IgG elisa kit product blog
2017-09-14 09:58:31 by mybiosource_staff
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|Tags: ELISA Kit; IgG elisa kit; IgG; Yersinia IgG;
|The IgG n/a (Catalog #MBS191293) is an ELISA Kit and is intended for research purposes only. The product is available for immediate purchase.
To buy or view more detailed product information and pricing, please click on the technical datasheet page below:
(Or you can also download the PDF Manual
for complete product instructions).
Y. enterocolitica and Y. pseudotuberculosis are important human pathogens which are responsible for a range of intestinal syndromes e.g. enteritis, diarrhea, pseudoappendicitis and, in immune compromised patients, haemopathies and sepsis.Post infection complications such as reactive arthritis and erythema nodosum are common mainly in carriers of the HLA-B27 antigen. Yersinia are ubiquitous but primarily distributed in temperate and sub-tropical areas. They are found in the intestinal tracts and faeces of warm blooded animals (wild and domesticated). Transmission is primarily through contaminated foodstuffs. The virulence of the bacterium is dependent upon plasmid-encoded proteins with molecular weights in the range of 23-51 kDa. These virulence-associated proteins, referred to as YOPs (Yersinia Outer Proteins) have been characterized and found to be important indicators of infection. Serion ELISA classic Yersinia IgG/IgM/IgA detect antibodies against all Yersinia serovars pathogenic for humans.
Test Principle: Microtiter wells are coated with antigens. This constitutes the solidphase. Sample is added to the wells and any antibodies specific for the antigen present will bind to the solid phase. After removal of unbound material, anti-human IgGorIgAorIgM conjugated to an enzyme (alkalinephosphatase) 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 coloredderivativeofthesubstrate. The color intensity is proportional to the level of specific antibody bound and can be quantified photometrically.
Intended Use: SERION ELISA classicYersinia (IgG, IgM, IgA) are quantitative and qualitative test systems for detection of human anti-Yersinia antibodies in serum or plasma directed against plasmid-encoded virulence markers. For sale in the U.S. for Research Use Only. Material required but not supplied: common laboratory equipment
for the IgM-ELISA: SERION Rf-Absorbent (Order no. Z200/20ml)
photometer for microtiter plates with filter, wave length 405 nm, recommended reference wave length 620 nm - 690 nm (e.g. 650 nm)
incubator 37 degree C
Test Procedure Serion ELISA Classic: Evidence of deterioration
Only use SERION ELISA classic 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. Dilution buffer, washing solution and substrate solution can be used for all SERION ELISA classic kits irrespective of the lot and the test.
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 SERION ELISA classic kits may be used up to the dates given on the labels, if stored at +2 degree C to +8 degree C. Complete stability and storage data are described under "6. Storage and Stability ".
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 SERION ELISA 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 SERION ELISA classic. 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.
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.
Before running the test, samples must be diluted in dilution buffer (V1 + V2) as follows:
SERION ELISA classic Campylobacter jejuni IgG/IgA
After dilution and before pipetting into the microtiter plate the samples must be mixed thoroughly to prepare a homogenous solution.
SERION ELISA classic Yersinia IgM
Rheumatoid factors are autoantibodies mainly of the IgM-class, which preferably bind to IgG-immune-complexes. The presence of non-specific IgM-antibodies (rheumatoid factors) an lead to false-positive results in the IgM-assay. Furthermore, the possibility exists, that weak-binding pathogen-specific IgM-antibodies are displaced by stronger-binding IgG-antibodies. In this case, IgM-detection can lead to false-negative results. Therefore it is necessary to pretreat samples with rheumatoid factor-absorbent prior to IgM detection (SERION Rheumatoid Factor-Absorbent, Order-No. Z200 (20 ml/100 tests)).
Before running the test,rheumatoid factor-absorbent (V1) must be diluted 1+4 indilution buffer (V2).
Samples (V4) must be diluted in this Rf-dilution buffer (V3)
The stoppered samples can be stored in a refrigerator up to 7 days at 2-8 degree C. Extended storage is possible at <= -20 degree C.
Avoid repeated freezing and thawing of samples.
Diluted samples can be stored at 2-8 degree C for one week.
Preparation of kit reagents
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.
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.
Anti-human-IgG-, IgM- or IgA-AP-conjugate (ready-to-use)
Please do not mix up conjugates from different kits. They are optimized for each lot. Conjugates are exchangeable as described in 7.1.
Avoid contamination of ready-to-use conjugates (please pour sufficient for test into a secondary container to avoid repeatedly pipetting from the original bottle).
Dilute washing buffer concentrate (V1) 1:30 with aqua dest. to a final volume of V2.
Dilution buffer for samples (ready-to-use)
To avoid contamination use gloves. For pipetting substrate solution use sterile tips only!
Stopping solution (ready-to-use)
Place the required number of cavities in the frame and prepare a protocol sheet.
Add each 100 mul of diluted sample or ready-to-use controls into the appropriate wells of microtest strips. Spare one well for substrate blank,
Sample incubation for 60 minutes (+/- 5 min) at 37 degree C (+/- 1 degree C) in moist chamber
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 mul 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
Addition of conjugate
Add 100 mul of IgG-/IgM-/IgA-conjugate (ready-to-use) to the appropriate well (except substrate blank)
Conjugate incubation for 30 minutes (+/- 1 min) * at 37 degree C (+/- 1 degree C) in moist chamber.
After incubation wash all wells with washing solution (see above)
Addition of substrate
Add 100 mul substrate solution (ready-to-use) to each well (including well for substrate blank!)
Substrate incubation for 30 minutes (+/- 1 min) * at 37 degree C (+/- 1 degree C) in moist chamber.
Stopping of the reaction
Add 100 mul stopping solution (ready-to-use) to each well, shake microtest plate gently to mix.
Read optical density
Read OD within 60 minutes at 405 nm against substrate blank, reference wave lengthbetween 620 nm and 690 nm (e.g. 650 nm).
Test Evaluation: SERION ELISA classic Yersinia IgG/IgM/IgA (quantitative)
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 SERION ELISA classic 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:
For each lot the standard curve is evaluated by Institut Virion\Serion GmbH (Würzburg, Germany) 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.
Criteria of validity
the substrate blank must be OD < 0.25
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.
SERION ELISA classic Yersinia IgG/IgM/IgA (quantitative)
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),
If the measured mean absorbance value of the standard serum is 0.64, the range of the cut-off 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 lot-specific standard curve.
First, daily deviations have to be corrected by calculating a factor (correction factor F):
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.
Calculation of the factor "F": the given reference value is divided by the mean of the extinction of the standard serum:
All measured values of samples are multiplied by "F".
Antibody activities in IU/ml or U/ml can be determined from the standard curve with the corrected values.
Automatic test evaluation with
SERION easy base 4PL-Software/SERION evaluate-Software
After input of the 4 parameters and the reference value of the standard serum, antibody activities are calculated online. If the optical density of the standard is out of the valid range, the following message will appear:
SERION easy base 4PL-Software:
"Standards are not in tolerance range" and/or "Distance between standards is greater than 20 %."
„Standard values out of ranges in following groups: Group 1-24. Standard value differ more than 20% in following groups: Group 1-24."
In these cases the test run is invalid and should be repeated.
Parameters and reference value need to be changed only if there is a change of lot (evaluation table shows parameters and reference values). Correct input of the lot specific data can be checked on the basis of the IU/ml or U/ml assigned to the standard serum. The calculated mean value of the units has to correspond to the unit value indicated on the lot specific certificate. There is an automatic correction of the measured values. In the standard version the printout displays the following:
Statements of warning: The SERION ELISA classic 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!
Please observe the relevant statutory requirements!.