DiaMetra AFP Elisa Kit

CLINICAL SIGNIFICANCE
Alpha Fetoprotein (AFP) is a 68 kDa glycoprotein, which is normally only produced in the fetus during its development. It is a normally produced by the liver and yolk sac of the fetus.
AFP levels decrease soon after birth and probably has no function in normal adults. It binds the hormone estradiol to keep it from affecting the fetal brain. Its measurement during pregnancy has been useful to detect certain abnormalities - specifically, if high levels of AFP are found in amniotic fluid, it can indicate a developmental defect in the baby.
In some patients who are not pregnant a tumor can produce AFP, thus it can be used as a tumour marker. AFP is the main tumour marker (along with HCG) to diagnose testicular cancer and its values over time can have significant effect on the treatment plan.
Like all tumour markers, the detection of AFP by itself is not diagnostic of anything, although if it is detected it is certainly advisable to rule out the diseases could cause levels to rise. The primary reason tumor markers are used are to measure the success of a treatment (e.g. chemotherapy), if levels of AFP are going down, it is an indication that a disease is improving. New research exhibits that an isoform of AFP which binds Lens culinaris agglutinin (AFP-L3) can be particularly useful in early identification of aggressive tumors associated with hepatocellular carcinoma (HCC).

PRINCIPLE
AFP ELISA test is based on simultaneous binding of human AFP to two monoclonal antibodies, one immobilized on microwell plates, the other conjugated with horseradish peroxidase (HRP).
After incubation, the separation bound-free is obtained with a simple solid-phase washing.
Then the enzyme HRP in the bound-fraction reacts with the Substrate (H2O2) and the TMB Substrate and develops a blu color that changes into yellow when the Stop Solution (H2SO4) is added.
The colour intensity is proportional to the AFP concentration in the sample.
The AFP concentration in the sample is calculated through a calibration curve.

QUALITY CONTROL
Each laboratory should assay controls at normal, high and low levels range of AFP for monitoring assay performance. These controls should be treated as unknowns and values determined in every test procedure performed. Quality control charts should be maintained to follow the performance of the supplied reagents. Pertinent statistical methods should be employed to ascertain trends. The individual laboratory should set acceptable assay performance limits. Other parameters that should be monitored include the 80, 50 and 20% intercepts of the calibration curve for run-to-run reproducibility. In addition, maximum absorbance should be consistent with past experience. Significant deviation from established performance can indicate unnoticed change in experimental conditions or degradation of kit reagents. Fresh reagents should be used to determine the reason for the variations.

PRECAUTIONS
Please adhere strictly to the sequence of pipetting steps provided in this protocol. The performance data represented here were obtained using specific reagents listed in this Instruction For Use.
All reagents should be stored refrigerated at 2-8°C in their original container. Any exceptions are clearly indicated. The reagents are stable until the expiry date when stored and handled as indicated.
Allow all kit components and specimens to reach room temperature (22-28°C) and mix well prior to use.
Do not interchange kit components from different lots. The expiry date printed on box and vials labels must be observed. Do not use any kit component beyond their expiry date.
If you use automated equipment, the user has the responsibility to make sure that the kit has been appropriately tested.
 

RESULTS
Mean Absorbance
Calculate the mean of the absorbance (Em) for each point of the calibration curve (C0-C4) and of each sample.
Calibration curve
Plot the values of absorbance (Em) of the calibrators (C0-C4) against concentration. Draw the best-fit curve through the plotted points. (Es: Four Parameter Logistic).
Calculation of Results
Interpolate the values of the samples on the calibration curve to obtain the corresponding values of the concentrations expressed in ng/mL.