Laboratory
Sample Preparation and Stability
Components
Test Procedure
Equipment
Shelf life and Statistics
Sample Preparation and Stability
What is the most important technical aspect of the specimen preparation for the test?
Good specimen preparation requires good pipetting techniques. It is important to have good pipetting technique, calibrated pipettes and to have the correct pipettes available for accurate dilutions. For example, it is not practical to use a 1250µl variable pipette for dispensing 10µL.
Can the same sample diluents be used for all of the tests?
No. There are different sample diluents necessary for each specific test. None of the components should be exchanged between kits.
If I run samples at a later date, do I have to run standards again?
Yes. Results of the Standards are necessary to analyze data for each use of the ELISA.
Are there special sample collection requirements for the Oncogene Science ELISAs?
See the individual Instructions for Use for the kit in question. Generally speaking there are no special sample collection and handling requirements. Testing with the ELISAs is performed with samples sent to laboratories following common sample collection procedures in serum and plasma.
Are there special sample collection requirements for the Oncogene Science ELISAs?
See the individual Instructions for Use for the kit in question. Generally speaking there are no special sample collection and handling requirements. Testing with the ELISAs is performed with samples sent to laboratories following common sample collection procedures in serum and plasma.
For Plasma Samples – EDTA “Purple Top” Tubes are recommended
For Serum Samples – “Red Top” tubes are recommended
uPA & PAI-1 ELISAs are also validated for Citrate “Blue Top” and Coagulation Tube. Remember, PAI-1 is only able to use plasma samples, not serum samples.
How long are diluted samples (analyte) stable?
For the HER-2/neu ELISA, diluted samples may be stored up to 1 week at 2-8°C. For the other Oncogene Science ELISA kits , samples should be diluted immediately before running in the test. For further information, see the individual IFU for the kit in question.
Can hemolyzed samples be used?
Measurement of hemolyzed samples should be avoided. We do not recommend use of samples of questionable quality (hemolyzed, cloudy, lipemic, containing flocculent material).
How long do you recommend that frozen serum/ plasma samples thaw and what are recommended storage volumes for the sample aliquots?
Aliquots of 100 to 1000 µL are ideal. One hundred µL aliquots should take approximately 10 minutes to thaw at room temperature. Thawed samples should be placed on ice prior to testing.
What is the stability of fresh serum or plasma samples submitted for an ELISA test? Should the serum or plasma sample be submitted at room temperature or frozen?
If the sample is to be used within 24-48 hours, it can be kept at room temperature or on ice packs at 2-8°C. For longer periods, samples should be kept frozen and brought to room temperature before use.
Components
What is the purpose of the Substrate Blank?
The Substrate Blank reading will not be used in calculating any data, as it serves as a quality check on the integrity of the reagent and proper procedure. It should not be assigned in the ELISA reader software as a Blank. It is used as a check to determine that the substrate solution used in the test has not undergone any degradation or contamination. If the Substrate Blank is within specifications, there has been no compromise of the reagent.
What components are included in an ELISA and can components be purchased individually?
Pre-coated 96 well microplate, Standards for calibration, Detector Antibody and/or Conjugate, Diluent buffers, Substrate and Platewash are included in the kits. None of the kit components are sold separately. A list of components provided in each kit can be found in the Instructions for Use. Instructions for Use can be downloaded directly from the Product section of this website. Controls are available for our ELISAs and are sold separately.
Do your ELISA controls contain multiple analytes?
No, the control materials only contain the analyte specific for the ELISA with which it is used.
I ran out of Plate Wash. Could I just use my laboratory supply of PBS?
No, it is not advisable to use non-supplied reagents.
If crystals can be seen in the Platewash Concentrate is it okay to still use the buffer?
At 4°C Platewash Concentrate may form salt crystals. The buffer should be warmed approximately 45 minutes prior to use. Place the bottle of Platewash Concentrate in a 37°C waterbath to help dissolve the crystals. Platewash Concentrate can be stored at room temperature during the indicated shelf life.
How long is Platewash stable after it has been diluted?
After diluting the wash buffers into a 1X solution, the solution should be used within 24 hours or discarded.
Can I use the same Platewash for all of the ELISA tests?
No. Our ELISA products employ more than one formulation of Platewash Concentrate. Use of the incorrect Platewash may invalidate results.
Is there enough Platewash Concentrate to wash the plate more than the required number of washes and run each microtiter plate multiple times through the wash cycle?
No. The Platewash Concentrate provided is sufficient for performing the required number of washes.
What kind of special ancillary components are required for this test?
Most of the materials and equipment are commonly found in the laboratory but the following is a checklist of items that are required and not included in the kits. (Information also available in all IFUs)
- For the CA IX ELISA, a Lab-Line Instruments titer plate shaker or equivalent, 600-1000rpm is required
- Dry heat incubator capable of maintaining 37°C (for some of the ELISAs)
- Pipettors: 2-20 µL, 20-200 µL, and 200-1000 µL pipettors with disposable tips
- Precision repeating pipettor
- Automated 96-well microtiter plate washer
- Tubes for sample preparation
- Vortex mixer
- Microtiter plate reader with proper wavelength filter capable of measuring absorbance in 96-well plates. Ideally, the reader should interface with a computer with dedicated ELISA software.
- 500- or 1000-mL graduated cylinder
- Reagent reservoirs
- Deionized water
- Plastic wrap or adhesive plate sealers
- Liquid household bleach for decontaminating Platewasher waste
- Disposable paper towels
Test Procedure
Can partial strips be run so I don’t waste an entire row if I only want to run a few samples?
Partial strips cannot be used. Once you insert an antibody coated 8-well strip into the plate frame, you will need to use the entire strip. Tests should be planned to make the most use of a kit. Standards and controls consume wells for each test. Duplicates should be placed into wells on different strips.
Is there a point during the test where I can stop the test without causing a shift in results?
The operating protocol for each ELISA has been carefully designed to provide reliable data from samples and should be carefully followed for all steps. Refer to the Instructions for Use.
How much time do I have after adding the stop reagent to read the plate? Do the ELISA plates need to be read right away?
Yes, the ELISA plates should be read as soon as possible. If the plate cannot be read immediately, it should be protected from light and read within 30 minutes.
What if a standard falls out of range and is not calculating consistent with assigned value?
Problems with duplicate agreement for standards, controls and samples may require the operator to repeat the test. Quality standards of each laboratory will determine the necessity of repeat tests.
Variable results are seen on the edges of the plate. What could be the cause of this?
Adding cold reagents to a plate may cause this so-called edge effect. Be sure that all reagents are equilibrated to room temperature before addition to the plate. We also suggest taping down the edges of the plate sealer/plastic wrap, especially if the antigen incubation is at 37°C.
What should I do if my unknown sample is out of range (higher than the highest concentration of standard?
The concentration of analyte in such a sample exceeds the concentration of the level 6 standard. This sample must be diluted to a level that can be read on the standard curve. See Instructions for Use on further sample dilutions.
Is it critical how long an ELISA plate sits immediately after tapping it dry?
Yes, the timing is important because excessive drying can damage the reagents on the plate and therefore compromise the test results. We recommend proceeding directly to the next step.
Should plates be tapped dry only on certain absorbent surfaces?
Yes! Use lint free material to avoid introducing particulate matter into the wells. Use fresh material for each test wash step.
How hard should a plate be tapped for sufficient drying after it is washed?
The plate may be blotted upside down with just a few short vigorous slaps. Avoid jarring the strips from the frame. Strips may be labelled with a laboratory marker on the outer finger tab to avoid any confusion if such an accident occurs.
Is there a checklist to ensure a valid test run?
- Check that the OD levels for standards fall within the expected dynamic range of the test.
- Check to be sure that the correct wavelength was used in the reader.
- OD values follow a trend consistent with standard order.
- Check that CVs are not >10%. Each laboratory must establish quality standards which determine the need for repeat testing.
- Check that the standard value assignments were input correctly into the ELISA software template.
- Check that the background is not elevated.
- Check that the R2 correlation coefficient of the standard curve is close to 1.0 (.999).
- Check that stop solution was added to plate.
Equipment
What Software Packages are recommended for use with the ELISA readers?
WILEX Inc. uses Softmax Pro, which is provided by Molecular Devices, Inc. as well as software provided by Bio-Tek Instruments, Inc. There are a number of reliable ELISA software packages available.
Can a 620nm-wavelength filter be used instead of a 650nm-wavelength filter for the Oncogene Science ELISAs?
In cases where one of our ELISA requires a 650nm-wavelength filter, a 620nm-wavelength filter is acceptable as long as the quality of the OD readings and control results meet test specifications.
How often do the Washers need to be primed (cleaned)?
All washers should be rinsed every night as part of their daily QC maintenance. See the equipment manufacturer recommended daily and periodic maintenance instructions.
Do you provide enough reagents in the kit to accommodate automated washers that may require more Wash Buffer Reagent in the tubing?
Reagents should be sufficient for semi-automated systems like the Bio-Tek washer. Washers that are “strip readers” and are setup to only wash the number of strips that have been run on the plate are also suitable.
Customers using the Oncogene Science ELISAs on automated ELISA instruments may find they do not have adequate volumes of reagent. These ELISAs are intended to be performed manually.
How often should the gaskets, o-rings and airline filter be checked on the plate washers?
See the recommendations of the company supplying the washer.
What chemicals are used in the decontamination protocol?
See the recommendations of the company supplying the washer.
How often should the washer tubing be replaced?
See the recommendations of the company supplying the washer.
What should be remembered when setting up a Reader with ELISA Software?
- All tests are read at a single wavelength.
- Select the appropriate wavelength filter for a particular test.
- Click “No” for the options of Auto mixing and Blanking. Always set “No” to Blanks.
- All tests are Endpoint (versus kinetic). Click on Template and assign wells the appropriate designation (standard, sample control).
- Standard 1 always defaults to 0. Concentrations for standards 2-6 must be entered using information from the package insert of each test. Assign standards the proper concentration (ng/mL, or pg/mL).
Does the plate reader have any setup prior to reading? What is required?
Refer to the specific manufacturer instructions for your particular plate reader.
What are some possible causes of variable results?
Lack of Washer maintenance, poor pipetting techniques, contamination of the plate by reusing plate sealers for subsequent steps, and reusing blotting material for subsequent steps can cause variable results. Use fresh plate sealers and paper towel for blotting.
Can I microwave my Platewash Concentrate to dissolve salts?
Place the Platewash Concentrate in a warm water bath to dissolve salt crystals. Refer to the Instructions for Use.
What kind of water should be used for this test and to dilute my Platewash Concentrate?
Distilled or Deionized water
Shelf life and Statistics
What is the coefficient of determination” R2 ” a measure of?
R2 is a measure of the curve’s quality. The curve-fitting algorithm attempts to fit a line as close to each of the measured standards points as possible. The error calculated from standards points to the fitted line is indicated as the coefficient of determination. The closer R2 is to 1.0, the better. R2 is ideally .997 or better. This is not unlike a least squares determination. Please refer to the product IFU for additional information.
What is an acceptable CV% (coefficient of variation) for standards and samples (unknowns)?
Acceptable CVs are 10% or less. If the CV% is greater than 10% determine outliers and mask those. If there is a high CV number and only 2 unknowns were run, it is necessary to re-test the sample because determination of which sample is the outlier is impossible… Each laboratory should establish its own methods and specifications for identifying and handling outlier results
What is the lot numbering system for reagent vials and kit? Are there lot numbers on each of the vials in the kits? Are the lot numbers on the vials the same as the lot numbers on the kit?
Each component reagent vial in the kit has a unique lot number. The various components may all have different lot numbers and dating. The complete kit has a unique lot number and expiration date on the outer box label of the kit. The kit’s expiration date is equivalent to the shortest dated component vial in the kit configuration.
