Carfentanil ELISA (Enzyme-Linked Immunosorbent Assay) kit is a qualitative one-step kit designed for use as a screening test for the detection of carfentanil. The kit was designed for screening purposes and is intended for forensic use only.
Specifications
Analyte Carfentanil
Application Urine, Blood, Oral Fluid, Forensic Matrices
Assay Sample Size 20 µL
Brand NEOGEN®
Drug Panel Narcotic Analgesics
Intended Use For the determination of trace quantities of Carfentanil and/or other metabolites in human urine, blood or oral fluid. Contact a NEOGEN representative for information on other matrices.
Microplate Size 96 well microplate
Platform ELISA
Result Type Qualitative
Sensitivity
Compound I-50 in EIA Buffer
Carfentanil 0.1 ng/mL

The term I-50 is used to define the sensitivity of the test. This number is derived from a standard curve generated with the drug in EIA Buffer. The drug concentration that shows 50% less color activity than the zero standard is considered to be the I-50.

Species Human, Research Animal
Storage Conditions Test kit 2-8°; store controls -20°C
Total Assay Incubation Time 75 minutes
Wavelength
  • 650 nm with Red Stop Solution
  • 450 nm with acid stop

Compound % Cross-Reactivity
Carfentanil 0.1% 100%
Remifentanil 3.7% 2.7%
Sufentanil 20% 0.5%
Alfentanil 50% 0.2%
β-Methylfentanyl 164% 0.06%
Fentanyl 166% 0.06%
Lofentanil 250% 0.04%
Norsufentanil <200%> <0.05%>
Cyclopropylfentanyl 410% 0.02%
Furanylethylfentanyl 479% 0.02%
Acrylfentanyl 523% 0.02%
a-Methylthiofentanyl 593% 0.02%
Acetylfentanyl 606% 0.02%
Furanylfentanyl 608% 0.02%
Methoxyacetylfentanyl 721% 0.01%
p-Chlorisobutyrylfentanyl 725% 0.01%
Butryfentanyl 740% 0.01%
  1. EIA Buffer
  2. Wash Buffer Concentrate (10X)
  3. K-Blue Substrate
  4. Drug-Enzyme Conjugate
  5. Antibody Coated Plate
  6. Red Stop Solution
  7. Qualitative QC Positive Control
  8. Qualitative QC Negative Control
  1. Deionized water.
  2. Precision pipettes that range from 10 μL - 1000 μL and disposable tips.
  3. Graduated cylinder to dilute and mix wash buffer.
  4. Plate cover or plastic film to cover plate during incubation.
  5. Clean glassware (i.e. test tubes) to dilute samples.
  6. Microplate reader with 450 nm filter.
  7. Cut-off calibrator.

Optional Materials

  1. 1N HCI if Red Stop Solution is not used
  2. Microplate shaker

Technology

ELISA Assay Theory

NEOGEN’s direct competitive ELISAs operate on the basis of competition between the horseradish peroxidase (HRP) enzyme conjugate and the analyte in the sample for a limited number of specific binding sites on the precoated microplate.

ELISA Assay Procedure

Samples, standards or calibrators are first added to the precoated antibody microplate. Next, the enzyme conjugate is added and the mixture is incubated at room temperature. During incubation, competition for binding sites on the microplate is taking place. The plate is then washed, removing all unbound material. The bound enzyme conjugate is detected by the addition of a TMB-based substrate.

ELISA test results may be obtained by measuring and comparing the absorbance reading of the wells of the samples against the standards with a microplate reader at 650 nm or 450 nm if acid stop is used. The extent of color development is inversely proportional to the amount of analyte in the sample or standard.

ELISA Assay Test Principle Procedure

  1. Plates are precoated with the antibody. The plate is ready for use. DO NOT WASH.
  2. A sample or control is added to each well. Next the drug-enzyme conjugate is added and the mixture is incubated at room temperature.
  3. Wash the plate to remove all unbound materials.
  4. The bound materials now remain in the microplate.
  5. Add TMB substrate to each well and allow the color to develop.
  6. Qualitative results are obtained by measuring the absorbance reading at 650 nm or 450 nm if acid stop is used.
AssayPrincipleIllustration_Forensic.png

Training

Our customers’ success is our shared success. Our experts are ready to train you and your team on our solutions, so you can rest easy knowing procedures are performed properly and yield accurate results. In addition, we provide certificates upon completion of training.


Support

The following are common examples of unexpected results when running NEOGEN's ELISA kits with possible explanations.

  1. Extreme deep blue color development with samples and standards.
    1. The plate was not properly washed (3 x 300 µL) with the diluted wash buffer. If using an automated washer, ensure the machine is working correctly and increase wash cycle to 5 x 300 µL.
    2. The enzyme conjugate concentrate was incorrectly diluted.

  2. Extremely low color development with samples and standards.
    1. The wash buffer was not diluted correctly before use.
    2. A poor quality of water was used to dilute the wash buffer. Deionized water should be used for dilutions.
    3. The enzyme conjugate concentrate was incorrectly diluted.
    4. The kit prematurely deteriorated, possibly from adverse shipping and storage conditions. Investigate the condition of the kit when it was received and how it was stored prior to use. Proper storage conditions are listed in the package insert.
    5. The kit has expired. Check the expiration dates on the test kit and reagents. No component of the kit should be used past the expiration date. Do not mix any reagents or components of one kit with the reagents or components of another kit.
    6. Contamination. Always use aseptic techniques when opening and removing reagents from vials and bottles. Keep the plate covered except when adding reagents, washing or reading. Always use different pipette tips for each reagent. When pipetting, do not allow the pipette tip to touch any of the reagents already in the well.

  3. No color development with samples and standards.
    1. Improper dilution of enzyme conjugate concentrate.
    2. The kit has expired. Check the expiration dates on the test kits and reagents. No components of the kit should be used past the expiration date. Do not mix any reagents or components of one kit with the reagents or components of another kit.
    3. Incorrect addition of kit reagents. Review assay procedure.

  4. Little to no displacement with the standard curve.
    1. Incorrect dilution of standards. Refer to the dilution scheme in the package insert.
    2. Contamination.
    3. The plate was not properly washed (3 x 300 µL) with the diluted wash buffer. If using an automated washer, ensure the machine is working correctly and increase wash cycle to 5 x 300 µL.
    4. Standard has deteriorated prematurely. Contact your NEOGEN representative and provide them with the kit name, lot number, expiration date and your OD readings for further investigation.

  5. The standard curve performed correctly, but the known negative samples gave low color development.
    1. Samples need to be diluted or extracted to eliminate interference. Refer to the extraction procedure in the kit insert.

  6. The standard curve performed correctly, but the extracted samples gave low color development.
    1. The concentration of the analyte in the extracted sample is too high. The extracted sample will need to be diluted before running it in the assay so the sample OD reading will fit in the standard curve. When a dilution is used, the concentration determined from the standard curve must be multiplied by the dilution factor.
    2. Inadequate extraction of samples resulting in the presence of a solvent. Refer to the recommended extraction procedure in the package insert.

  7. Variability with duplicates.
    1. Inconsistent and/or inadequate pipetting technique when adding reagents. Improve pipetting technique.
    2. Inconsistent washing. The wells should be washed 3 x 300 µL with the diluted wash buffer. If using an automated washer, ensure the machine is working correctly and increase wash cycle to 5 x 300 µL.
    3. Inadequate aspiration during washing. The wells should be dumped and tapped between each washing. Tap out excess liquid in wells before adding substrate. Add substrate immediately to the wells once the excess wash buffer has been removed. If using an automated washer, ensure the machine is aspirating correctly.
    4. Interruption during assay set-up. Have all reagents prepared before assay set-up commences. Reagent addition should be performed in a timely and accurate manner.