Fetoprotein alpha Lens Culinaris Agglutinin 3 (aFPL3) ELISA Kit

Antigen: Fetoprotein alpha Lens Culinaris Agglutinin 3 (aFPL3)

Synonyms: FETA, HPAFP, AFP, MGC128267

Reactivity: Human

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN366920

Additional Information

Characteristics: This immunoassay kit allows for the in vitro quantitative determination of human AFP-L3 concentrations in cell culture supernates, serum, plasma and other biological fluids.

Alternative name: alpha-fetoprotein Lens culinaris agglutiin 3 (AFP-L3)

Sample Type: Serum, Plasma

Description: AFP-L3 is one of AFP fractions which is synthesized by malignant cell and comprise special oligosaccharide. It is a new generation of tumor marker for HCC and yields useful information on HCC for clinical decision making. AFP-L3 bears strong affinity to the sugar chain of Lens culinaris, a type of lentil. Reports showed that if the ratio of AFP-L3 increased over 10%, the risk of catching HCC had increased highly, and non-malignant diseases hardly synthesize this AFP fraction (AFP-L3).Comparing with ultrasonography(US), AFP-L3 can be detected 9-12 months earlier and shows the remarkable precaution effect to HCC.

Specificity: This assay recognizes recombinant and natural human AFP-L3. No significant cross-reactivity or interference was observed.

Sensitivity: The minimum detectable dose of human AFP-L3 is typically less than 0.2 ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. Detection Range: The minimum detectable dose of human AFP-L3 is typically less than 0.2 ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero.

Application Details

Principle: The microtiter plate provided in this kit has been pre-coated with an antibody specific to AFP-L3. Standards or samples are then added to the appropriate microtiter plate wells with a Horseradish Peroxidase (HRP)-conjugated monoclonal antibody preparation specific for AFP-L3 and incubated. Then substrate solutions are added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of AFP-L3 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protocol: Assay Procedure:
Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate. All the reagents should be added directly to the liquid level in the well. The pipette should avoid contacting the inner wall of the well. 1. Set a Blank well without any solution. 2. Add 5 0μl of Standard or Sample per well. Cover with the adhesive strip. Incubate for 20 min at 37° C. 3. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with ddH 2 O (20 0μl) using a squirt bot tle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining solution by aspirating or decanting. Invert the plate and blot it against clean paper towels. 4. Add 50 μl of HRP-conjugate solution to each well. Not to Blank ！ Cover the microtiter plate with a new adhesive strip. Incubate for 20 min at 37°C. 5. Repeat the aspiration and wash three times as before. 7 6. Add 50 μl of Substrate A and 5 0μl of Substrate B to each well. Incubate for 15 minutes at 37°C. Keeping the plate away from drafts and other temperature fluctuations in the dark. 7. Add 50 μl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 8. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Calculation of Results:
Using the professional soft ",Curve Exert 1.3", to make a standard curve is recommended, which can be downloaded from our web. Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the AFP-L3 concentrations versus the log of the O.D. 8 and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
Limitations of the Procedure:
The kit should not be used beyond the expiration date on the kit label.Do not mix or substitute reagents with those from other lots or sources.Any variation in operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding.This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
Sample collection and storage:
Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles. Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at-20°C. Centrifuge the sample again after thawing before the assay. Avoid repeated freeze-thaw cycles. Note: Grossly hemolyzed samples are not suitable for use in this assay. If samples generate values higher than the highest standard, dilute the samples with the Sample Diluent and repeat the assay.

Assay Procedure: Assay Time: 1-3h
Sample Volume: 50-100ul

Application Notes: Detection Wavelength: 450 nm

Components: Reagent (Quantity): Assay plate (1), Standard 6x1ml HRP-conjugate (1x6ml), Sample Diluent 1 x11ml Substrate A (1x7ml), Substrate B (1x7ml), Stop Solution (1x7ml)

Material not included: Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm. Pipettes and pipette tips. Deionized or distilled water. Squirt bottle, manifold dispenser, or automated microplate washer. An incubator which can provide stable incubation conditions up to 37°C ± 0.5°C.

Storage: 1. Unopened test kits should be stored at 2-8 C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit, provided it is stored as prescribed above. Refer to the package label for the expiration date. 2. Opened test plate should be stored at 2-8 C in the aluminum foil bag with desiccants to minimize exposure to damp air. The kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

Restrictions: For Research Use only