NEOGEN's Lipoxin A4 ELISA is used for the quantitative analysis of Lipoxin A4 levels in biological fluid.

Lipoxin A4 (LXA4) is a biologically active lipoxygenase interaction product derived from arachidonic acid. Arachidonic acid is first oxygenated by 15-lipoxygenase to form 15-HETE which is then converted by 5-lipoxygenase and epoxide hydrase to generate LXA4. LXA4 stimulates leukocyte chemotaxis without aggregation and inhibits natural killer cell cytotoxicity. It also provokes contraction of parenchymal strips and stimulates microvascular changes. Recent findings indicate that LXA4 inhibits leukocyte-dependent inflammation. Determination of LXA4 levels may provide new understanding of the role of LXA4 in basic cellular reactions and in pathophysiology of inflammation and other disease processes.

Specifications
Analyte Lipoxin A4
Application Urine, Oral Fluid, Tissue Culture Supernatant, Plasma, Serum
Assay Range 0.02 - 2 ng/mL
Assay Sample Size 50 µL
Brand NEOGEN®
Drug Panel Specialty
Intended Use For the determination of Lipoxin A4 in urine, oral fluid, tissue culture supernatant, plasma, or serum samples. Contact a NEOGEN representative for information on other matrices.
Microplate Size 96 well microplate
Platform ELISA
Result Type Quantitative
Species Non-Species Specific
Storage Conditions -20°C for lyophilized conjugate, 2-8°C for all other kit components
Total Assay Incubation Time 90 minutes
Wavelength 650 nm or 450 nm with acid stop

Analytes Cross-Reactivity
Lipoxin A4 100.0%
15-epi-Lipoxin A4 24.0%
5(S), 6(R)-DiHETE 5.0%
Lipoxin B4 1.0%
15-HETE 0.10%
5-HETE <0.10%>
12-HETE < 0.10%
Leukotriene B4 < 0.01%
Leukotriene C4 < 0.01%
Leukotriene D4 < 0.01%
Leukotriene E4 < 0.01%
  1. EIA Buffer
  2. Wash Buffer (10X)
  3. K-Blue Substrate
  4. Extraction Buffer (5X)
  5. Lipoxin A4 Enzyme Lyophilized Conjugate
  6. Lipoxin A4 Standard
  7. Lipoxin A4 Antibody Coated Microplate

NOTE: If all or several strips are to be used at one time, it is suggested that a multichannel pipette be used.

  1. Deionized water
  2. Precision pipettes that range from 10 μL-1000 μL and disposable tips. NOTE: If all or several strips are to be used at one time, it is suggested that a multichannel pipette be used.
  3. Clean test tubes used to dilute the standards and conjugate.
  4. Graduated cylinders to dilute and mix wash buffer and extraction buffer.
  5. Microplate reader with 650 nm filter.
  6. Plate cover or plastic film to cover plate during incubation.

Additional Information

Calculation of Results

NEOGEN has partnered with MyAssays.com to provide an online tool for data calculations.

Follow the simple step-by-step guide to generate your data.

Calculate Results

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Technology

The sample or standard solution is first added to the microplate. Next, the diluted enzyme conjugate is added, and the mixture is shaken and incubated at room temperature for one hour. During the incubation, competition for binding sites is taking place. The plate is then washed removing all the unbound material. The bound enzyme conjugate is detected by the addition of substrate which generates an optimal color after 30 minutes. Quantitative 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. The extent of color development is inversely proportional to the amount of Lipoxin A4 in the sample or standard. For example, the absence of Lipoxin A4 in the sample will result in a bright blue color, whereas the presence of Lipoxin A4 will result in decreased or no color development.
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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.