Bp IgG/IgM gemeinsam
Contents
1. v FIRST INCUBATION 60 MIN v FIRST WASH STEP y After incubation wash 4 times with WASHBUF v Pipette 1000 pl CONJ ALK each chamber vy SECOND INCUBATION 30 MIN y SECOND WASH STEP see 1 Wash step v Pipette 1000pl SUBSJCH each chamber v THIRD INCUBATION SUBSTRATE INCUBATION 15 MIN v After 15 min stop the Enzyme Substrate reaction 2 times 5 min with 2 ml aqua dest v VISUEL EVALUATION OR WITH VIRO BLOT SOFTWARE mall CE Viro Immun Labor Diagnostika GmbH In der Au 29 D 61440 Oberursel Germany Tel 49 6171 6281 00 Fax 49 6171 6281 12 email info viro immun de2. 1 weak antigen band questionable 2 weak antigen bands positive at least 1 positive antigen band particulary for Flagellin p41 or p39 and Osp C p25 13 Performance Characteristics Specificity Sensitivity 216 samples IgG and 62 samples IgM were tested parallel in VIRO BLOT Anti Borrelia IgG and IgM and comparison methods ELISA The sensitivity and specificity are based on the results found Specificity IgG 97 9 Sensitivity IgG 87 8 Specificity IgM 100 Sensitivity IgM 82 6 Borderline results were not taken into account in the calculation 14 Cross reactivity e In the early infection phase there may be an insufficient number of antibodies and therefore a negative test result is possible e When there is a clinical suspicion of Borrelia infection with a negative or questionable test result a repeat test two weeks later is recommended e Antibiotic therapy may suppress the production of detectable antibodies in the early stages e Despite the presence of detectable IgG antibodies an active Borrelia infection need not be present since IgG antibodies may persist for a long time after a previous infection e Leptospira and Treponema infections are known to show cross reaction with Borrelia antigens e An acute EBV infection may lead to the stimulation of Borrelia antibodies In the case of an unclear clinical picture this infection should be excluded with appropriate tests e The serological data s
3. Distilled water Pipettes Timer Measuring cylinder Tweezers Test tubes for sample and conjugate dilution Water jet vacuum pump to remove fluid from the incubation dish Rocking platform or horizontal shaker 6 Precautions The VIRO BLOT test is for use only 1 Do not mix lot specific reagents such as N Controls and from different kit lots The doesn t have to be from the original test kit But the lot of the should be the same as indicated on the kit label The SPE DIL the WASHBUF 25x can be used for all VIRO BLOT tests irrespective of the lot and the nature of the VIRO BLOT 2 Seal all bottles properly after use in order to avoid bacterial contamination All samples and kit components should be considered potentially infectious All control samples have been tested for Hepatitis Bs antigen anti HIV and II anti HCV and found to be negative 3 The are coated with inactive antigen However normal laboratory precautions should maintained when handling with infectious material Do not pipette by mouth 4 Avoid contact with skin and mucous membranes when handling reagents which contain preservatives see kit contents Wash thoroughly with water in case of contact and possibly look up a doctor 5 Controls containing sodium azide may react with lead and copper plumbing building explosive metal acids Flush with sufficient water when disposing of reagents 6 For disposal the legal regulations have to
4. both see control strip The test procedure is valid if serum control as well as the conjugate control appears claerly visible on the developed strip In case not only one conjugate control band appears the more reactive gives notice to the used conjugate Refer to the kit specific positive control strip card for information of the exact position of the serum and conjugate control Usage evaluation of the cut off control Cut off band IgG VIsA Cut off band IgM p25 Bands with an intensity weaker than the cut off control are not considered for the interpretation The proteins p41 and p39 are only high specific for IgM The width of the bands as well as their intensity are variable and depend on the concentration of the detected Borrelia antibodies in the sample The band pattern in the IgG determination is generally more intense and diverse than IgM Evaluation of the strips manuell Match the dry at the edge of the protocol sheet and fix the with tape or similar at the numbered ends of the NTS The identification of the bands is possible with the kit specific positive control strip card The high specific bands are signed on the positive control strip card The control card will be matched at the incubated fixed patient NTS the black line have to agree Now it is possible to read directly the immuno specific bands Record the recognisable bands according to their molecular weight and intensity always in consideration to
5. specific in VIRO BLOT complex important IgG marker conserved within B afzelii p18 kDa genus specific early acute infection more frequent detection of IgM antibodies late chronic infection more frequent detection of IgG antibodies rarer detection of IgM antibodies 12 Diagnostic Relevance and interpretation of results Serological findings in a Borrelia infection depend on the stage and duration of the disease the severity of the symptoms as well as previous antibiotic therapy To obtain a final diagnosis the patient history and clinical symptoms should be taken into consideration IgM antibodies are primarily detectable in Stage although some patients remain IgM negative In the case of long disease duration and in the late stage almost all detectable antibodies are IgG A sharp increase in IgG antibodies is observed during reinfections Where neurological symptoms are present CSF should be examined in addition BORRELIA IgG Both the intensity and position of bands should be taken into consideration when assessing VIRO BLOT bands p41 and 39kD is certainly a genus specific antigen protein but p41 is also cross reactive and thus to be considered with care Impotant Only one band in a complex will be considered for the result Exclusion double band p59 p62 are in combination more positive Bands occurring highly specific and specific_antigen bands p100kD p59 u 62kD complex p45kD p35kD OspA p30kD p25
6. VIRO BLOT ANTI BORRELIA IgG IgM Instruction for use 1 Intended use Immunoblot for qualitative determination of antibodies to Borrelia burgdorferi Lyme in human serum and plasma IgG BG 111 24 Y 24 complete kit IgG BG 1118 Y8 complete kit IgM BM 111 24 Y 24 complete kit IgM BM 111 8 Y8 complete kit 2 Antigen Characterisation Antigen Borrelia burgdorferi whole cell antigen Strain Borrelia afzelii Borrelia garinii sensu stricto Purification Ultrasonication centrifugation filtration Cultivation Kelly Medium 3 Introduction Lyme disease Lyme borreliosis is a bacterial infection due to a tick borne spirochete Borrelia burgdorferi This organism was first isolated from Ixodes dammini in the USA 1982 and later from Ixodes ricinus in Europe Virtually all human cases of Lyme disease follow tick bite exposure Lyme disease is a multi system disease manifested in three stages and can occur in different organs skin joints heart eye and nervous system and either separately or in a combination of symptoms Stage I Early Appears after some days or a few weeks in the form of Erythema migrans accompanied by flu like symptoms such as fever vomiting chills headache and occasionally Lymphadenosis benigna cutis At this stage IgM antibodies are present in 50 90 of patients The prevalence of IgG antibodies is low so that frequently a negative result is obtained for an IgG antibody determination Stage Il Become
7. be followed 7 Storage and stability Store all reagents at 1 2 8 C protected from intense light The expiration date of each component is indicated on the respective vial label Do not use reagents beyond the expiration date The diluted is stable up to 4 weeks when stored at 4 2 8 C 8 Specimen collection and general handling 1 The assay procedure should be carried out only by qualified and well trained employees 2 The instruction for use describes the applicable test method In case of modification or applications others then the intended use the user has to validate the procedure and take the responsibility for it 3 Lipaemic icteric haemolysed or microbially contaminated specimen may cause interference 4 Suitable specimens are serum or plasma heparinized EDTA Citrat samples obtained by standard laboratory techniques 5 The samples should not be heat inactivated since non specific results may occur Avoid repeated freeze thaw cycles 6 For ethical reasons the use of CSF samples was only evaluated by testing pairs of CSF and serum samples only if available distributed by the WHO Collaborating Centre of Quality Assurance and Standardization in Laboratory Medicine in Germany Therefore the use of CSF should be proved by testing a sufficient number of samples 7 Patient samples should be stored at X 2 8 C For long term storage 1 20 C or lower is recommended 8 Note Diluted patient samples mu
8. hould be taken into consideration with the clinical history for a full diagnosis Where the results are questionable repeated testing is recommended 15 Precision and reproducibility Intra assay reproducibility was determined by testing samples of different levels of antibody activities in one test run In determination of the samples there is no significant difference of detected antigen bands and their intensity constant level Inter assay reproducibility was determined by testing samples of different levels of antibody activities in different test runs In determination of the samples there is no significant difference of detected antigen bands and their intensity constant level 16 Internal quality control The cut off control should be carried out with every test run Recommendation For every new Lot it is recommended to perform an internal quality control by testing one or more internal control samples which should be clearly specified 17 Literature Karlsson M J Clin Microbiol 28 5 2148 2150 WilskeB FEMS Immunol Med Microbiol 291 2007 13 21 B GB 03 2009 18 Troubleshooting ERROR POSSIBLE CAUSES no colourimetric reaction no CONJ ALK pipetted contamination of after addition substrate possibly with control sera during pipetting may cause an inactivation incorrect CONJ ALK i e not from original test kit incubation generally time too long or incubation temperature too high water quali
9. kD p21 u p20kD complex p19 u p17kD complex highly specific and antigen band VIsA Recommended BORRELIA IgG interpretation a weak antigen negative 1 positive band only one positive band from p59 p62 complex no or very weak band VIsA a positive antigen double band from p59 p62 1 heavy antigen band 2 positive bands 1 positive band and at least 2 further weak bands co control weak VIsA band intensity VIsA co control borderline questionable positive VIsA band intensitiy gt co control a positvs double band p59 p62 and at least 1 further weak band at least 3 positive antigen bands at least 1 heavy band and at least 1 positive band at least 2 heavy bands positive In patients in a late phase of Borrelia infection particularly many antigen bands from classes A and B may be observed BORRELIA IgM Both the intensity and position of bands should be taken into consideration when assessing VIRO BLOT bands Although p41kD is a genus specific antigen protein and is cross reactive in VIRO BLOT a p41 and 39kD band reaction should be judged for Borrelia IgM as a positive reaction IgM antibodies which are considered early markers of Borrelia infection are frequently observed against Flagellin and Osp C Bands occurring highly specific antigen bands p41kD p39kD p21 u p20kD complex p19 u p17kD complex Recommended BORRELIA IgM interpretation negative no antigen bands
10. n RTU 1 Evaluation diagram with positive Control strip IgG optional 1 Protocol sheet 1 extra sheet for BLOTrix software 4 Principle of the WESTERN BLOT The WESTERN BLOT may be used for the qualitative determination of human antibodies to Borrelia burgdorferi antigens For this Immunoblot two European strains of Borrelia burgdorferi complete antigen was treated with SDS and then electrophoretically separated in Polyacrylamide gel according to molecular weight of the proteins This was then transferred to Nitrocellulose blotted and cut into individual NTS The were then incubated with blocking buffer For the assay the are incubated with the control and the samples to be investigated antibodies present will bind specifically to the protein bands and form antigen antibody complexes Unbound serum or plasma is removed by means of washing During the second incubation Anti human IgG or IgM Conjugate binds to the antibody antigen complex In the final wash step excess Conjugate is removed and substrate is added to the chamber B GB 03 2009 The specific protein bands appear as purple bands The reaction is then stopped by the addition of distilled water and the may be dried Optional With the aid of a kit specific evaluation diagram the band pattern may be assessed and evaluated The interpretation of the band pattern provides evidence of the antibody response 5 Materials required but not provided with the Kit
11. ncubation 60 min Carefully pipette 1000 ul diluted Control or 1020 ul pre diluted patient sample into the incubation chamber to coat the strip and incubate 60 min on the rocking platform e First wash step Completely remove patient samples with the vaccum pump and wash each strip 1 x with 2ml prepared and immediately drain Next wash the a further 3 x with 2 ml for 5 min each time Incubation on the rocking platform Second incubation conjugate incubation 30 min Completely remove the WASHBUF and pipette 1 mi pre diluted CONJJALK in each chamber Incubate for 30 min on the rocking platform e Second wash step see first wash step e Third incubation Substrate incubation 15 min Completely remove the WASHBUF and pipette 1 ml in each well Incubate from 10 min to 15 min on the rocking platform Remove the completely from each compartment Stop the enzyme substrate reaction with 2x 2 ml for 5 min distilled water each Remove the carefully from the compartment and dry completely on filter paper The protein bands may be identified and evaluated with aid of the Evaluation diagram For the purposes of documentation the should be kept dry and protected from the light 11 Test results Every has two controls a serum control and a conjugate control IgG IgM separat If the assay handled right you can see on top of the the purple coloured control bands of
12. s clinically apparent after some weeks or months Cardiological complications as well as neurological disease e g meningitis or encephalitis may occur Cross over to multi system disease Antibodies to Borrelia burgdorferi are detectable in 70 90 of patients in this second stage In the first weeks of the disease IgM is more prevalent later IgG is more prevalent Stage Ill Late Months or years later some patients disease may progress to a third stage In this stage arthritis neurological disturbances progressive encephalopathy or Acrodermatitis chronica athrophicans may develop IgM antibodies are barely detectable in this late stage while IgG antibodies are observed in 90 100 of patients Recovery is possible in Stages and II even without treatment However due to the possibility of progression of the infection antibiotic treatment of Lyme disease is recommended for all stages Kit content Y 24 or Y 8 Nitrocellulose test strips with purified INTS 2418 separated B afzelii garinii s s proteins 3 1 Incubation dish with 8 chambers WASHBUF 25x 25ml 25ml__ Wash solution concentrate 25x SPE DIL 2x25ml 21 ml Dilution buffer with 0 05 microbial preservative CONJJALK 10x Anti human IgG or IgM Alkaline IgG ARA sane O OAS Thimieidsa Si Cut off control human preservative 0 095 sodium azid 5x 1 0 ml 0 4ml SUBS CH 25ml 9ml Chromogen Substrat solutio
13. st be used on the same day 9 Preparation of the test Bring all reagents to room 4 prior to use room X up to maximally 25 C WASHBUF Dilute the WASHBUF 25x 1 25 with distilled water e g add 10ml of WASHBUF 25x to 240 ml distilled water and mix well Control Dilute the CONTROL 1 5 with Specimen diluent 800 ul Specimen diluent 200 ul cut off Control Dilution of samples Dilute patient samples 1 51 with e g 20ul sample 1000p SPE DIL mix thoroughly Conjugate dilution Dilute the during the first wash step with e g for 5 strips 4 5ml 0 5ml 5ml Conjugate dilution Please note e Carefully remove the using tweezers holding the strip by the numbered end e Close the tube immediately after taking the NTS e The should be used in number order from low to high e Lay the required number of NTS one per chamber of the incubation dish placing them with the number facing up e Ensure that the remain covered with fluid and that they do not dry out between the incubation or wash steps The have to be used for every run IgG and IgM determination The cut off control band in IgG and IgM is always detected as a weak band the developed sample bands interpreted in relation to the intensity of this band of the cut off control Only more strong positive bands than the cut off control will be evaluated 10 WESTERN BLOT Assay procedure Incubation at room 4 First incubation sample i
14. the co control on the protocol sheet Interpretation of result by using BLOTrix software from VIRO IMMUN The result of a can be interpreted by using the BLOTrix software B GB 03 2009 Evaluation mode is described in the user manual Borrelia_immune reactive proteins Table 1 Molecular Antigen Specificity weight In kDa gt 101 not specific 92 100 Borrelia surface high specific late p100 membrane vesicle in VIRO BLOT protein p83 91 breakdown products specific late p59 p62 heat shock protein highly specific complex membrane protein in VIRO BLOT p45 Important IgG marker specific Flagellin protein Not specific cross reactive a associated with Flagellin complex p41 43 p41 p39 40 p39 heavy band may indicate a Borrelia infection highly specific for IgM p36 38 probably specific Osp B high specific detectable at all p35 external stages of the infection patients membrane protein often show only a weak reaction Specific man made less sensitive p31 Osp A serological marker p30 Frequent positive IgG highly specific marker in stage Il and Ill in VIRO BLOT p28 29 probably specific Plasmid coded high specific in VIRO BLOT p23 26 p25 ijpoprotein important and most common Osp C IgM marker IgM Marker in stage ll p20 p21 Frequent positive IgG high specific in VIRO BLOT complex marker in stage II and III P17 p19 Plasmid coded Osp high
15. ty too high reaction for WASHBUF insufficient low grade of deionization generally incorrect CONJ ALK i e not from original test kit incubation too low reaction time too short incubation temperature too low incorrect dilution of samples lipaemic haemolytic or icteric samples contamination of pipettes tips or containers or with metals iron copper etc incubation conditions not constant time temperature high variation of room temperature controls and samples are not carried out at same time same intervals check pipetting order person related variation surface damaging of because of improper treatment NTS dried out after washing unreproducible results Reagents not pre warmed to room temperature prior to use INTs Insufficient washing false positive negative samples unexplainable outliers high variation from series to series generel source of error that may leads to incorrect results The close observance of the test procedure will help to avoid these errors WESTERN BLOT SHORT ASSAY PROCEDURE Dilute patient sample 1 51 20 ul patient sample 1000p SPEJDIL Dilute controls 1 5 800u 200 ul control Dilute 1 10 f e 4 5ml 0 5 ml Take required holding with tweezers by the numbered end out of the tube All inkubations wash steps on the rocking platform M PIPETTE 1 ml DILUTED SAMPLES DILUTED CONTROLS EACH AND CHAMBER
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