Product Description

AFP ELISA Kit

Catalog No. L5E1001

Intended use

This immunoassay kit allows for the in vitro quantitative determination of α-Fetoprotein (AFP) in human serum, plasma and other biological fluids.

Introduction

Alpha-fetoprotein (AFP),the foetal equivalent to albumin, is a 67 kDa glycoprotein.AFP is normally produced during fetal and neonatal development by the liver, yolk sac, and in small concentrations by the gastrointestinal tract. After birth, serum AFP concentrations decrease rapidly, and by the second year of life and thereafter only trace amounts are normally detected in serum. Elevation of serum AFP to abnormally high values occurs in several malignant diseases, most notably nonseminomatous testicular cancer and primary hepatocelluar carcinoma. In the case of nonseminomatous testicular cancer, a direct relationship has been observed between the incidence of elevated AFP levels and the stage of disease. Elevated AFP levels have also been observed in patients diagnosed with seminoma with nonseminomatous elements, but not in patients with pure seminoma. In addition, elevated serum AFP concentrations have been measured in patients with other noncancerous diseases, including ataxia telangiectasia, hereditary tyrosinemia, neonatal hyperbilirubinemia, acute viral hepatitis, chronic active hepatitis and cirrhosis. Elevatedserum AFP concentrations are also observed in pregnant women. Therefore, AFP measurements are not recommended for use as a screening procedure to detect the presence of cancer in the general population. Normally, status shows that 95% of the healthy subjects had assay value below 25ng/ml.

Test principle

The LINC-BIO AFP ELISA is a solid-phase, non-competitive immunoassay based on the direct sandwich technique. Calibrator, controls and patient samples are incubated together with anti-AFP monoclonal antibody coated micro titer strips. AFP present in calibrators, controls or samples is absorbed to the microtiter wells during the incubation. The strips are then washed and incubated with horseradish peroxidase (HRP) labeled anti-AFP McAb. After washing, a TMB (3, 3’5, 5′ tetramethyl-benzidine) substrate solution is added to each well and the enzyme reaction is allowed to proceed. During the enzyme reaction a blue colour will develop if antigen is present. The intensity of the colour is proportional to the amount of AFP antigen present in the samples.

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. Calibration curves are constructed for each assay by plotting absorbance value versus the concentration for each calibrator. The concentration of AFP in the samples is then determined by comparing the O.D. of the samples to the calibration curve.

Materials and components

Reagent                                                       Quantity

Assay plate                                                      1

Calibrators                                                      5 x 500ul

With concentrations of 0 ng/ml，5 ng/ml，20 ng/ml，80 ng/ml，320 ng/ml.

AFP-HRP conjugate                                                                  1 x 10ml

Wash Buffer  (20 x concentrate)                                    1 x 30ml

Substrate Solution                                                                          1 x 10ml

Stop Solution                                                                                   1 x 5ml

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 approximately 1000 x g. Remove serum and assay immediately.

Plasma – Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2 – 8° C within 30 minutes of collection.

Other biological fluids – Remove particulates by centrifugation and assay immediately。

Note: Serum, plasma, and cell culture supernatant samples to be used within 7 days may be stored at 2-8 ° C, otherwise samples must stored at -20°C (≤ 3 months) or -80°C (≤ 6 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature.

It is recommended that all samples be assayed in duplicate.

DO NOT USE HEAT-TREATED SPECIMENS.

Assay procedure

Allow all reagents to reach room temperature. All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Arrange and label required number of strips. Prepare all reagents, working standards and samples as directed in the previous sections.

1. Add 40 uL of Calibrators, Blank, or Sample per well. Cover with the adhesive strip. Incubate for 40mins at 37° C.
2. Aspirate the liquid of each well and wash. repeating the process five times.

Wash by filling each well with Wash Buffer (350 uL) using a squirt bottle, 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 Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.

3. Add 100 uL of HRP conjugate solution to each well. Incubate for 40mins at 37°C.
4. Aspirate each well and wash, repeating the process five times for a total of three washes. Refer to step2.
5. Add 100 uL of Substrate Solution to each well. Incubate for 5-10 minutes at 37°C. Protect from light.
6. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
7. Determine the optical density of each well within 10-15 mins, using a microplate reader set to 450 nm.

Important Note:

1. 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. When mixing or reconstituting protein solutions, always avoid foaming.
2. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.
3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
4. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.
5. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
6. Duplication of all standards and specimens, although not required, is recommended.
7. Do not substitute reagents from one kit lot to another. Standard, conjugate  a microtiter plates are matched for optimal performance.  Use only the reagents supplied by manufacturer.

Calculation of results

Average the duplicate readings for each calibrator, control, and sample and subtract the average zero standard optical density. Create a calibration curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit or liner curve. As an alternative, construct a calibration curve by plotting the mean absorbance for calibrator 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 concentrations versus the log of the O.D. 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.

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

Sensitivity  The minimum detectable dose of human AFP is typically less than 1 ng/ml.

Detection Range    5-320 ng/ml..

Storage of test kits and instrumentation

1. Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag with desiccants to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit (six months from the date of manufacture). Refer to the package label for the expiration date. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above.
2. Do not remove microtiter plate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.
3. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.
4. Substrate Solution is easily contaminated. If bluish prior to use, do not use.

Limitations of the procedure

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Precaution

The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.