ELISA-VIDITEST anti-mycoplasma pneumoniae IgG assay Lot:
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1 ELISA-VIDITEST anti-mycoplasma pneumoniae IgG assay Lot: Instruction manual PRODUCER: VIDIA Ltd., Nad Safinou II 365, Jesenice, Czech Republic, Tel.: , Fax: TITLE: ELISA-VIDITEST anti-mycoplasma pneumoniae IgG assay ELISA kit for detection of IgG antibodies to Mycoplasma pneumoniae. 2. INTENTED USE: The kit is intended for diagnosis of diseases caused by bacterium Mycoplasma pneumoniae. This test is used in diagnosis of all inflammations in the respiratory system from mild catarrh to severe pneumonia and their complications such as pericarditis, meningoencephalitis, otitis and erythema nodosum. It is recommended to monitor the dynamics of antibodies levels to improve the diagnostic value of the test. In such case, two serum samples are withdrawn from the same patient. The first sample is taken during the acute phase of the disease and the second is withdrawn ten to fifteen days later at which time the changes of the particular antibodies level are expected. Considering the antibody isotype switching during infection, it is recommended to test all antibody isotypes (IgG, IgA and IgM) simultaneously in each sample. 3. TEST PRINCIPLE: ELISA-VIDITEST anti-m. pneumoniae IgG assay is a solid-phase immunoanalytical test. The strips are coated with specific antigen. The anti-mycoplasma pneumoniae antibodies, if present in the tested sera, bind to the immobilized antigens and the antibodies being in complexes with antigen are later on recognised by animal anti-human IgG antibodies labelled with horseradish peroxidase. The labelled antibodies are revealed by an enzymatic reaction with a chromogenic substrate. Negative sera do not react - a mild change in colour, if present, may be attributed to the reaction background. 4. KIT COMPONENTS: ELISA break-away strips (green) coated with specific antigen 1 microplate 1,3 ml Calibrator r.t.u. 1 1 vial 1,3 ml Positive control serum r.t.u. 1 vial 1,3 ml Negative control serum r.t.u. 1 vial 0,2 ml Anti-human IgG antibodies labelled with horseraddish peroxidase 101x concentrated (Px-conjugate) 1 vial 125 ml Wash buffer 10x concentrated 1 vial 125 ml Dilution buffer (DB) r.t.u. 1 vial 15 ml Chromogenic substrate (TMB substrate) r.t.u. 1 vial 15 ml Stop solution r.t.u. 1 vial Sealable pouch for unused strips Instruction manual Certificate of quality 1) ready to use 1
2 Wash buffer, Chromogenic substrate (TMB substrate) r.t.u and Dilution buffer are intended only for ELISA-VIDITEST anti-mycoplasma pneumoniae IgG, IgM, IgA kits and THEY ARE NOT COMPATIBLE with other ELISA-VIDITEST kits produced by VIDIA Ltd. 5. MATERIAL REQUIRED BUT NOT PROVIDED WITH THE KIT 1. Distilled or deionised water for dilution of the Wash buffer concentrate. 2. Appropriate equipment for pipetting, liquid dispensing and washing. 3. Spectrophotometer/colorimeter (microplate reader wavelenght 450 nm). 6. PREPARATION OF REAGENTS AND SAMPLES a. Allow all kit components to reach room temperature. b. Mix Dilution buffer r.t.u and Chromogenic substrate r.t.u well. c. Vortex samples, Calibrator and the Controls in order to ensure homogeneity and mix all solution well prior use. d. Dilute serum samples 1:100 in Dilution buffer and mix (e.g. 5 μl of serum sample μl of Dilution buffer). Do not dilute the Control sera and Calibrator, they are ready to use. e. Prepare Wash buffer by diluting the Wash buffer concentrate 10 times with an appropriate volume of distilled or deionised water (e.g. 100 ml of the concentrated Wash buffer ml of distilled water). If there are crystals of salt present in the concentrated Wash buffer, warm up the vial to +32 to +37 C in a water bath. Diluted Wash buffer is stable for one month at laboratory temperature. f. Dilute concentrated Px-conjugate 1:100 with Dilution buffer (e.g. 0.1 ml Px-conjugate + 10 ml Dilution buffer). (Note: For one microtitre plate you will need approx. 12 ml of diluted Px-conjugate., i.e. 0,12 ml of Px-conjugate + 12 ml of Dilution buffer). g. Do not dilute TMB substrate and Stop solution, they are ready to use. 7. ASSAY PROCEDURE a. Allow the vacuum-closed aluminium bag with strips to reach a room temperature. Withdraw the adequate number of strips and put unused strips into the provided pouch and seal it carefully with the desiccant kept inside. b. Pipette 100 µl of Dilution buffer, Controls and serum samples to the wells according to the pipetting scheme in Figure 1 (page 3): start with filling the first well with Dilution buffer (DB) to estimate reaction background, fill the next three wells with Calibrator (Cal) and then fill one well with Positive control serum (PCS), one well with Negative control serum (NCS). Fill the remaining wells with diluted serum samples (S1, S2, S3...). It is satisfactory to apply samples as singles, however, if you want to minimize a laboratory error then apply the controls and samples in doublets. c. Incubate 30 minutes (±2 min) at room temperature. d. Aspirate the liquid from the wells into a collecting bottle containing appropriate disinfectant (see Safety Precautions). Wash and aspirate the wells four times with 250 µl/well of Wash buffer. Avoid cross-contamination between wells! If some liquid remains in the wells, invert the plate and tap it on an adsorbent paper to remove the last remaining drops. e. Add 100 µl of diluted Px-conjugate into each well. f. Incubate 60 minutes (±5 min) at room temperature. g. Aspirate and wash four times with 250 µl/well of Wash buffer. 2
3 h. Dispense 100 µl of TMB substrate into each well. i. Incubate for 15 minutes (+/-30 seconds) at room temperature. The time measurement must be started at the beginning of TMB dispensing. Cover the strips with an aluminium foil or keep them in the dark during the incubation with TMB substrate. j. Stop the reaction by adding 100 µl of Stop solution. Use the same pipetting rhythm as with the TMB substrate to ensure the same reaction time in all wells. Tap gently the microplate for a few times to ensure complete mixing of the reagents. k. Read the absorbance at 450 nm with a microplate reader within 20 minutes. It is recommended to use reference reading at 630 nm. Fig 1: Pippetting schema a b c d e f g h DB Cal Cal Cal NCS PCS S1 S2 S3 S4. 8. ASSAY EVALUATION First, subtract the absorbance of the background (absorbance of the DB well) from the absorbances of all other wells. 8.1 Qualitative evaluation 1. Compute the mean absorbance of the three parallels of Calibrator (Cal). If any value of the three parallels is different more from the mean than 20 then exclude the deviating well from the calculation and compute a new mean using the remaining two wells. 2. Compute the cut-off value by multiplying the Calibrator mean with a Correction factor. The Correction factor of Calibrator for this Lot. (010810) is Assign the samples with absorbances less than the cut-off value as negative and the samples with absorbances higher than the cut-off value as positive Semiquantitative evaluation Determination of sample Positivity Index: 1. Compute the cut-off value (see previous paragraph 8.1) 2. Compute the Positivity Index for each sample according to the folowing formula: 3
4 sample absorbance Sample Positivity Index = cut-off value 3. Express serum reactivity in accordance with data in Table 1 (Evaluation of results) Table 1 Evaluation of results: Index value Interpretation < 1,00 Negative / > Note! Indifferent sample reactivity, i.e. interpreted as +/-, requires repetition of the sample testing. If the result is repetitively indifferent then it is recommended to use an alternative testing method or to obtain another sample from the patient withdrawn 1-2 weeks later. Example of calculation: Calibrator absorbances = 1.360; 1.474; Calibrator mean = Correction factor = 0.20 Cut-off value = 1,400*0.20 = Sample absorbance = Sample Positivity Index = 0.900/0.280 = INTERPRETATION OF RESULTS: During result interpretation, it is suitable to determine all 3 isotypes of antibodies (IgG, IgM, IgA) in two samples from one patient (first in acute phase and second later). IgG IgM IgA Result interpretation - + (+) Early acute infection Acute infection Early acute infection without IgM antibodies production (or solitary persistence of IgA) Late acute infection + - (+) Primoinfection or reinfection Infection underwent in the past Seronegativity (without contact with Mycoplasma pneumoniae ) 4
5 10. VALIDITY, SPECITITY AND SENSITIVITY OF THE TEST 10.1 Validity of the test The test is valid if: a. The background of the reaction (the absorbance of the Dilution buffer) is less than b. The Calibrator mean is higher than c. Sample Positivity Index of NCS should be < 1.00 d. The Control absorbances can be lined up as follows: NCS < CAL < PCS Precision of the test The intraassay variability (within the test) and the interassay variability (between tests) evaluation was performed with samples of variable absorbance values. Example of evaluation with positive serum samples (n=13): Absorbance range Intraassay variability (n = number of parallels): n A ±δ CV repro % % Interassay variability (n = number of paralells): n A ±δ Range (min max) CVrepro % % % Accuracy of the test Spiking recovery test The percentage of recovery was between 99% - 116%. The spiked samples were prepared by mixing a sample A with a spike (sample B), where the sample A had a concentration of anti- VCA EBV IgG antibodies corresponding to the central linear part of the calibration curve and the sample B had either antibody concentration ten times higher or ten times lower than the concentration in the sample A. 11. SAFETY PRECAUTIONS All ingredients of the kit are intended for laboratory use only. Controls contain human sera that has been tested negative for HBsAg, anti-hiv-1,2 and anti-hcv. However they should be regarded as contagious and handled and disposed of according to the appropriate regulations. Autoclave all reusable materials that were in contact with human samples for 1 hour at 121 C, burn disposable ignitable materials, decontaminate liquid wastes and nonignitable materials with 3% chloramine. Liquid wastes containing acid (Stop solution) should be neutralized in 4% sodium bicarbonate solution. Handle Stop solution with care. Avoid contact with skin or mucous membranes. In case of contact with skin, rinse immediately with plenty of water and seek medical advice. 5
6 Do not smoke, eat or drink during work. Do not pipette by mouth. Wear disposable gloves while handling reagents or samples and wash your hands thoroughly afterwards. Avoid spilling or producing aerosol. 12. HANDLING PRECAUTIONS: Manufacturer guarantees performance of the entire ELISA kit.wash buffer, Chromogenic substrate (TMB substrate) r.t.u and Dilution buffer are compatible only with ELISA-VIDITEST anti- Mycoplasma pneumoniae IgG, IgM, IgA kits. Follow the assay procedure indicated in the Instruction manual. Controls, TMB substrate, Dilution buffer, Urea solution and Px-conjugate contain preservative ProClin 300. Avoid microbial contamination of serum samples and kit reagents. Avoid cross-contamination of reagents. Avoid contact of the TMB substrate with oxidizing agents or metal surfaces. Variations in the test results are usually due to: * Insufficient mixing of reagents and samples * Inaccurate pipetting and inadequate incubation times * Poor washing technique or spilling the rim of well with sample or Px-conjugate * Use of identical pipette tip for different solutions 13. STORAGE AND EXPIRATION: Store the kit and the kit reagents at +2 to +10 C, in a dry place and protected from the light. Store unused strips in the sealable pouch and keep the desiccant inside. Store serum samples at +2 to +10 C up to one week. For longer period make aliquots and keep them at -20 C. Avoid repeated thawing and freezing. Do not store diluted samples and a diluted Px-conjugate. Always prepare fresh. Kits are shipped in cooling bags, the transport time of 72 hours have no influence on expiration. If you find damage at any part of the kit, please inform the manufacturer immediately. Expiration date is indicated at the ELISA kit label and at all reagent labels. 6
7 13. FLOW CHART: Step 1. Step 2. Step 3. Step 4. Prepare reagents and samples Dispense 100 μl/well controls and samples Incubate 30 minutes at room temperature Wash 4 times (250 μl/well), aspirate Dispense 100 μl/well of Px-conjugate Incubate 60 minutes at room temperature Wash 4 times (250 μl/well), aspirate Dispense 100 μl/well of TMB substrate Incubate 15 minutes at room temperature Step 5. Step 6. Dispense 100 μl/well of Stop solution Read the absorbance at 450 nm References: M Karppelin, K Hakkarainen, M Kleemola, and A Miettinen: Comparison of three serological methods for diagnosing Mycoplasma pneumoniae infection. Clin Pathol.; 46(12): , December M. Granstrom, T. Holme, A. M. Sjogren, A. Ortqvist and M. Kalin: The role of IgA determination by ELISA in the early serodiagnosis of Mycoplasma pneumoniae infection, in relation to IgG and mucapture IgM methods. The Journal of Medical Microbiology, Vol 40, Issue , Ken B. Waites and Deborah F. Talkington: Mycoplasma pneumoniae and Its Role as Human Pathogen. Clinical Microbiology Reviews, p , Vol. 17, No. 4, October Date of the last revision of this manual: 12/2010 Next recommended revision: 04/2011 7
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