PNEUMOBACT ELISA IgG STORAGE OF REAGENTS ONCE OPENED: STABILITY AND STORAGE. Refer to package label for expiration date (at 2-8ºC) Rest of reagents - PDF

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0318 EN 1 G1040: Indirect immunoenzyme assay for the simultaneous screening in human serum of IgG antibodies of the main ethiological bacterial agents of infectious diseases of the respiratory tract: Legionella

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0318 EN 1 G1040: Indirect immunoenzyme assay for the simultaneous screening in human serum of IgG antibodies of the main ethiological bacterial agents of infectious diseases of the respiratory tract: Legionella pneumophila serogroup 1, Mycoplasma pneumoniae, Coxiella burnetii and Chlamydophila pneumoniae. 96 tests. INTRODUCTION: A large number of ethiological agents of atypical pneumonia has been described among which those most commonly encountered are: Legionella pneumophila Although more than 30 species have been reported, Legionella pneumophila serogroup 1 is responsible for most infections in humans. Atypical pneumonia is often associated with systemic manifestations. It is responsible for 10% cases of pneumonia acquired in both the community and hospitals. Mycoplasma pneumoniae Atypical pneumonia produced by Mycoplasma pneumoniae is most frequently found in children and adolescents. The isolation in culture is tedious and consequently serological diagnosis is most frequently performed. Coxiella burnetii Q fever is a systemic disease caused by Coxiella burnetii that can produce fever, atypical pneumonia, hepatitis or endocarditis. The diagnosis is based on serological methods since isolation from clinical samples is difficult. Chlamydophila pneumoniae Chlamydophila has a great ability to cause respiratory infections, particularly bronchitis and pneumonia. The higher incidence takes place in elderly people and it is considered responsible of 10% of all the cases of pneumonia, although it has been considered by some authors as the most frequent cause of those cases of known ethiology. PRINCIPLE OF THE TEST: The ELISA method is based upon the reaction of antibodies in the sample tested with the antigen adsorbed on the polystyrene surface. Unbound immunoglobulins are washed off. An enzyme-labelled anti-human globulin binds the antigen-antibody complex in a second step. After a new washing step, bound conjugate is developed with the aid of a substrate solution (TMB) to render a blue coloured soluble product which turns into yellow after adding the acid stopping solution. Coloured product PNEUMOBACT ELISA IgG Peroxidase detergent-soluble antigens (containing P1 protein) of M. pneumoniae, strain FH (ATCC 15531), 3 strips (blue) with antigen of C. burnetii phase II, strain Nine Mile Q (ATCC VR-616) and 3 strips (green) with purified antigens (complexes of outer membrane proteins) of C. pneumoniae strain CM-1 (ATCC VR-1360). 2 VIRCELL SERUM DILUENT: 25 ml of serum dilution solution: a blue coloured phosphate buffer containing protein stabilizers and Proclin. Ready to use. 3A VIRCELL LEGIONELLA IgG POSITIVE CONTROL: 500 µl of positive control serum containing Proclin. 3B VIRCELL MYCOPLASMA IgG POSITIVE CONTROL: 500 µl of positive control serum containing Proclin. 3C VIRCELL COXIELLA IgG POSITIVE CONTROL: 500 µl of positive control serum containing Proclin. 3D VIRCELL CHLAMYDOPHILA IgG POSITIVE CONTROL: 500 µl of positive control serum containing Proclin. 4A VIRCELL LEGIONELLA IgG CUT OFF CONTROL: 500 µl of cut off control serum containing Proclin. 4B VIRCELL MYCOPLASMA IgG CUT OFF CONTROL: 500 µl of cut off control serum containing Proclin. 4C VIRCELL COXIELLA IgG CUT OFF CONTROL: 500 µl of cut off control serum containing Proclin. 4D VIRCELL CHLAMYDOPHILA IgG CUT OFF CONTROL: 500 µl of cut off control serum containing Proclin. 5 VIRCELL IgG NEGATIVE CONTROL: 500 µl of negative control serum containing Proclin. 6A VIRCELL IgG CONJUGATE I: 9 ml of anti-human IgG peroxidase conjugate dilution in a red-coloured Proclin-containing buffer. Ready to use. 6B VIRCELL IgG CONJUGATE II: 9 ml of anti-human IgG peroxidase conjugate dilution in a red-coloured Proclin-containing buffer. Ready to use. 7 VIRCELL TMB SUBSTRATE SOLUTION: 15 ml of substrate solution containing tetramethylbenzidine (TMB). Ready to use. 8 VIRCELL STOP REAGENT: 15 ml of stopping solution: 0.5 M sulphuric acid. 9 VIRCELL WASH BUFFER: 50 ml of 20x washing solution: a phosphate buffer containing Tween R -20 and Proclin. Store at 2-8ºC and check expiration date. Colourless substrate Anti-human IgG globulin Anti-L. pneumophila, M. pneumoniae, C. burnetii or C. pneumoniae human IgG L. pneumophila, M. pneumoniae, C. burnetii or C. pneumoniae antigen Materials required but not supplied: Precision micropipettes 5 and 100 µl. Eight channel micropipette 100 µl. ELISA plate washer. Thermostatized incubator/water bath. ELISA plate spectrophotometer with a 450 nm measuring filter and a 620 nm reference filter. Alternatively, an ELISA automated processor. Distilled water. KIT FEATURES: All reagents, except for the washing solution, are supplied ready to use. Serum dilution solution and conjugate are coloured to help in the performance of the technique. Sample predilution is not necessary. Break-apart individual wells are supplied, so that the same number of wells is consumed than the number of tests performed. KIT CONTENTS: 1 VIRCELL PNEUMOBACT PLATE: 1 96-wells plate coated with the following antigens: 3 strips (orange) with LPS antigens of L. pneumophila, serogroup 1, strain Philadelphia-1 (ATCC 33152), 3 strips (red) with STORAGE REQUIREMENTS: Store at 2-8ºC. Do not use the kit reagents beyond the expiration date. This will be valid only if reagents are stored closed and at 2-8ºC. STORAGE OF REAGENTS ONCE OPENED: REAGENT STABILITY AND STORAGE 1x washing solution 4 months at 2-8ºC Rest of reagents Refer to package label for expiration date (at 2-8ºC) STABILITY AND HANDLING OF REAGENTS: Handle reagents in aseptic conditions to avoid microbial contaminations. Do not let the plate dry between washing and reagent addition. 2 Substrate solution is light sensitive. Avoid light exposure and discard if blue colour develops during storage. Substrate solution should not get in contact with oxidizers such as bleach solutions or metals. Make sure that no metal components come in contact with the substrate. Use only the amount of washing, serum dilution, conjugate and TMB solutions required for the test. Do not return the excess solution into the bottles. VIRCELL, S.L. does not accept responsibility for the mishandling of the reagents included in the kit. RECOMMENDATIONS AND PRECAUTIONS: 1. For in vitro diagnosis use only. For professional use only. 2. Use kit components only. Do not mix components from different kits or manufacturers. Only the serum dilution, washing, stopping and substrate solutions are compatible with the equivalents in other VIRCELL ELISA references and lots. 3. Clean pipette tips must be used for every assay step. Use only clean, preferably disposable material. 4. Do not use in the event of damage to the package. 5. Never pipette by mouth. 6. Serum dilution solution, plate, conjugates and controls in this kit include substances of animal origin. Controls include as well substances of human origin. Although the human serum controls of this kit have been tested and found negative for Hepatitis B Surface Antigen (HBsAg), Hepatitis C antibodies and Human Immunodeficiency Virus antibodies, control sera and patient specimens should be handled as potentially infectious. The wells are coated with inactivated L. pneumophila, M. pneumoniae, C. burnetii or C. pneumoniae antigen. Nevertheless, they should be considered potentially infectious and handled with care. No present method can offer complete assurance that these or other infectious agents are absent. All material should be handled and disposed as potentially infectious. Observe the local regulations for clinical waste disposal. 7. Susbstrate solution may be irritant to skin and mucus. In case of contact with this solution, rinse thoroughly with water and seek medical attention. For further information a Material Safety Data Sheet is available. 8. Before incorporating this product onto an automatic processing system, we strongly recommend the performance of a pre-evaluation assay. To this purpose, VIRCELL counts with sets of samples reserved for evaluation in parallel with the manual technique. These sets of samples are available on request, as well as a list of commercial systems which have already been validated for use with the VIRCELL ELISA range. 9. During incubation times, an adequate sealing of the plates with the adhesive film included in the kit avoids the desiccation of the samples, and guarantees the repeatability of the results. 10. The antigens corresponding to each conjugate are indicated in the conjugate label. SPECIMEN COLLECTION AND HANDLING: Blood should be collected aseptically using venipuncture techniques by qualified personnel. Use of sterile or aseptic techniques will preserve the integrity of the specimen. Serum samples are to be refrigerated (2-8ºC) upon collection or frozen (-20ºC) if the test cannot be performed within 7 days. Samples should not be repeatedly frozen and thawed. Do not use hyperlipemic, hemolysed or contaminated sera. Samples containing particles should be clarified by centrifugation. Do not use plasma. PRELIMINARY PREPARATION OF THE REAGENTS: Only the washing solution must be prepared in advance. Fill 50 ml of 20x washing solution up to 1 litre with distilled water. Should salt crystals form in the washing concentrate during storage, warm the solution to 37ºC before diluting. Once diluted, store at 2-8ºC. ASSAY PROCEDURE: 1.-Set incubator/water bath to 37±1ºC. 2.-Bring all reagents to room temperature before use (approximately 1 hour), without removing the plate from the bag. 3.-Shake all components. 4.-Remove the plate 1 from the package. Determine the numbers of wells of each antigen to be employed counting in four wells for the controls: two for the cut off serum and one each for the negative and positive sera. Wells not required for the test should be returned to the pouch, which should then be sealed (ml). 5.-Add 100 µl of serum diluent 2 to all wells. Add 5 µl of each sample, 5 µl of the corresponding positive control 3A, 3B, 3C or 3D, 5 µl of the corresponding cut off serum 4A, 4B, 4C or 4D (in duplicate) and 5 µl of negative control 5 into the corresponding wells. If the assay is performed manually, shake the plate in a plate shaker (2 min) in order to achieve a homogenous mixture of the reagents. If for some reason correct shaking cannot be guaranteed, a pre-dilution of the sample in a separate tube or plate should be made, using double volume of serum diluent 2 and sample. Mix homogenously with the pipette and dispense 105 µl of each diluted sample to the wells Cover with a sealing sheet and incubate at 37±1ºC for 45 min. 7.-Remove the seal, aspirate liquid from all wells and wash five times with 0.3 ml of washing solution 9 per well. Drain off any remaining liquid. 8.-Immediately add 100 µl of the corresponding IgG conjugate solution 6A or 6B into each well. 9.-Cover with a sealing sheet and incubate in incubator/water bath at 37±1ºC for 30 min. 10.-Remove the seal, aspirate liquid from all wells and wash five times with 0.3 ml of washing solution 9 per well. Drain off any remaining liquid. 11.-Immediately add 100 µl of substrate solution 7 into each well. 12.-Incubate at room temperature for 20 min. protected from light. 13.-Add immediately 50 µl of stopping solution 8 into all wells. 14.-Read with an spectrophotometer at 450/620 nm within 1 hour of stopping. INTERNAL QUALITY CONTROL: Each batch is subjected to internal quality control (Q.C.) testing before batch release complying with specifications stricter than validation protocol for users. Final Q.C. results for each particular lot are available. The control material is traceable to reference sera panels internally validated. VALIDATION PROTOCOL FOR USERS: Positive, negative and cut off controls must be run with each test run. It allows the validation of the assay and kit. Optical densities (O.D.) must fall in the following ranges. Otherwise, the test is invalid and must be repeated. CONTROL O.D. POSITIVE CONTROL 0.9 NEGATIVE CONTROL 0.55 0.7 x(o.d. POSITIVE CONTROL) CUT OFF CONTROL 1.5 x(o.d. NEGATIVE CONTROL) INTERPRETATION OF RESULTS: Calculate the mean O.D. for cut off serum. Antibody index=(sample O.D./ cut off serum mean O.D.) x 10 INDEX INTERPRETATION 9 Negative 9-11 Equivocal 11 Positive Samples with equivocal results must be retested and/or a new sample obtained for confirmation. Samples with indexes below 9 are considered as not having IgG specific antibodies against the antigen. Samples with indexes above 11 are considered as having IgG specific antibodies against the antigen. LIMITATIONS: 1.-This kit is intended to be used with human serum. 2.-The user of this kit is advised to carefully read and understand the package insert. Strict adherence to the protocol is necessary to obtain reliable test results. In particular, correct sample and reagent pipetting, along with careful washing and timing of the incubation steps are essential for accurate results. 3.-The results of samples should be used in conjunction with clinical evaluation and other diagnostic procedures. A definitive diagnosis should be made by isolation techniques. 4.-This test will not indicate the site of infection. It is not intended to replace isolation. 3 5.-Lack of significant rise in antibody level does not exclude the possibility of infection. 6.-Samples collected very early in the course of an infection may not have detectable levels of IgG. In such cases, it is recommended an IgM assay be performed or a second serum sample be obtained 14 to 21 days later to be tested in parallel with the original sample to determine seroconversion. 7.-Results in IgG detection in neonates must be interpreted with caution, since maternal IgG is transferred passively from the mother to the foetus before birth. IgM assays are generally more useful indicators of infection in children below 6 months of age. 8.-The results of a single-specimen antibody determination should not be used to aid in the diagnosis of recent infection. Paired samples (acute and convalescent) should be collected and tested concurrently to look for seroconversion or a significant rise in antibody level. 9.-The serologic results must be evaluated in the clinical context of the patient to reach an adecuate diagnosis. In primoinfections by C. pneumoniae IgM is generally found, and 3-6 weeks after the onset of the disease high nivels of IgG appear. Otherwise, in reinfections the IgM response is not usually present and there is a quick rise of the levels of IgG and IgA. The seroprevalence to C. pneumoniae is high in adults and therefore the presence of antibodies is not indicative of recent infection. Titration of positive samples by microimmunofluorescence can help to confirm the diagnosis. This assay is not designed to measure the immune state to C. pneumoniae, but to detect levels of antibodies to the infection. 10.-Cross-reaction with C. psittaci positive samples have not been tested for this assay due to the low prevalence of the disease and the lack of samples. 11.-The performance of this assay has not been evaluated for therapy follow-up. 12.-ELISA assays with a single dilution do not present a lineal relation with titers detected by IFA. 13.-The performance of the technique for diseases by C. pneumoniae other than pneumonia has not been evaluated. 14.-The test is intended to use as screening of IgG in human serum. Positive sera must be retested with the corresponding specific test. The values of the results obtained can vary slightly. PERFORMANCE SENSITIVITY AND SPECIFICITY: 110 serum samples were assayed against an immunofluorescence kit. The results were as follows: IgG % 97% 85 serum samples were assayed against another commercial ELISA kit. The results were as follows: IgG 85 98% 97% Discordant results were solved by means of an immunofluorescence test. TEST 1 92 serum samples were assayed against another commercial available ELISA kit. The results were as follows: IgG 92 98% 100% Discordant results were solved by means of an immunofluorescence test. TEST serum samples were assayed against a IFA test. The results were as follows: IgG % 97% 178 serum samples were assayed against an IFA kit. The results were as follows: IgG % 83% PC NC CO PC NC CO PC NC CO SERUM N %CV PC NC CO INTER-ASSAY PRECISION: 3 sera were individually pipetted on 5 consecutive days by 2 different operators. The results were as follows: PC NC CO PC NC CO PC NC CO SERUM N % CV PC NC CO CROSS REACTIVITY AND INTERFERENCES: 16 samples known to be positive for other bacteria of the syndromic group (Coxiella burnetii, Chlamydophila pneumoniae and Mycoplasma pneumoniae) and Rickettsia conorii were assayed. 6 samples known to be positive for other bacteria of the syndromic group (Legionella pneumophila, Chlamydophila pneumoniae and Coxiella burnetii) were assayed. 6 samples known to be positive for other bacteria of the syndromic group (Legionella pneumophila, Chlamydophila pneumoniae and Mycoplasma pneumoniae) were assayed. 22 samples known to be positive for other bacterial members of the syndromic group (Legionella pneumophila, Coxiella burnetii and Mycoplasma pneumoniae) and Rickettsia conorii and Chlamydia trachomatis were assayed. The negative results of the test demonstrated the specific reaction of the kit with no cross-reaction or interferences with the referred specimens. INTRA-ASSAY PRECISION: 3 sera were individually pipetted 10 times each serum in a single assay performed by the same operator in essentially unchanged conditions. The results were as follows: 4 SYMBOLS USED IN LABELS: In vitro diagnostic medical device 2ºC x 8ºC Use by (expiration date) Store at 2-8ºC Contains sufficient for X tests Batch code Catalogue number Consult instructions for use X wells SUMMARY OF THE ASSAY PROCEDURE 100 μl serum diluent 2 5 μl serum and controls 3A, 3B, 3C or 3D 4A, 4B, 4C or 4D 5 Wash 5x (washing solution) μl conjugate 6A or 6B Wash 5x (washing solution) μl substrate 7 45 min. at 37ºC 50 μl stopping solution 8 30 min. at 37ºC 20 min. at room temperature Read at 450/620 nm LITERATURE: 1. Bangsborg, J. M., G. H. Shand, K. Hansen, and J. B. Wright Performance of four different indirect enzyme-linked immunosorbent assays (ELISAs) to detect specific IgG, IgA, and IgM in Legionnaires' disease. APMIS 102: Barka, N., J. P. Tomasi, and S. Stadtsbaeder ELISA using whole Legionella pneumophila cell as antigen. Comparison between monovalent and polyvalent antigens for the serodiagnosis of human legionellosis. J Immunol Methods 93: Edelstein, P. H. Laboratory diagnosis of Legionella disease: an update from Pelaz, C., L. Garcia Albert, and C. M. Bourgon Cross-reactivity among Legionella species and serogroups. Epidemiol Infect 99: Busolo, F., E. Tonin, and G. A. Meloni Enzyme-linked immunosorbent assay for serodiagnosis of Mycoplasma pneumoniae infections. J Clin Microbiol 18: Lieberman, D., P. Shvartzman, D. Lieberman, M. Ben-Yaakov, Z. Lazarovich, S. Hoffman, R. Mosckovitz, B. Ohana, M. Leinonen, D. Luffy, and I. Boldur Etiology of respiratory tract infection in adults in a general practice setting. Eur J Clin Microbiol Infect Dis 17: Thacker, W. L. and D. F. Talkington Analysis of complement fixation and commercial enzyme immunoassays for detection of antibodies to Mycoplasma pneumoniae in human serum. Clin Diagn Lab Immunol 7: Uldum, S. A., J. S. Jensen, J. Sondergard-Andersen, and K. Lind Enzyme immunoassay for detection of immunoglobulin M (IgM) and IgG antibodies to Mycoplasma pneumoniae. J Clin Microbiol 30: Cowley, R., F. Fernandez, W. Freemantle, and D. Rutter Enzyme immunoassay for Q fever: comparison with complement fixation and immunofluorescence tests and dot immunoblotting. J Clin Microbiol 30: Kovacova, E., J. Gallo, S. Schramek, J. Kazar, and R. Brezina Coxiella burnetii antigens for detection of Q fever antibodies by ELISA in human sera. Acta Virol 31: Lieberman, D., P. Shvartzman, D. Lieberman, M. Ben-Yaakov, Z. Lazarovich, S. Hoffman,
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