendale Road, Croft Business Park, Bromborough, Wirral, CH62 3QL, U.K. Tel: +44 151 346 1234, Fax: +44 151 346 1223 e-mail: [email protected]

© Copyright 2006 Dynal Biotech Ltd., U.K. All rights reserved Printed: 27.09.06 Rev. 000

Dynal AllSet+TM Worksheet

Minor Histocompatibility Antigen (mHA) Tube Minor b.p. Polymorphism Typing Kit: No.

Result Result Prod. No: 511.21 1 190 H HA-1 Kit Batch No. TK0676 2 190 R Primer Lot No. 523-x 3 271 V HA-2 DynaMix PlusTM no: T0640 4 271 M Expiry Date: 2008-01 5 128 T HA-3 6 317 M

7 186 R HA-8 Institution: 8 186 P Sample ID: 9 213 H HB-1 10 210 Y Patient Name:

11 136 Y ACC-1 Patient ID: 12 136 C Date of Birth: 13 197 D ACC-2 14 197 G

15 185 R HwA-9 16 185 G Comments:

17 207 R HwA-10 18 207 stop

19 UGT2B17 353 Ex1a 20 HY 136 HY Neg Ctrl 21 1009

Tested By: Date:

Approved By: Date:

Gel Picture