RIDA FLUOR Borrelia burgdorferi sensu lato IgG, IgM

Transcription

1 RIDA FLUOR Borrelia burgdorferi sensu lato IgG, IgM Art. No.: I3621 (IgG) I3631 (IgM) R-Biopharm AG, An der neuen Bergstraße 17, D Darmstadt, Germany Tel.: +49 (0) / Fax: +49 (0)

2 1. Intended use For in-vitro diagnostic use. The RIDA FLUOR Borrelia burgdorferi sensu lato test is an indirect immunofluorescence test (IFT) for the qualitative detection of IgG or IgM antibodies against Borrelia burgdorferi sensu lato (Borrelia burgdorferi (s.l.)) in human serum and for IgG antibodies against Borrelia burgdorferi (s.l.) in liquor. 2. Summary and explanation of the test In Mideuropean regions, Lyme Disease is the most common disease caused by tick-bites. The causative organism is a helical-formed bacterium of the genus Borrelia. It was named Borrelia burgdorferi according to its discoverer Willy Burgdorfer. Meanwhile three genospecies are known combined to the term B. burgdorferi (s.l.). In particular there are B. burgdorferi sensu stricto (s.s.), B. afzelii and B. garinii. In Europe, the transmission to humans and animals is caused mainly by bites of the tick species Ixodes ricinus. The infection rate of ticks with Borrelia burgdorferi changes from 5 % to 40 % depending on the area. As a result, much more than 10,000 infections per year are reported in Germany alone. Next to humans, domestic animals can also be infected. The clinical course of Lyme Disease can be divided into three stages. These stages can turn into each other, but they can also be separated by periods of a symptom-free latency. The course of the disease is not obligate. Symptoms generally found in specific stages can be missed and spontaneous recovery may occur at any point. With serological diagnostics it has to be considered, that in stage I % as well as in stage II % of the patients with clinical manifestations have no sufficient antibody titers against Borrelia burgdorferi. In stage III of the disease high antibody titers can be detected generally. Furthermore, it has to be considered, that an early antibiotic therapy can prevent the occurrence of serious and chronic disease manifestations. But, it reduces also the production of antibodies. Therefore, a prophylactic antibiotic therapy should be avoided if only a tick bite has been reported. Due to the fact that the cultivation of Borrelia from skin samples or blood is very difficult routine laboratories use tests for the detection of antibodies (IFT, ELISA, Western Blot). The RIDA FLUOR Borrelia burgdorferi sensu lato is covered with a mixture of B. burgdorferi sensu stricto, B. afzelii and B. garinii. RIDA FLUOR Borrelia burgdorferi sensu lato IgG, IgM

3 3. Test principle Inactivated Borrelia are fixed in defined wells on the surface of test slides. Diluted serum samples are given to the wells. Present anti-borrelia-antibodies bind to cell surfaces and form antigen-antibody-complexes. After a washing step for removing unbound antibodies, the complexes become visible by adding FITC-conjugated anti-human-immunoglobulines (anti-igg or anti-igm-conjugate). After a further washing step for removing excess conjugate, the test can be analyzed by a fluorescence microscope with 400x magnification. 4. Reagents provided Tab. 1: Contents of the kit (All reagents in this kit are sufficient for 10 x 10 reactions.) Slide 10 x 10 reactions slides with 10 wells each; sealed separately; coated with a German isolate of B. burgdorferi (s.s.), B. afzelii, B. garinii Buffer 50 ml Washing/Dilution Buffer (20x conc.); contains 0.2 % NaN 3 and Thimerosal each; for 1 l ready to use PBS-Buffer ph 7.5 Control IgG ml Positive Control IgG; Control IgM ml Positive Control IgM; Control 0.5 ml Negative Control; Conjugate IgG 2.5 ml Anti-human-IgG-FITC-Conjugate; FITC-conjugated antibody (goat); ready to use, with Evans Blue for counterstaining; contains 0.05 % NaN 3 and Thimerosal each Conjugate IgM 2.5 ml Anti-human-IgM-FITC-Conjugate; FITC-conjugated antibody (goat); ready to use, with Evans Blue for counterstaining; contains 0.05 % NaN 3 and Thimerosal each MF 2.5 ml Mounting Fluid; contains Antifade, ready to use I3621 I3631 RIDA FLUOR Borrelia burgdorferi sensu lato IgG, IgM

4 Tab. 2: Additional components Slide 10 x 10 reactions slides with 10 wells each; sealed separately; coated with a German isolate of B. burgdorferi (s.s.), B. afzelii, B. garinii Control IgG ml Positive Control IgG; Control IgM ml Positive Control IgM; Control 0.5 ml Negative Control; Conjugate IgG 2.5 ml Anti-human-IgG-FITC-Conjugate; FITC-conjugated antibody (goat); ready to use, with Evans Blue for counterstaining; contains 0.05 % NaN 3 and Thimerosal each Conjugate IgM 2.5 ml Anti-human-IgM-FITC-Conjugate; FITC-conjugated antibody (goat); ready to use, with Evans Blue for counterstaining; contains 0.05 % NaN 3 and Thimerosal each MF 2.5 ml Mounting Fluid; contains Antifade, ready to use Buffer 50 ml Washing/Dilution Buffer (20x conc.); contains 0.2 % NaN 3 and Thimerosal each; for 1 l ready to use PBS-Buffer ph 7.5 I3608 I3224 I3234 I0005 I0026 I0036 I0000 I Storage instructions All reagents have to be stored at 2 8 C and can be used up to the expiry date printed on the labels. If stored at 2 8 C the diluted buffer ca n be used up to four weeks, if stored at RT (20 25 C) it can be used up to five days. A quality w arranty cannot be given beyond the kit expiration date. Contamination must be avoided. 6. Materials required but not provided 6.1. Reagents - Distilled or deionized water - RIDA RF-Absorbens (Art. No. Z0202) - Possibly FTA absorbent RIDA FLUOR Borrelia burgdorferi sensu lato IgG, IgM

5 6.2. Accessories - Test tubes - Micropipettes for volumes of µl and µl - Vortex mixer - Moist chamber/incubator at 37 C - Washing trays for slides - Coverslips - Fluorescence microscope 7. Precautions for users For in vitro diagnostic use only. This test must only be carried out by trained laboratory personnel. The guidelines for working in medical laboratories must be followed and the instructions for carrying out the test must be strictly adhered to. Samples or reagents must not be pipetted by mouth and contact with injured skin or mucous membranes must be prevented. When handling the samples, wear disposable gloves and when the test is finished, wash your hands. Do not smoke, eat or drink in areas where samples or test reagents are being used. The control sera (positive control and negative control) in the kit have been tested for HIV- and HCV-Ab as well as HbsAg with negative results. Nevertheless, they must be treated as potentially infectious in the same way as the patient samples and all other materials with which they come into contact and they must be handled in accordance with the relevant national safety regulations. All reagents and materials coming into contact with potentially infectious samples must be treated with suitable disinfectants or autoclaved at 121 C for at least 1 hour. CAUTION: To prevent the formation of poisonous gases, any liquid waste containing stop reagent must be neutralized before adding it to hypochlorite solution. Slides and reagents must not be used if their pouch is damaged or the vials are leaking. 8. Specimen collection and storage The RIDA FLUOR Borrelia burgdorferi sensu lato IgG, IgM tests have been developed for the investigation of human serum and liquor samples. After blood collection, the blood should be separated from blood clots as soon as possible in order to prevent haemolysis. The samples must be stored cold or frozen until they are tested. Repeated freezing and thawing of the samples and microbial contamination must be prevented at all costs. Using heat-inactivated, lipaemic, haemolytic, icteric or turbid samples can lead to false results. RIDA FLUOR Borrelia burgdorferi sensu lato IgG, IgM

6 Tab. 3: Sample storage Undiluted serum/liquor Diluted serum 2 8 C 20 C 2 8 C 1 week > 1 week 7 hours 9. Test procedure 9.1. General information Bring all reagents and the test slides to room temperature before use. Take the slides out of the aluminum foil after they have reached room temperature and mark them on the mask. The reagents must be thoroughly mixed immediately before use. After use, the kit must be immediately returned to storage between 2 8 C. Take only the volume of reagents that is needed for test procedure. Do not pour reagents back into vials as reagent contamination may occur. Do not pour reagents back into vials as this may lead to reagent contamination Preparing the wash buffer 1 part of the concentrated washing/dilution buffer Buffer is diluted with 19 parts of distilled water. Add 50 ml of the concentrated washing buffer to a 1000 ml graduated cylinder. Bring the final volume up to 1000 ml with distilled or deionized water. Any crystals present in the concentrate must be solubilized (e.g. by warming in a water bath at 37 C). The diluted buffer can be used for a maximum of 4 weeks provided it is stored at 2-8 C Preparation the samples Before starting the test, serum and liquor samples have to be diluted with the washing/dilution buffer (see table 4). For IgM-determination an IgG absorption should be made with a rheumatoid factor absorbent (RIDA RF-Absorbens, Art. No. Z0202). Eventually, high positive sera should be further titrated with the buffer. Attention! Positive and negative controls are ready to use and can be used without dilution. Corresponding to patient sample dilution, the ready-to-use positive controls are prediluted 1:32 (IgM) and 1:64 (IgG). The titer specification on the label of the positive controls is the endpoint titer including the predilution. RIDA FLUOR Borrelia burgdorferi sensu lato IgG, IgM

7 Tab. 4: Sample dilution Serum Liquor IgM-determination IgG-determination IgG-determination Sample 5 µl 5 µl 20 µl RF-Absorbens 50 µl - - Incubation at RT* - - Dilution buffer 105 µl 315 µl 20 µl Final concentration 1:32 1:64 1:2 *RT: room temperature; for further information see instructions of RIDA RF-Absorbens 9.4. First incubation The slides are subdivided into single wells. Place one sample (patient or control) per well. Hydrophobic slide coating between the wells avoids a mixture of the samples. Add one drop of positive control Control IgG + or Control IgM + and negative control Control -. Pipette 20 µl of the diluted serum samples are pipetted. Incubate the slides in a moist chamber or incubator at 37 C for 30 minutes Washing Rinse the slides shortly with the washing buffer. Then, put the slides two times for 5 minutes into a container with fresh washing buffer. Rinse again shortly with washing buffer and remove all liquid by tapping the slides vertically on an absorbent paper. Do not allow the wells to dry at any time! 9.6. Second incubation Add one drop of the anti-human-fitc-conjugate (IgG or IgM) Conjugate IgG or Conjugate IgM to corresponding wells. Incubate the slides for 30 min at 37 C in a moist chamber or incubator Washing Wash according to step Microscopy Place the mounting fluid MF concentric in longitudinal direction on the coverslips. Put them on the wells without generating air bubbles. Remove access of mounting fluid by slightly pressing the coverslips on the slides. Read the result in a fluorescence microscope at 400x magnification. RIDA FLUOR Borrelia burgdorferi sensu lato IgG, IgM

8 10. Quality control indications of instability or deterioration For the quality control, positive control and negative control must be carried along with each test run. The test was carried out correctly if the negative control shows no specific fluorescence and the positive control shows a 4+ reaction (see also point 8.). If the positive control does not show a (4+)-reaction, which is manifested in just a weak fluorescence of the cell surface and a non-homogenous distribution of the fluorescence within the well, it may indicate that the reagents have expired. With slides containing a mixture of the three genospecies (B. burgdorferi s.l.) it can happen, that due to the different species not all Borrelia show a fluorescence signals. If the conditions are not fulfilled, please check the following before repeating the test: - kit expiry - functional capability of the instruments used (pipettes, microscope) - correct text execution - visual examination of kit components for contamination, deterioration or leakage If the test criteria are not fulfilled after repeating, please contact your local distributor of R-Biopharm. All additional reagents and slides are evaluated with the single components of the R-Biopharm kits. If slides and reagents from other companies are used the user has to validate these components. 11. Evaluation and interpretation Specific fluorescence is decisive for positive evaluation of a sample. The specific fluorescence is a bright green coloration visible on the cell surfaces of the Borrelia in a homogenous distribution within the wells. When using slides with a mixture of the three genospecies (B. burgdorferi sensu lato) it is possible, that due to the different species not all Borrelia show a fluorescence signal. Intensity of the fluorescence can vary from weak (1+ reaction) to very strong (4+ reaction). If no specific fluorescence is visible, the test is valuated negative. In this case Borrelia may be visible grey-green colored. Tab. 5: Validation of fluorescence Reaction Fluorescence Serum Titer Liquor Titer Remark 1:64 (IgG) 1:32 (IgM) 1:2 (IgG) 4+ very strong positive positive positive further titration is recommended 3+ strong positive positive positive further titration is recommended 2+ moderate positive positive positive further titration is recommended 1+ weak equivocal equivocal equivocal endpoint titer - not visible negative negative negative RIDA FLUOR Borrelia burgdorferi sensu lato IgG, IgM

9 12. Limitations of the method The RIDA FLUOR Borrelia sensu lato IgG, IgM tests detect antibodies against Borrelia. They should be used in reasonable suspected cases of Borrelia infections. The fluorescence intensity does not correlate with the clinical relevance. The results obtained should always be interpreted in consideration to clinical data. In the samples antibodies against Treponema may be included that may influence the test result false positive, it is recommended to absorb sera with a commercially available FTA-absorbent. Titers of 1:>64 (IgG) and 1:>32 (IgM) should be considered positive in non-endemic areas. However, in endemic areas with a high incidence of Borrelia an initial dilution of 1:128 for IgGdetermination is possibly recommended. Serum samples showing just a weak fluorescence (1+) with the recommended initial dilution are considered equivocal. Serum samples with titers of 1:<64 (IgG) and 1:<32 (IgM) are considered negative. A positive IgM result as well as a four fold IgG titer increase indicates an acute infection. To improve the diagnostic statement, two consecutive sera of a patient should be generally examined. Titer development is important for right interpretation. A positive result does not exclude the presence of other pathogens as cause for an illness. Negative antibody findings cannot exclude a Borrelia infection. Due to low antibody titers at the beginning of an infection, the tests can show negative results. Even in stage II of the disease, no detectable antibody levels are found in up to 30% of cases. If the clinical suspicion for a Borrelia infection subsists, a further patient sample should be taken and tested. A directly after a tick bite taken serum sample is unsuitable for a fresh infection even if the IgGtest shows a positive result (antibodies of a former infection). IgM-antibodies are detectable five days after a tick bite with Borrelia infection at the earliest. If antibiotics are given directly after a tick bite, the immune response can be reduced so that despite of an infection, antibodies will not become detectable with diagnostic tests. If the IFA is used for therapy control, take into consideration, that clear antibody titer decreases are often detectable not before months after a successful therapy. Additionally, IgM-antibodies may persist in these cases. RIDA FLUOR Borrelia burgdorferi sensu lato IgG, IgM