attomol HLA-B*27-Realtime LT 2 Assay for the detection of the human HLA-B*27-locus using LightCycler (Do not use for tissue typing!) For in vitro diagnostic use only! 50 determinations Order number: 95 C. Introduction The Human Leukocyte-Antigens (HLA) are glycoproteins of the major histocompatibility complex (MHC), which are expressed on nearly all nucleated cells. These antigens define the immunological individuality (differentiation between endogenic, i.e. internally generated and exogenic, i.e. foreign ). The HLA-B antigens belong together with HLA-A and HLA-C to the MHC class I molecules and their genes are located on the short arm of chromosome 6 (Thomas, 2000, Labor und Diagnose, 5. erweiterte Auflage, TH-Books Verlagsgesellschaft mbh, S. 878-888). There exist different forms of HLA-B antigens, the so-called determinants (e.g. HLA-B7, -B8, -B5,...). One of these determinants - HLA-B27 - was found in 973 to be closely related to certain rheumatic diseases, especially to Spondylitis ancylosans (SPA, Morbus Bechterew) (Brewerton et al., 973, Lancet :904-907). Carriers of HLA-B27 bear an 87-fold increase in the risk to suffer from SPA as carriers of other determinants. The disease manifests very often between the 5th and 30th year of life and in 90 % of all cases is found in men. HLA-B27 occurs in about 7 % of the Western Europe population. Patients with SPA carry HLA-B27 in 80-90 % of all cases (Isselbacher et al., 995, Harrisons Innere Medizin, 3. Auflage, Blackwell-Wissenschaftsverlag, S. 453-460). However, the pathogenesis is not completely understood (Taurog, J Rheumatol 200, 37(2):2606-6). Until now, HLA-B27 was detected serologically. Recently, this method is increasingly replaced by molecular biological techniques, because molecular tests work without living cells and are cheaper and easier to perform. 2. Important Remarks Keep the kit at the indicated storage temperature. This kit should only be used for in vitro diagnostic applications. Only use the latest provided manual for test realization. Do not use this kit after the expiration date. To carry out the assay take care for an appropriate laboratory equipment and well-trained staff. Wear gloves during all steps of the test application. Patient s samples should be considered as a potentially infectious agent and should therefore be handled according to the current law. Store reagents for the PCR apart from patient DNA samples and amplification products. 3. Materials included Order Number 326 Reagent Volumes Storage Temperature Oligomix HLA-B*27 LT 2 (blue cap) tube with 00 µl primer/probe mix, ready to use -20 C until first use, after first use store tubes dark at 4 C* * Freezing of oligomix after first use is recommended only if you do not plan to use it during the next 4 weeks.
2 4. Additional Reagents and Materials not included Patient s DNA samples (use genomic DNA dissolved in aqua dest. or TE-buffer) Control template (HLA-B*27 positive DNA) Micropipettes (volume range 0.5-000 µl) and sterile filter tips From the kit LightCycler Faststart-DNA-Master HypProbe from Roche (catalog number: 2 239 272 00 or 03 003 248 00): mix vial no. a (red cap) with vial no. b (colorless cap) according to instructions of Roche, resulting in 0x ready to use mix (contains Taq polymerase, nucleotides, MgCl 2 ) hereinafter called Roche mix PCR-water LightCycler -capillaries (20 µl, for LightCycler 2; order no. 04 929 292 00) or suitable PCR tubes for LightCycler 480 LightCycler 2.0 or 480 with appropriate control and evaluation software Color compensation file generated with the attomol color compensation kit 2 (order no. 97) 5. Test Principle The kit described here can be used to determine whether a patient carries the HLA-B27 allele or not. More detailed HLA-B27 subtyping or typing of other HLA-B determinants is not possible by using this test kit. This test only can evaluate the risk for rheumatic diseases (refer to paragraph ) but it is not allowed to use this test for tissue typing. After isolating DNA from patient s samples, the HLA-B*27-locus and a reference gene used as an internal standard become amplified parallel within one PCR reaction. Detection of amplification products takes place by hybridisation of the specific mutation-covering LoopTag* ) probe. The LoopTag probe hybridises to the elongated primer forming a primer-probe loop. The formation of a loop enables FRET between the fluorescence donor (D) and the fluorescence acceptor (A) as shown in fig.. Dehybridisation of LoopTag probe takes places during melting curve analysis and thereby confirms the amplification of the HLA-B*27-locus through specific melting temperatures (see subsection 6 Manual, subheading Protocol, sub item Evaluation ). The amplification of the internal standard indicates the proper process of PCR especially in the case of HLA-negative samples. A clinical diagnosis should not be concluded from results of one diagnostic method alone. The doctor has to consider all clinical and laboratory results available. 3 A primerelongation FRET A D 3 Fig. : LoopTag detection principle probe hybridisation * ) LoopTag is patented by Attomol (patent-no. PCT/EP2008/05752)
3 6. Manual In advance to the first usage of this test kit a color compensation file has to be generated with attomol color compensation kit 2 (order no. 97). It is necessary to include the following set of control PCR reactions in addition to patient s samples:. Negative control (PCR-blank) 2. Positive control (HLA-B*27 positive sample) The positive control may be ordered separately from the manufacturer (order number 06). Patient s samples that were genotyped correctly by the customer can also be used as positive control. Protocol: DNA-preparation from patient s blood according to standard procedures; resulting DNA samples (app. 20 ng/µl) can be used without dilution. Attention: If you have any problems (poor amplification, not evaluable melting curves) at first repeat the assay with diluted DNA samples (e.g. :0). Thaw tubes with oligomix, shake and centrifuge the tubes to pull down condensed water. Storage remark: After taking off the needed amount of oligomix you can store the rest of it in the refrigerator (4 C, dark). Freezing of oligomix is recommended only if you do not plan to use it during the next days. Before you take it again please centrifuge and mix it well. Generally avoid repeated freezing-thawing-cycles of the oligomix! Preparation of the PCR mix (8 µl PCR mix und 2 µl sample DNA are required for one PCR reaction) According to the number of samples pipette PCR water, Roche-Mix, and oligomix in an Eppendorf tube for the preparation of PCR mix (see following table). Mix it well after preparation. number of samples PCR water Roche-Mix oligomix number of samples PCR water Roche-Mix 2.4 2.2 4 28 59.6 30.8 56 4 22.8 4.4 8 30 7.0 33.0 60 6 34.2 6.6 2 32 82.4 35.2 64 8 45.6 8.8 6 34 93.8 37.4 68 0 57.0.0 20 36 205.2 39.6 72 2 68.4 3.2 24 38 26.6 4.8 76 4 79.8 5.4 28 40 228.0 44.0 80 6 9.2 7.6 32 42 239.4 46.2 84 8 02.6 9.8 36 44 250.8 48.4 88 20 4.0 22.0 40 46 262.2 50.6 92 22 25.4 24.2 44 48 273.6 52.8 96 24 36.8 26.4 48 50 285.0 55.0 00 26 48.2 28.6 52 oligomix Transfer of reagents to LightCycler -capillaries or -tubes Negative control: Positive control: Patient s sample: 8 µl PCR mix 8 µl PCR mix 8 µl PCR mix + 2 µl PCR-H 2 O + 2 µl DNA (HLA-B*27 positive) + 2 µl DNA Seal the capillaries/tubes and centrifuge. PCR incubation of the samples is performed in the LightCycler according to the program listed in appendix. Evaluation of the samples is done by analysis of amplification signals and melting peaks according to the following tables. A second human gene is amplified in all samples serving as an internal standard (IS). For HLA- B*27-negativ or not clearly identifiable samples it has to be verified if an amplification signal of the internal standard is existent. If for a sample neither a melting curve signal nor an amplification curve signal can be detected an inhibition of the PCR can be concluded. In this case the relevant sample has to be tested again (dilute the sample or repeat the DNA extraction).
4 Evaluation criteria: HLA-B*27 positive samples: LC 2.0 LC 480 HLA-B*27 amplification signal at 70 nm with clearly sigmoid or exponential growth, c t -value < 40, peak maximum of melting curve at 65 C ± 2 C amplification signal at 660, 670 or 705 nm with clearly exponential or sigmoid growth, c t-value < 40, peak maximum of melting curve at 65 C ± 2 C HLA-B*27 negative samples: LC 2.0 LC 480 HLA-B*27 no amplification signal at 70 nm, straight curve, slight, continuous slope possible, without melting curve peak or melting curve peak outside of the tolerance region at 65 C ± 2 C (e.g. in the case of other HLA-types) no amplification signal at 660, 670 or 705 nm, straight curve, slight, continuous slope possible, without melting curve peak or melting curve peak outside of the tolerance region at 65 C ± 2 C (e.g. in the case of other HLA-types) choose wavelengths according to the applied set of filters Internal standard amplification signal at 560 nm with sigmoid or exponential growth, c t-value < 36, can drop out in the case of a strongly HLA-B*27-positive sample amplification signal at 568 or 580 nm with sigmoid or exponential growth, c t-value < 36, can drop out in the case of a strongly HLA-B*27-positive sample Internal standard amplification signal at 560 nm with sigmoid or exponential growth, c t-value < 36 amplification signal at 568 or 580 nm with sigmoidal or exponential growth, c t-value < 36 Fig. 2: Examples for the evaluation of a positive HLA-B*27 sample (red curve), a negative sample (blue curve), and a blank (negative control, black curve) The example data were obtained by using LightCycler 480. A: amplification curves for HLA-B*27 B: melting curves for HLA-B*27 C: amplification curves for the internal standard In the case of problems during the test application do not hesitate to contact the manufacturer or the distributor.
5 Annex The wavelengths necessary for excitation and detection can be found in the following table: excitation detection for HLA-B*27 detection for internal standard (IS) LC 2.0 automatic 70 nm (F6) 560 nm (F2) LC 480 HLA-B*27: 483 or 498 nm 660, 670 or 705 nm IS: 523 or 533 nm 568 or 580 nm choose wavelengths according to the applied set of filters To perform the PCR and melting curve analysis use the programs in the following tables. Program for LC 2.0 Program for LC 480 Program: Activation; Type: None; Cycles: Program: Activation; Analysis : None; Cycles: Temperature Hold Time Slope Target Hold Ramp Rate Target ( C) (sec) ( C/sec) ( C) (s) ( C/s) 95 420 20 none 95 none 420 4.4 Program: Amplification; Type: Quantification; Cycles: 45 Program: Amplification; Analysis : Quantification; Cycles: 45 Temperature Target ( C) Hold Time (sec) Slope ( C/sec) Target ( C) Hold (s) Ramp Rate ( C/s) 95 3 20 none 95 none 3 4.4 62 30 20 single 62 single 30 2.2 72 30 20 none 72 none 30 4.4 Program: melting curve; Type: Melt. Curves; Cycles: Program: melting curve; Analysis : Melt. Curves; Cycles: Temperature Target ( C) Hold Time (sec) Slope ( C/sec) Target ( C) Hold (s) Ramp Rate ( C/s) 95 7 20 none 95 none 7 4.4 50 5 20 none 50 none 5 2.2 40 25 20 none 40 none 25 2.2 80 0 0.2 continuous 80 continuous 0 0.0 3 Program: Cooling; Type: None; Cycles: Program: Cooling; Analysis : None; Cycles: Temperature Hold Time Slope Target Hold Ramp Rate Target ( C) (sec) ( C/sec) ( C) (s) ( C/s) 40 20 20 none 40 none 20 2.2 Annex 2 Overview about symbols used for labeling of tubes and kit. explanation of symbols h manufacturer l batch number v expiry date 2 t 6 store between +2 C and +6 C o -20 store at a maximum of -20 C b catalogue number i in vitro diagnostic medical devices pn content sufficient for <n> determinations g consult instructions for use