Oncogene FAQ

General

Which Oncogene Science ELISA products are FDA-cleared?
Only the IVD (In Vitro Diagnostic) ELISA products are FDA-cleared. RUO (Research Use Only) products are not. See list below.

Product	Item No.	Approval Status
HER-2/neu ELISA	06489876	In Vitro Diagnostic
EGFr ELISA	06489930	Research Use Only
uPA ELISA	06489892	Research Use Only
PAI-1 ELISA	06489914	Research Use Only
VEGF₁₆₅ ELISA	06489957	Research Use Only
TIMP-1 ELISA	06489973	Research Use Only
Ras p21 ELISA	06490009	Research Use Only
MN/CAIX ELISA	06490025	Research Use Only

Are your ELISA Products Reimbursable?
Third party payers will only reimburse kits that are FDA-cleared for their intended use. RUO assays do not generally qualify for reimbursement.

Product	CPT Code	NLA*
HER-2/neu ELISA	83950	$89.99

* Center for Medicare and Medicaid Services National Limitation Amount. This reimbursement information is being provided as a service to Siemens Healthcare Diagnostics Inc. customers to assist in accurate reporting and billing for Siemens Diagnostics tests. This information is based upon information from coding authorities and/or insurers on how it is appropriate to bill and report Siemens Diagnostics assays in claims to third party insurers, such as Medicare. Specifically, this information was retrieved from the Centers for Medicare and Medicaid Services website at www.cms.gov, and is subject to change. Siemens Diagnostics makes no representations or warranties regarding the accuracy or completeness of the information on this website. There is no guarantee that any test will be covered and paid. Coverage varies depending upon the health insurer's policies and the patient's medical condition. It is the responsibility of the treating physician to indicate the patient's condition, which is the basis for determining medical necessity and coverage. Actual payment amounts for covered tests can vary from state to state, and from payor to payor. Please contact your local Medicare carrier or other health plan for additional details on local policies and to ensure compliance with specific plan requirements.

How many tests does a kit generate?
There are 40 test determinations per kit when samples are tested in duplicate. Generally, assays use 6 standards and 3 controls, each in duplicate. Test wells dedicated to these reagents reduce the number of wells available for samples.

What sample types are your ELISA products intended for?
All of the ELISA products we offer are intended for HUMAN serum and/or plasmas samples only. Our EGFr ELISA can also be run using cell culture fluids. However, refer to the Instructions For Use (found on this website) for the specific ELISA product in question.

Product Specific

HER-2/neu ELISA

Can urine samples be run on the HER-2/neu ELISA?
Urine samples are not validated for use in the HER-2/neu ELISA.

Can plasma samples be run on the HER-2/neu ELISA?
Yes, the HER-2/neu ELISA can be used on plasma samples, but for research purposes only. The intended use of the HER-2/neu ELISA for clinical purposes is for measurement of HER-2/neu protein in serum samples when monitoring patients with metastatic breast cancer.

Does the HER-2/neu ELISA measure phosphorylated or non-phosphorylated versions of the HER-2/neu oncoprotein?
The HER-2/neu ELISA reagents are directed to the p105 extracellular domain (ECD) of the HER-2/neu oncoprotein, and are not able to distinguish phosphorylation states of the full length molecule.

Can I run an overnight HER-2/neu assay?
No, the HER-2/neu Instructions for Use describe a one-day procedure only.

EGFr ELISA

What is measured in the EGFr ELISA?
The EGFr ELISA reagents are directed to the p110 extra cellular domain (ECD) of the EGFr oncoprotein.

What is the clinical utility of EGFr ELISA?
The EGFr ELISA is a Research Use Only product. There is no established clinical utility at this time.

Does the HER-2/neu p105 interfere with the EGFr ELISA?
The EGFr ELISA does not detect HER-2/neu p105. The EGFr ELISA has been challenged with concentrations of HER-2/neu that are ten-fold higher than the upper end of the assay dynamic range with no interference detected.

What is the stability of EGFr in frozen sera, which has already been defrosted once?
Our evaluation of freeze/thaw stability of EGFr p110 indicates that the molecule is stable through at least six freeze-thaw cycles.

What are the normal ranges for the EGFr ELISA?
The levels listed should be used as a guideline only. The determination of normal ranges should be carried out by each laboratory using appropriate samples.

Sample Type	Mean Value (ng/mL)	Range (ng/mL)
Serum	61	48–72
EDTA Plasma	62	52–75

uPA ELISA

What are the normal ranges for the uPA ELISA?
The levels listed should be used as a guideline only. The determination of normal ranges should be carried out by each laboratory using appropriate samples.

SAMPLE GROUP: HEALTHY MALES AND FEMALES (N=118)
Mean recovery of uPA	Recovery range
1192 pg/mL serum	459–1924 pg/mL serum

PAI-1 ELISA

What are the normal ranges for the PAI-1 ELISA?
The levels listed should be used as a guideline only. The determination of normal ranges should be carried out by each laboratory using appropriate samples.

SAMPLE GROUP: HEALTHY MALES AND FEMALES (N=100)
Mean recovery of PAI-1:	Recovery range:
25.9 ng/mL plasma	5.6–150 ng/mL plasma

Has Oncogene Science performed any comparison studies on what kind of plasma is most suited for the PAI-1 ELISA?
In comparative in-house PAI-1 studies, no differences were seen in results obtained from plasma samples under Heparin, EDTA, or Citrate sample collection conditions. Therefore, the customer should have confidence in their PAI-1 ELISA results no matter what anticoagulant/preservative is used with the plasma samples.

TIMP-1 ELISA

What is measured in the TIMP-1 ELISA?
The TIMP-1 ELISA is intended for the quantitation of total (free and complexed) tissue inhibitor of metalloproteinase-1 (TIMP-1).

What are the normal ranges for the TIMP-1 ELISA?
The levels listed should be used as a guideline only. The determination of normal ranges should be carried out by each laboratory using appropriate samples.

TIMP-1 LEVELS IN NORMAL HUMAN EDTA PLASMA (N=35)
Range	Mean;	Mean + 2SD	95% fall below:
94–250 ng/mL	150 ng/mL	220 ng/mL	216 ng/mL

VEGF₁₆₅ ELISA

What does the "165" represent in the product name?
The VEGF165 ELISA is a research-use-only, enzyme immunoassay for the quantitation of the 165 amino acid length human vascular endothelial growth factor (VEGF165). This product is specific for detecting VEGF165 Ievels, the most prominent isoform found in circulation, in human serum and plasma.

What are the normal ranges for the VEGF₁₆₅ ELISA?
A number of human serum and plasma samples have been tested to determine sample values. In general, plasma recoveries are significantly lower than serum recoveries when measuring matched samples.

The levels listed below should be used as a guideline only. The determination of normal ranges should be carried out by each laboratory using appropriate samples.

VEGF₁₆₅ LEVELS IN NORMAL HUMAN MALE SERUM (N=101) (ND = Nondetectable)
Range	95% fall below:
ND-303 pg/mL	155 pg/mL

VEGF₁₆₅ LEVELS IN NORMAL HUMAN FEMALE SERUM (N=121)
Range	95% fall below:
ND-835 pg/mL	163 pg/mL

VEGF₁₆₅ LEVELS IN NORMAL HUMAN EDTA PLASMA (N=76)

Range

ND-60 pg/mL

Ras p21 ELISA

What is measured in the Ras p21 ELISA?
The Ras p21 ELISA is an enzyme-linked immunoassay used for the quantitation of circulating ras p21 in human serum and plasma.

What are the normal ranges for the Ras p21 ELISA?
A number of human serum and plasma samples have been assayed to determine expected values. Plasma values may be significantly higher than serum values when measuring matched samples. Therefore it is suggested that sample type be kept consistent within a study.

The levels listed in the following tables should be used as a guideline only. The determination of normal ranges should be carried out by each laboratory using appropriate samples.

Ras p21 IN MATCHED NORMAL FEMALE SERUM AND PLASMA SAMPLES (N=55) < 40 pg/mL = Nondetectable (ND)
	Plasma (pg/mL)	Serum (pg/mL)
Mean	98.2	62.7
SD	65.3	32.3
Mean-2SD	ND	ND
Mean+2SD	195.8	96.8
Range	ND-291.2	ND-218.4
95% fall below	259.2	104.1

Ras p21 IN MATCHED NORMAL MALE SERUM AND PLASMA SAMPLES (N=54) < 40 pg/mL = Nondetectable (ND)
	Plasma (pg/mL)	Serum (pg/mL)
Mean	98.7	70.8
SD	62.8	29.0
Mean-2SD	ND	ND
Mean+2SD	224.4	128.8
Range	53.7-478.0	ND-147.3
95% fall below	167.0	132.2

Ras p21 IN MATCHED PROSTATE CANCER SAMPLES (N=18) < 40 pg/mL = Nondetectable (ND)
	Plasma (pg/mL)	Serum (pg/mL)
Mean	1451.1	171.0
SD	1150.8	72.0
Mean-2SD	ND	ND
Mean+2SD	3752.8	315.0
Range	91.1-4077.1	77.7-323.2
95% fall below	2940.9	272.2

MN/CAIX ELISA

What is measured in the MN/CAIX ELISA?
The MN/CAIX ELISA is an enzyme-linked immunoassay used for the quantitation of MN protein, also known as CA IX, in human serum and plasma.

What are the normal ranges for the MN/CAIX ELISA?
The levels listed in the following tables should be used as a guideline only. The determination of normal ranges should be carried out by each laboratory using appropriate samples.

MN/CAIX pg/ mL IN NORMAL HUMAN SERUM AND EDTA PLASMA SAMPLES
	Serum (N=34)	Plasma (N=10)
Range	25–1795.0	58–432.0
Mean	269.0	178.0
Mean+2SD	648.0	397.0
95% fall below	678.0	ND

MN/CAIX IN MATCHED NORMAL HUMAN SERUM AND PLASMA SAMPLES (N=10)
Sample	Serum (pg/mL)	Plasma (pg/mL)
1	136.0	142.0
2	166.0	172.0
3	175.0	183.0
4	37.0	62.0
5	323.0	280.0
6	106.0	161.0
7	54.0	58.0
8	121.0	124.0
9	150.0	164.0
10	398.0	432.0

Laboratory Questions

Sample Preparation and Stability

What is measured in the MN/CAIX ELISA?
The MN/CAIX ELISA is an enzyme-linked immunoassay used for the quantitation of MN protein, also known as CA IX, in human serum and plasma.

What is the most important technical aspect of the specimen preparation for the assay?
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 250ul variable pipette for dispensing 10ul.

Can the same sample diluents be used for all of the assays?
No. There are different sample diluents necessary for each specific assay. 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.

For Plasma Samples- EDTA "Purple Top" Tubes are recommended

For Serum Samples- "Red Top" tubes are recommended

uPA & PAI-1 Assays are also validated for Citrate "Blue Top" and Coagulation Tube. Remember, uPA:PAI-1 Complex and PAI-1 are only able to use plasma samples, not serum samples.

How long are diluted samples (analyte) stable?
See the individual Instructions for Use for the kit in question. In some cases, samples may be diluted in Sample Diluent the day before an assay is to be performed and stored at 4 °C.

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 uL are ideal 100 uL aliquots should take less than 5 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 serum 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, serum 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 assay 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 4C Platewash Concentrate may forms 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 assays?
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. Careful planning may enable a laboratory to perform more than one assay run with the same kit.

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 Assay Protocols)

Dry heat incubator capable of maintaining 37°C
Pipettors: 2-20 µL, 20-200 µL, and 200-1000 µL pipettors with disposable tips
Precision repeating pipettor
Automated 96-well microtiter plate washer
12 x 75 mm culture 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 inactivating clinical specimens and decontamination of plate washer
Disposable paper towels

Assay 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. Assays should be planned to make the most use of a kit. Standards and controls consume wells for each assay. Duplicates should be placed into wells on different strips.

Is there a point during the assay where I can stop the assay 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 assay. Quality standards of each laboratory will determine the necessity of repeat assays.

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 assay 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 assay 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 labeled 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 assay run?

Ask customer to see raw data including the standard curve fit.
Check that the OD levels for standards fall within the expected dynamic range of the assay.
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 is close to 1.0 (.999)

Equipment

What Software Packages are recommended for use with the ELISA readers?
Oncogene Science uses Softmax Pro, which is provided by Molecular Devices, Inc.. Bio-Tek Instruments, Inc. sells a software package called KC4. 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 assay 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 assays are read at a single wavelength.

Select the appropriate wavelength filter for a particular assay.
Click "No" for the options of Auto mixing and Blanking. Always set "No" to Blanks.
All assays 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 assay. 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. 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 assay 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.

What is an acceptable CV% (coefficient of variation) for standards and patient 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 so-called "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.

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