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Ec114.00_ec114.08e

Mycoplasma pneumoniae ELISA
(recombinant)
IgG / IgM Testkit
Order No.: EC114.00 (IgG/IgM Testkit)
Order No.: EC114.08 (IgA-Set)
Color Coding: dark blue
FOR IN VITRO DIAGNOSIS ONLY
Sekisui Virotech GmbH
Löwenplatz 5
65428 Rüsselsheim - Germany
Tel.: +49-6142-6909-0
Fax: +49-6142-966613
http://www.sekisuivirotech.com
REV 22 / Mycoplasma pneumoniae ELISA IgG/IgM/IgA GB Contents
1. Intended Use. 3
2. Diagnostic Relevance. 3
3. Test Principle. 3
4. Package Contents . 3
5. Storage and Shelflife of the testkit and the ready to use reagents . 4
6. Precautions and Warnings. 4
7. Material required but not supplied . 4
8. Test Procedure . 5
9. Test Evaluation. 6
9.2 Calculation of the Virotech Units (VE) . 6 10. Performance Data . 7
10.5 Intra-assay-Coefficient of Variation (Repeatability). 10 10.6 Inter-assay-Coefficient of Variation (Reproducibility) . 10 11. Literature. 10
12. Test Procedure Schemata. 11
Mycoplasma pneumoniae ELISA IgG/IgM/IgA GB Intended Use
The Mycoplasma pneumoniae ELISA is intended for the semiquantitative and qualitative detection of IgG-, IgM- and IgA- antibodies in human serum. The IgG-antibody detection test is especially suitable for the detection of an acute infection. To detect a re-infection or past infection it is recommended to test serum pairs at an interval of 5-10 days. Diagnostic Relevance
The bacteria Mycoplasma pneumoniae, which is lacking cell wall components, is the cause of atypical pneumonia and tracheobronchitis of humans and affects mostly children, young adults and immunodeficient people (1,2,3,4). So called adhesins (6), enable the bacteria to attach to the epithelial cells, against which the host develops antibodies. Studies made by Foy show, that in the USA 15 to 20% of all pneumonia cases are caused by Mycoplasma pneumoniae (8). The ELISA detects Mycoplasma-antibodies with a defined antigen fraction of the strain M129, which is defined via monospecific sera. It includes membrane proteins, cytoskeleton proteins and recombinant proteins. The incubation time during an infection with Mycoplasma pneumoniae is 10 – 21 days: Specific IgM-antibodies occur 6-10 days after infection. Basically, about 80% of the patients younger than 20 years develop IgM-antibodies and 40% of the patients that are older than 20 years. This means a specific IgM-response can be missing especially in older patients. IgM-antibodies may be detected, referring to literature, still at least one Specific IgG-antibodies appear 9-14 days after infection. They may persist up to 4 years. Specific IgA-antibodies appear one week after start of the infection and decrease about 5 weeks after start of the infection again. As a rule, the IgA-titer exceeds the IgM-titer. Considering the fact that IgM-antibodies persist very long in some persons and are missing in others completely, it is important to detct beside the IgM- also the specific IgG- and IgA-titer. Re-infections often take place without any production of IgM-antibodies but under significant increase of IgG- and IgA-antibody titers. Two patient sera, taken at an interval of 5-10 days allow a proper statement concerning the rise of the antibody titer (5). It is important to consider that a first attack of Mycoplsma pneumoniae does not leave a sufficient protection against a new colonization. For diagnosis it is necessary in any case to consider the clinical picture in addition to the serological results. Mycoplasma infections are generally treated successfully with antibiotics like Tetracycline and Macrolide. The treatment with non-suitable, w.g. cell-wall-specific antibiotics (penicillin) leads to a serological advantage for Mycoplasma against all Penicillin-sensitive microorganisms. Test Principle
The antibody searched for in the human serum forms an immune complex with the antigen coated on the microtiter-plate. Unbound immunoglobulins are removed by washing processes. The enzyme conjugate attaches to this complex. Unbound immunoglobulins are again removed by washing processes. After adding the substrate solution (TMB), a blue dye is produced by the bound enzyme (peroxidase). The color changes to yellow when the stopping solution is added. Package Contents
IgG/IgM Testkit
1 Microtiter-Plate consisting of 96 with antigen coated, breakable single wells, lyophilised
PBS-Dilution Buffer, (blue, ready to use), 2x50ml, pH 7,2, with preservative and Tween 20
PBS-Washing Solution, (20x concentrated) 50ml, pH 7,2, with preservative and Tween 20
IgG negative Control, 1300µl, human serum with protein-stabilizer and preservative, ready to use
IgG cut-off Control, 1300µl, human serum with protein-stabilizer and preservative, ready to use
IgG positive Control, 1300µl, human serum with protein-stabilizer and preservative, ready to use
IgM negative Control, 1300µl, human serum with protein-stabilizer and preservative, ready to use
IgM cut-off Control, 1300µl, human serum with protein-stabilizer and preservative, ready to use
IgM positive Control, 1300µl, human serum with protein-stabilizer and preservative, ready to use
IgG-Conjugate (anti-human), 11ml, (sheep or goat)-horseradish-peroxidase-conjugate with protein-stabilizer and
preservative in Tris-Buffer, ready to use Mycoplasma pneumoniae ELISA IgG/IgM/IgA GB IgM-Conjugate (anti-human), 11ml, (sheep or goat)-horseradish-peroxidase-conjugate with FCS and preservative in
Tetramethylbenzidine substrate solution (3,3’,5,5’-TMB), 11ml, ready to use
Citrate-Stopping Solution, 6ml, contains an acid mixture
IgA negative Control, 1300µl, human serum with protein stabilizer and preservative, ready to use
IgA cut-off Control, 1300µl, human serum with protein stabilizer and preservative, ready to use
IgA positive Control, 1300µl, human serum with protein stabilizer and preservative, ready to use
IgA-Conjugate (anti-human), 11ml, (sheep or goat)-horseradish-peroxidase with FCS and preservative in Tris-Buffer,
Storage and Shelflife of the testkit and the ready to use reagents
Store the testkit at 2-8°C. The shelf life of all components is shown on each respective label; for the kit shelf life please see Microtiter strips/single wells are to be resealed in package after taking out single wells and stored with desiccant at 2-8°C. Reagents should immediately be returned to storage at 2-8°C after usage. The ready to use conjugate and the TMB-substrate solution are sensitive to light and have to be stored in dark. Should there be a color reaction of the substrate dilution due to incidence of light, it is not useable anymore. Take out only the amount of ready to use conjugate or TMB needed for the test insertion. Additional conjugate or TMB taken out may not be returned but must be dismissed. Material
Shelflife
Precautions and Warnings
Only sera which have been tested and found to be negative for HIV-1 antibodies, HIV-2 antibodies, HCV antibodies and Hepatitis-B surface-antigen are used as control sera. Nevertheless, samples, diluted samples controls, conjugate and microtiter strips should be treated as potentially infectious material. Please handle products in accordance with laboratory Those components that contain preservatives, the Citrate Stopping Solution and the TMB have an irritating effect to skin, eyes and mucous. If body parts are contacted, immediately wash them under flowing water and possibly consult a doctor. The disposal of the used materials has to be done according to the country-specific guidelines. Material required but not supplied
Mycoplasma pneumoniae ELISA IgG/IgM/IgA GB ELISA handwasher or automated EIA plate washing device ELISA plate spectrophotometer, wavelength = 450nm, reference length = 620nm (Reference Wavelength 620-690nm) Test Procedure
Working exactly referring to the Sekisui Virotech user manual is the prerequisite for obtaining correct results. Examination Material
Either serum or plasma can be used as test material, even if only serum is mentioned in the instructions. Any type of Always prepare patient-dilution freshly. For a longer storage the sera must be frozen. Repeated defrosting shall be avoided. Only fresh non-inactivated sera should be used. Hyperlipaemic, haemolytic, microbially contaminated and turbid sera should not to be used (false positive/negative Preparation of Reagents
The Sekisui Virotech System Diagnostica offers a high degree of flexibility regarding the possibility to use the dilution buffer, washing solution, TMB, citrate stopping solution as well as the conjugate for all parameters and for all different lots. The ready to use controls (positive control, negative control, cut-off control) are parameter specific and only to use with the
plate lot indicated in the Quality Control Certificate. Set incubator to 37°C and check proper temperature setting before start of incubation. Bring all reagents to room temperature before opening package of microtiter strips. Shake all liquid components well before use. Make up the washing solution concentrate to 1 L with distilled or demineralised water. If crystals have formed in the concentrate, please bring the concentrate to room temperature before use and shake well before use. High IgG-titers or rheumatoid-factors may disturb the specific detection of IgM-antibodies and lead to false positive respetively false negative results. For a correct IgM-determination it is therefore necessary to treat the sera with
RF-SorboTech (VIROTECH adsorption). The IgM-controls must not be treated with the pre-absorption.
Virotech ELISA Test Procedure
For each test run, pipette 100µl each of ready to use dilution buffer (blank), IgG-, IgM- and IgA-positive, negative and cut- off controls as well as diluted patient sera. We propose a double insertion (blank, controls and patient sera); for cut-off control a double insertion is absolutely necessary. Working dilution of patient sera: 1+100; e.g. 10µl serum + 1ml dilution After pipetting start incubation for 30 min. at 37°C (with cover). End incubation period by washing microtiter strips 4 times with 350 – 400µl washing solution per well. Do not leave any washing solution in the wells. Remove residues on a cellulose pad. Pipette 100µl of ready to use conjugate into each well. Incubation of conjugates: 30 min. at 37°C (with cover). Stop conjugate incubation by washing 4 times (pls. refer to point 3 above). Pipette 100µl of ready to use TMB into each well. Incubation of substrate solution: 30 min. at 37°C (with cover, keep in dark). Stopping of substrate reaction: pipette 50µl of citrate stop solution into each well. Shake plate carefully and thoroughly until liquid is completely mixed and a homogeneous yellow color is visible. Measure extinction (OD) at 450/620nm (Reference Wavelength 620-690nm). Set your photometer in such a way that the blank value is deducted from all other extinctions. Extinctions should be measured within 1 hour after adding the stopping Pls. refer to last page for Test Procedure Schemata Mycoplasma pneumoniae ELISA IgG/IgM/IgA GB Usage of ELISA processors
All Sekisui Virotech ELISAs can be used on ELISA processors. The user is bound to proceed a validation of the devices Sekisui Virotech recommends the following procedure: Sekisui Virotech recommends to proceed the validation of device referring to the instructions of the device manufacturer during the implementation of the ELISA processor respectively after bigger reparations. It is recommended to check the ELISA-processor with the Validationkit (EC250.00) afterwards. A regular check using the Validationkit shall be proceeded minimum once a quarter to test the accuracy of the processor. The release criteria of the Quality Control Certificate of the product must be fulfilled for each testrun. With this procedure, your ELISA processor will function properly and this will support quality assurance in your laboratory. Test Evaluation
The ready to use controls serve for a semiquantitative determination of specific IgG-, IgM- and IgA-antibodies. Their concentration can be expressed in Virotech units = VE. Fluctuations resulting from the test procedure can be balanced with this calculation method and a high reproducibility is achieved in this way. Use the means of the OD values for calculation of Test function control
The OD of the blank should be < 0.15. The OD-values of the negative controls should be lower than the OD-values mentioned in the Quality Control Certificate. The OD-values of the positive controls as well as of the cut-off controls should be above the OD-values mentioned in the Quality The Virotech Units (VE) of the cut-off controls are defined as 10. The calculated VE of the positive controls should be within the ranges mentioned in the Quality Control Certificate. If those requirements (OD-values, VE) are not fulfilled, the test has to be repeated. Calculation of the Virotech Units (VE)
The extinction of the blank value (450/620nm) has to be subtracted from all other extinctions. Interpretation of Results
a) IgM and IgA all patients, IgG age of patient > 14 years Result (VE)
Evaluation
IgG infants (0-14 years) or suspected primary infection, IgM and / or IgA positive For infants between 0-14 years of age it is allowed to correct the c.o. for IgG 2 VEs downwards, as the Virotech-ELISA has been adjusted in IgG to detect mainly acute infections. Precondition for the use of this method is that the serum has provided a positive IgM- and / or a positive IgA-result. Result (VE)
Evaluation
Mycoplasma pneumoniae ELISA IgG/IgM/IgA GB If the measured values are above the defined borderline range, they are considered to be positive. For the secure detection of an infection it is necessary to determine the antibody concentration of two serum samples.One sample shall be taken directly at the beginning of the infection and a second sample 5 – 10 days later (convalescent serum). The antibody concentration of both samples have to be tested in parallel, that means in one test run. A correct diagnosis based on the evaluation of a single serum sample is not possible. The highest sensitivity is reached if all 3 antibody classes (IgG, IgM and IgA) are tested, as it has to be considered that some persons do not If the measured values are below the defined borderline range, no measurable antigen specific antibodies are present in the sample. The samples are considered to be negative. Interpretation Scheme
No contact with Mycoplasma pneumoniae or antibody level has already Very early stage of an acute infection or re-infection Very early stage of an acute infecction; either primary infection or reinfection without IgM or IgM-titer is still to come Acute infection, primary infection as a rule; later stage, IgG- and IgM already developed, IgA not yet decreased Acute infection, primary infection as a rule; late stage, IgG and IgM already developed, IgA already decreased Re-infection, very late stage, IgA still present, IgM no longer present, or reactivation or infection without development of IgM Re-infection, very late stage, IgA already decreased or not developed at all (happens in some adults) or reactivation or infection without development of IgM or persistent IgG-titer after past infection Acute early infection, IgA still missing or already decreased, IgG-titer still too low (false positive IgMs can not be excluded completely due to the high cross-reactivity of some Mycoplasma pneumoniae-proteins. This Limits of the Test
1. The interpretation of serological results shall always include the clinical picture, epidemiological data and all further 2. Cross-reactions with M. genitalium or M. hominis can not be excluded. Also EBV-positive sera can cross-react. 10. Performance Data
Analytic specificity
For the determination of the analytic specificity the following amounts of sera have been tested in comparison to a Western Blot: 129 sera for IgG, 133 sera for IgA and 132 sera for IgM. The sera are from patients with different respiratory diseases. Mycoplasma ELISA
Serum collection (n=129)
Virotech
Mycoplasma WB
Mycoplasma pneumoniae ELISA IgG/IgM/IgA GB For IgG arises an analytic specificity of 94,3%. Mycoplasma pneumoniae ELISA IgG/IgM/IgA GB Mycoplasma ELISA
Serum collection (n=133)
Virotech
Mycoplasma WB
For IgA arises an analytic specificity of 86,1%. Mycoplasma ELISA
Serum collection (n=132)
Virotech
Mycoplasma WB
For IgM arises an analytic specificity of 98,4%. Analytic sensitivity
For the determination of the analytic sensitivity 40 sera with suspected Mycoplasma infection have been tested in Mycoplasma ELISA
Serum collection (n=40)
Virotech
Mycoplasma WB
For IgG arises an analytic sensitivity of 95,5%. Mycoplasma ELISA
Serum collection (n=40)
Virotech
Mycoplasma WB
Due to the low amount of positive sera a percental statement of the analytical sensitivity is inappropriate. Mycoplasma ELISA
Serum collection (n=40)
Virotech
Mycoplasma WB
For IgM arises an analytic sensitivity of 96,2%. Case Studies
The university of Heidelberg described 2 patients (31 and 37 years old) with a severe Mycoplasma pneumoniae infection in a case study. As serological tools for a Mycoplasma diagnostic, the Virotech ELISA and Virotech Westernblot have been used beside the CFA. In both cases the Mycoplasma pneumoniae infections have been recognized by the ELISA and the Mycoplasma pneumoniae ELISA IgG/IgM/IgA GB Prevalence (Expected Values)
80 blooddonor sera have been tested for IgG, IgA and IgM. negative
borderline
positive
Intra-assay-Coefficient of Variation (Repeatability)
In one assay strips of different plates of one batch have been tested with a serum (known to be at cut-off value). The obtained variation co-efficient values was <9% (n=96). Inter-assay-Coefficient of Variation (Reproducibility)
Three sera each have been tested in 10 independent test runs. The obtained variation co-efficient values was <15%. 11. Literature
Clyde WA.J.: Clinical overview of typical Mycoplasma pneumoniae infections. J. Clin Infect. Dis. 1993, 17 (suppl. 1) 32-37 Hu, P.-C., Collier, A.M. and Baseman, J.B. (1977): Surface parasitism by Mycoplasma pneumoniae of respiratory epithelium. J. of Experimental med. 145, 1328-13343. Razin, S. (1992): Peculiar properties of mycoplasmas: the smallest self-replicating prokaryotes. FEMS Microbiol. Lett. Taylor-Robinson, D. (1996): Infections due to species of Mycoplasma and Ureaplasma: an update. Clin. Infect. Dis. 23, Jacobs, E.: Mycoplasmen-Infektionen. mta. 1997, 12: 236-239 Jacobs, E.: Das Adhäsin von Mycoplasma pneumoniae: Seine Bedeutung als Virulenzfaktor in der Pathogenese und in der Diagnostik. Klin. Lab. 1994: 40: 228-229 Baum, H.V., Strubel, A., Nollert, J., Layh-Schmitt, G.:Two Cases of Fulminant Mycoplasma Pneumoniae Pneumonia Foy, HM: Infections caused by Mycoplasma pneumoniae and possible carrier state in different populations of patients. J Clin Infect Dis 1993, 17(suppl. 1) 37-47. Mycoplasma pneumoniae ELISA IgG/IgM/IgA GB 12. Test Procedure Schemata
Preparation of Patient Samples and Washing Solution
Washing Solution: Fill up concentrate to 1 liter with aqua dest./demin.
IgG-/IgA-Samples – Dilution
IgM-Samples – Dilution
Rheumafactor-absorption with RF-
SorboTech
10 µl serum/plasma + 1000 µl Dilution Buffer 5 µl serum/plasma + 450 µl Dilution Buffer + 1 drop RF-SorboTech, incubate for 15 min. at room Testprocedure
30 minutes at 37°C
100 µl Patient Samples
blank value (Dilution Buffer) and controls
400 µl Washing Solution
Remove Residues on a Cellulose Pad
30 minutes at 37°C
100 µl Conjugate
IgG, IgM, IgA
400 µl Washing Solution
Remove Residues on a Cellulose Pad
30 minutes at 37°C
100 µl Substrate
50 µl Stop Solution
shake carefully
Photometer at 450/620nm
(Reference Wavelength 620-
690nm)

Mycoplasma pneumoniae ELISA IgG/IgM/IgA GB

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Egr. Dott. Marco Tronchetti ProveraPirelli & C spaVia G. Negri 1020123 MilanoFusione Pirelli S.p.a. – Pirelli & C Luxembourg S.p.a. - Pirelli & C S.a.p.a. La presente per richiederle, a nome e per conto della signora XXXXXXXX, già azionista di Sip e di Pirellis.p.a. il risarcimento dei danni subiti dalla incongrua determinazione del rapporto di cambio proposto evoluto dal consigli

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Final Draft of the original manuscript: Jung, F.; Franke, R.-P.: Extreme reduction of the capillary lumen in segments of the venular legs of human cutaneous capillaries In: Microvascular Research (2009) Elsevier DOI: 10.1016/j.mvr.2009.02.010 Extreme reduction of the capillary lumen in segments of the venular legs of human cutaneous capillaries F. Jung1*, R.P. Franke1 1 GK

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