Human Aspergillus Fumigatus IgA ELISA Kit

Sample

Serum, plasma

Species Reactivity

Human

Detection Method

iELISA

Intended Use

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

Contents of Kit

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: < 0.1 % sodium azide, colouring: Amaranth O, 2 × 2 ml
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: < 0.1 % sodium azide, colouring: Lissamin green V, 2 ml
4. Anti-human-IgA-conjugate (ready-to-use) APC, Anti-human-IgA 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: < 0.1 % sodium azide, 33.3 ml
6. Dilution buffer DILB, Phosphate buffer with protein and Tween 20; preservative: < 0.1 % sodium azide 0.01 g/l Bromphenol blue sodium salt, 2 × 50 ml
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
9. Quality control certificate with standard curve and evaluation table INFO (quantification of antibodies in IU/ml or U/ml), 1

Storage

1. Microtiter strips (antigen): after opening at 2-8°C in closed aluminum bag with desiccant (4 weeks). Strips which are not used must be stored in the press-seal bag of aluminum compound foil under dry and airtight conditions!
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)
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! Avoid contamination (sterile tips!) Discard when solution turns yellow (extinction against distilled water > 0.25). (Until expiry date)
7. Stopping solution: after opening at room temperature (until expiry date).

General Description

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.

Standard Curve

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 ELISA 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 Creative Diagnostics 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
a. the substrate blank must be OD < 0.25
b. the negative control must be negative
c. 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!)
d. 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
Aspergillus fumigatus IgA (quantitative) - 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 × MW(STD) with upper cut-off
OD = 0.352 × 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.