ELISA/assay

Human Aspergillus Fumigatus IgG ELISA Kit

DEIA312

96T

Human Aspergillus Fumigatus IgG ELISA Kit

A. fumigatus

Human Aspergillus Fumigatus IgG ELISA Kit

iELISA

A. fumigatus

Human

Quantitative

Serum, plasma

96T

1. Microtiter strips (antigen). After opening at 2-8°C in closed aluminum bag with desiccant. Strips which are not used must be stored in the press-seal bag of aluminum compound foil under dry and airtight conditions! 4 weeks. 2. Control sera / standard sera. After opening at 2-8°C. Until expiry date; 24 months from date of production. 3. Conjugate ready-to-use solution, at 2-8°C. Avoid contamination (sterile tips!). Until expiry date 28 months from date of production 4. Dilution buffer. After opening at 2-8°C, 24 months. Discard cloudy solutions! Unopened, until expiry date; 36 months from date of production. 5. Washing solution. Concentrate after opening at 2-8°C, until expiry date. Working dilution at 2-8°C, 2 weeks. Working dilution at room temperature, 1 week. Bottles used for the working dilution should be cleaned regularly, discard cloudy solutions. 6. Substrate, ready-to-use solution at 2-8°C, protected from light! Until expiry date 24 months from date of production. Avoid contamination (sterile tips!) Discard when solution turns yellow (extinction against distilled water. > 0.25). 7. Stopping solution, after opening at room temperature, until expiry date.

A. fumigatus

Human

Aspergillus fumigatus, A. fumigatus, Fungi, Ascomycota, Eurotiomycetes, Eurotiales, Trichocomaceae, Aspergillus

The Aspergillus fumigatus IgG is quantitative and qualitative test for the detection of human antibodies in serum or plasma against Aspergillus fumigatus. For Research Use Only. Not for use in diagnostic procedures.

The fungus Aspergillus belongs to the group of ascomycetes. These organisms develop branched mycelia. They spread through conidiospores which are released from mycelia as extremely resistant lasting spores. 20 species of Aspergillus are human pathogens being opportunistic infectious agents. The most important species is Aspergillus fumigatus. Aspergillus fumigatus can cause multiple allergic and invasive diseases. The pathogenesis of an invasive Aspergillus infection takes place in several steps: The fungal spores are transmitted through the air as a so called "bioaerosol" and are absorbed by the lungs. Due to their size of about 3 μm they reach the lung alveoli. In case of an invasive aspergillosis fungus hyphae penetrate the bronchial mucosa and the surrounding lung parenchyma with the aid of released proteinases. The Aspergillus spores tend to penetrate the blood vessels and settle down in remote tissues after a hematogen dissemination.

Microtest plates are coated with antigens. This constitutes the solid phase. Sample is added to the plates and any antibodies specific for the antigen present will bind to the solid phase. After removal of unbound material, anti-human IgG conjugated to an enzyme (alkaline phosphatase) is allowed to react with the immune complex. After removal of excess conjugate by washing, an appropriate substrate (paranitrophenylphosphate) is added, with which the conjugated enzyme reacts producing a coloured derivative of the substrate. The colour intensity is proportional to the level of specific antibody bound and can be quantified photometrically.

1. Break apart microtiter test strips each with 8 antigen coated single wells (altogether 96) MTP, 1 frame, the coating material is inactivated, 12 2. Standard serum (ready-to-use) STD Human serum in phosphate buffer with protein; negative for anti-HIVAb, HBs-Ag (Hepatitis B-Virus-surface antigen) and anti-HCV-Ab; preservative: 3. Negative control serum (ready-to-use) NEG Human serum in phosphate buffer with protein; negative for anti-HIV-Ab, HBs-Ag (Hepatitis B-Virus-surface antigen) and anti-HCV; preservative: 4. Anti-human-IgG-conjugate (ready-to-use) APC Anti-human-IgG from goat (polyclonal), conjugated to alkaline phosphatase, stabilized with protein stabilization solution, preservative: 0.01 % methylisothiazolone, 0.01 % bromnitrodioxane, 13 ml 5. Washing solution concentrate (sufficient for 1 litre) WASH Sodium chloride solution with Tween 20, 30 mM Tris, preservative: 6. Dilution buffer DILB Phosphate buffer with protein and Tween 20; preservative: 7. Stopping solution STOP 1.2 N sodium hydroxide, 15 ml 8. Substrate (ready-to-use) pNPP Para-nitrophenylphosphate, solvent free buffer, preservative: < 0.1 % sodium azide, (Substrate in unopened bottle may have a slightly yellow coloring. This does not reduce the quality of the product!), 13 ml

1. common laboratory equipment 2. photometer for microtiter plates with filter, wavelength 405 nm, recommended reference wavelength 620 nm - 690 nm (e.g. 650 nm) 3. incubator 37°C 4. moist chamber 5. distilled water

1. Sample preparation and storage Lipaemic, hemolytic or icteric samples should only be tested with reservations although in our testing no negative influence has been found. Obviously contaminated samples (serum or plasma) should not be tested due to the risk of wrong results. Serum or plasma (EDTA, citrate, heparin) collected according to standard laboratory methods are suitable samples. Samples must not be thermally inactivated. 2. Sample preparation Before running the test, samples must be diluted in dilution buffer (V1 + V2) as follows: 3. Sample storage The stoppered samples can be stored in a refrigerator up to 7 days at 2-8°C. Extended storage is possible at ≤ -20°C. Avoid repeated freezing and thawing of samples. Diluted samples can be stored at 2-8°C for one week.

1. Microtest strips Microtest strips in frame are packed with desiccant in an aluminum bag. Take unrequired cavities out of the frame and put them back into the press-seal bag. Close press-seal bag carefully to ensure airtight conditions. 2. Control sera / standard sera Control and standard sera are ready-to-use and must not be diluted any further. They can be used directly for the test run. For each test run and for each test system - independent of the number of microtest strips to be used - control and standard sera must be included. The cut-off-control should be set up in duplicate. With the quantitative tests the standard serum should also be set up in duplicate. Do not treat control sera with Rf-absorbent. 3. Anti-human-IgG-conjugate (ready-to-use) Please do not mix up conjugates from different kits. They are optimized for each lot. Avoid contamination of ready-to-use conjugates (please pour sufficient for test into a secondary container to avoid repeatedly pipetting from the original bottle). 4. Washing solution Dilute washing buffer concentrate (V 1 ) 1:30 with distilled water to a final volume of V 2 . Example: 5. Dilution buffer for samples (ready-to-use) 6. Substrate (ready-to-use) To avoid contamination use gloves. For pipetting substrate solution use sterile tips only! 7. Stopping solution (ready-to-use)

1. Place the required number of cavities in the frame and prepare a protocol sheet. 2. Add each 100 μl of diluted sample or ready-to-use controls into the appropriate wells of microtest strips. Spare one well for substrate blank, e.g.: well A1 substrate blank well B1 negative control well C1 standard serum well D1 standard serum well E1 sample 1. 3. Sample incubation for 60 minutes (+/- 5 min) at 37°C (+/- 1°C) in moist chamber 4. After incubation wash all wells with washing solution (by automated washer or manually): aspirate or shake out the incubation solution fill each well with 300 μl washing solution aspirate or shake out the washing buffer repeat the washing procedure 3 times (altogether 4 times!) dry by tapping the microtest plate on a paper towel 5. Addition of conjugate: Add 100 μl of IgG-conjugate (ready-to-use) to the appropriate well (except substrate blank) 6. Conjugate incubation for 30 minutes (+/- 1 min) * at 37°C (+/- 1°C) in moist chamber. 7. After incubation wash all wells with washing solution (see above) 8. Addition of substrate: Add 100 μl substrate solution (ready-to-use) to each well (including well for substrate blank!) 9. Substrate incubation for 30 minutes (+/- 1 min) * at 37°C (+/- 1°C) in moist chamber. 10. Stopping of the reaction: Add 100 μl stopping solution to each well, shake microtest plate gently to mix. 11. Read optical density: Read OD within 60 minutes at 405 nm against substrate blank, reference wave length between 620 nm and 690 nm (e.g. 650 nm). *Please note, that under special working-conditions internal laboratory adaptations of the incubation times could be necessary.

1. Single-point quantification with the 4PL method Optimized assignment of extinction signals to quantitative values is guaranteed by using non-linear functions, which adjust a sigmoide curve without any further transformation to OD-values. Determination of antibody concentrations with the kit is carried out by the logistic-log-model (4 PL; 4 parameter) which is ideal for exact curve-fitting. It is based on the formula: The parameters A, B, C, and D are representative for the exact shape of the curve: lower asymptote → parameter A slope of the curve → parameter B turning point → parameter C upper asymptote → parameter D For each lot the standard curve is evaluated by CD in several repeated test runs under optimal conditions. Time consuming and cost intensive construction of the standard curve by the user is not necessary. For evaluation of antibody concentrations a lot specific standard curve as well as a lot specific evaluation table is included with each test kit. Appropriate evaluation software is available on request. To compensate for normal test variations and also for test run control a standard serum is used in each individual test run. For this control serum a ''reference value'' with a validity range is determined by the quality control of the producer. Within this range a correct quantification of antibody concentration is ensured. Since the standard serum is not necessarily a positive control, the value of the standard serum may be borderline or negative in some ELISA tests. 2. Criteria of validity The substrate blank must be OD The negative control must be negative Quantitative ELISA: the mean OD-value of the standard serum must be within the validity range , which is given on the lot specific quality control certificate of the kit (after subtraction of the substrate blank!) Qualitative ELISA: the mean OD-value of the positive control must be within the validity range, which is given on the lot specific quality control certificate of the kit (after subtraction of the substrate blank!) The variation of OD-values may not be higher than 20%. If these criteria are not met, the test is not valid and must be repeated. 3. Calculation ELISA classic Aspergillus fumigatus IgG/IgM/IgA (quantitative) a. Non-automated evaluation For the test evaluation a standard curve and an evaluation table are included in the test kit so that the obtained OD-values may be assigned to the corresponding antibody activity. The reference value and the validity range of the standard serum is given on the evaluation table (quality control certificate). The blank (A1) must be subtracted from all OD-values prior to the evaluation. Method 1: Qualitative Evaluation To fix the cut-off ranges please multiply the mean value of the measured standard-OD with the numerical data of the certificate of quality control (see special case formulas), e.g.: OD = 0.502 x MW(STD) with upper cut-off OD = 0.352 x MW(STD) with lower cut-off If the measured mean absorbance value of the standard serum is 0.64, the range of the cutoff is in between 0.225-0.321. Method 2: Continuous determination of antibody activities using the standard curve. So called interassay variations (day to day deviations and laboratory to laboratory deviations) are compensated by multiplication of the current measured value obtained with a sample with the correction factor F. This factor is calculated as follows: The procedure is necessary to adjust the current level of the test of the user with the lotspecific standard curve. First, daily deviations are to be corrected by calculating a factor (correction factor F): 1. The mean of the two OD-values of the standard serum has to be calculated and checked that it is within the given validity range. 2. Calculation of the factor "F": the given reference value is divided by the mean of the extinction of the standard serum: F = reference value extinction standard serum / mean value extinction standard serum. 3. All measured values of samples are multiplied by "F". 4. Antibody activities in IU/ml or U/ml can be determined from the standard curve with the corrected values.

1. Evidence of deterioration Only use ELISA reagents for test procedure, since all reagents are matched. In particular standard and control sera are defined exclusively for the test kit to be used. Do not use them in other lots. There are three different conjugate concentrations for each immunoglobulin class: LOW, MEDIUM, HIGH The classification is written on each label as follows: e.g. IgG + lowly concentrated IgG conjugate IgG ++ medium concentrated IgG conjugate IgG +++ highly concentrated IgG conjugate In rare cases the use of special conjugate is necessary to guarantee consistent quality for our products. Special conjugates are produced in a separate lot and do not wear the "+" sign. Therefore, special conjugates are not exchangeable with other conjugates. Please pay close attention to notifications on labels! Unopened, all components of the kits may be used up to the dates given on the labels, if stored at +2°C to +8°C. Complete stability and storage data are described under "Storage". Each reagent has been calibrated and optimized for the test. Dilution or alteration of these reagents may result in a loss of sensitivity. Avoid exposure of reagents to strong light during storage and incubation. Reagents must be tightly closed to avoid evaporation and contamination with microorganisms since incorrect test results could occur due to interference from proteolytic enzymes. To open the press-seal bag please cut off the top of the marked side, only. Do not use the strips if the aluminum bag is damaged or if the press-seal bag with remaining strips and desiccant was not properly reclosed. Bring all reagents to room temperature before testing. Use aseptic techniques for removing aliquots from the reagent tubes to avoid contamination. To avoid false positive results ensure not to contact or sprinkle the top-walls of wells while pipetting conjugate. Be careful not to mix the caps of the bottles and/or vials. Reproducibility depends on thorough mixing of the reagents. Shake the flasks containing control sera before use and also all samples after dilution (e.g. by using a monomixer). Be sure to pipette carefully and comply with the given incubation times and temperatures. Significant time differences between pipetting the first and last well of the microtiter plate when filling samples/control sera, conjugate or substrate may result in different 'pre incubation' times, which may influence the precision and reproducibility of the results. Optimum results can only be achieved if classic instructions are followed strictly. The test is not valid, if the lot-specific validation criteria on the quality control certificate are not fulfilled. Inadequate washing will affect the test results: The washing procedure should be carried out carefully. If the washing procedure is carried out automatically follow the instruction manual of the respective washer. Flat bottom wells are used for ELISA. All wells should be filled with equal volumes of washing buffer. At the end of the procedure ensure that the wells are free of all washing buffer by tapping the inverted microtest plate on a paper towel. Avoid foam! Do not scratch coated wells during washing and aspiration. If using an automated washer, ensure it is operating correctly. 2. Statements of warning The ELISA is only designed for qualified personnel who are familiar with good laboratory practice. All kit reagents and human specimen should be handled carefully, using established good laboratory practice. This kit contains human blood components. Although all control- and cut-off-sera have been tested and found negative for HBs-Ag-, HCV- and HIV-antibodies, they should be considered potentially infectious. Do not pipette by mouth. Do not smoke, eat or drink in areas in which specimen or kit reagents are handled. Wear disposable gloves, laboratory coat and safety glasses while handling kit reagents or specimen. Wash hands thoroughly afterwards. Samples and other potentially infectious material should be decontaminated after the test run. Reagents should be stored safely and be unaccessible to unauthorized access e.g. children. Stopping solution: corrosive (C); cause acid burn (R34) use safety glasses, gloves and laboratory coat while handling!

The Aspergillus fumigatus IgG is quantitative and qualitative test for the detection of human antibodies in serum or plasma against Aspergillus fumigatus. For Research Use Only. Not for use in diagnostic procedures.

1. Break apart microtiter test strips each with 8 antigen coated single wells (altogether 96) MTP, 1 frame, the coating material is inactivated, 12 2. Standard serum (ready-to-use) STD Human serum in phosphate buffer with protein; negative for anti-HIVAb, HBs-Ag (Hepatitis B-Virus-surface antigen) and anti-HCV-Ab; preservative: 3. Negative control serum (ready-to-use) NEG Human serum in phosphate buffer with protein; negative for anti-HIV-Ab, HBs-Ag (Hepatitis B-Virus-surface antigen) and anti-HCV; preservative: 4. Anti-human-IgG-conjugate (ready-to-use) APC Anti-human-IgG from goat (polyclonal), conjugated to alkaline phosphatase, stabilized with protein stabilization solution, preservative: 0.01 % methylisothiazolone, 0.01 % bromnitrodioxane, 13 ml 5. Washing solution concentrate (sufficient for 1 litre) WASH Sodium chloride solution with Tween 20, 30 mM Tris, preservative: 6. Dilution buffer DILB Phosphate buffer with protein and Tween 20; preservative: 7. Stopping solution STOP 1.2 N sodium hydroxide, 15 ml 8. Substrate (ready-to-use) pNPP Para-nitrophenylphosphate, solvent free buffer, preservative: < 0.1 % sodium azide, (Substrate in unopened bottle may have a slightly yellow coloring. This does not reduce the quality of the product!), 13 ml

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet