CDSimple™ AFP Chemiluminescent ELISA Kit

Sample

Serum

Species Reactivity

Human

Detection Method

Chemiluminescence

Intended Use

The Quantitative Determination of Alpha-Fetoprotein (AFP) Concentration in Human Serum by a Microplate Enzyme Immunoassay, Chemiluminescence

Contents of Kit

A. AFP Calibrators – 1 ml/vial
Six (6) vials of references AFP antigen at levels of 0(A), 5(B), 25(C), 50(D), 250(E) and 500(F) ng/ml. Store at 2-8C. A preservative has been added. Note: The calibrators, human serum based, were calibrated using a reference preparation, which was assayed against the WHO 1st IRP# 72-225.
B. AFP Tracer Reagent – 13 ml/vial
One (1) vial containing enzyme labeled antibody, biotinylated monoclonal mouse IgG in buffer, dye, and preservative. Store at 2-8°C.
C. Light Reaction Wells - 96 wells 
One 96-well white microplate coated with streptavidin and packaged in an aluminum bag with a drying agent. Store at 2-8°C.
D. Wash Solution Concentrate – 20ml/vial
One (1) vial containing a surfactant in buffered saline. A preservative has been added. Store at 2-8°C (see Reagent Preparation Section).
E. Signal Reagent A – 7ml/vial
One (1) vial containing luminol in buffer. Store at 2-8°C (see Reagent Preparation Section).
F. Signal Reagent B – 7ml/vial
One (1) vial containing hydrogen peroxide (H₂O₂) in buffer. Store at 2-8°C (see Reagent Preparation Section).
G. Product Insert
Note 1: Do not use reagents beyond the kit expiration date.
Note 2: Avoid extended exposure to heat and light. Opened reagents are stable for sixty (60) days when stored at 2-8°C. Kit and component stability are identified on the label.
Note 3: Above reagents are for a single 96-well microplate.

Performance Characteristics

The AFP test system was compared with a reference method. Biological specimens from low, normal, and elevated concentrations were assayed. The total number of such specimens was 235. The least square regression equation and the correlation coefficient were computed for this test in comparison with the reference method. The data obtained is displayed in Table 5.

Only slight amounts of bias between the AFP test system and the reference method are indicated by the closeness of the mean values. The least square regression equation and correlation coefficient indicates excellent method agreement.

Precision

The within and between assay precision of the AFP test system were determined by analyses on three different levels of control sera. The number, mean value, standard deviation and coefficient of variation for each of these control sera are presented in Table 3 and Table 4.

Sensitivity

The AFP Test System has a sensitivity of 0.003 ng. This is equivalent to a sample containing 0.134 ng/ml AFP concentration. The sensitivity was ascertained by determining the variability of the 0 ng/ml serum calibrator and using 2σ (95% certainty) statistic to calculate the minimum dose.

General Description

Alpha-Fetoprotein (AFP) is a glycoprotein with a molecular weight of 70 kDA. AFP shares considerable sequential homology with albumin and is normally produced during fetal development by the hepatocytes, yolk sac and to a lesser extent by the gastrointestinal tract. AFP appears as a major serum protein in the fetus, but its concentration declines rapidly towards birth. Serum concentrations reach a peak level of up to 10 mg/ml at twelve weeks of gestation.
This peak level gradually decreases to less than 25 ng/ml after one year of postpartum. Thereafter, the levels reduce further to less than 10 ng/ml.
Ever since its first reported association with tumors, by Tatarinov in 1964 based on his work with liver carcinomas, AFP has been a subject of discussion with relation to many different tumors. Abnormal AFP levels have been associated with hepatocellular carcinoma, ovarian cancer, gastrointestinal cancer and pulmonary cancer and, most recently, with non-seminomatous testicular cancer. Elevated levels of AFP are found in patients with primary heptatoma and yolk sac-derived germ tumors. AFP is the most useful marker for the diagnosis and management of hepatocellular carcinoma.
AFP is also elevated in pregnant women. Presence of abnormally high AFP concentrations in pregnant women provides a risk marker for Down syndrome.
In this method, AFP calibrator, patient specimen or control is first added to a streptavidin coated well. Biotinylated monoclonal and enzyme labeled antibodies (directed against distinct and different epitopes of AFP) are added and the reactants mixed. Reaction between the various AFP antibodies and native AFP forms a sandwich complex that binds with the streptavidin coated to the well. After the completion of the required incubation period, the enzyme-AFP antibody bound conjugate is separated from the unbound enzyme-AFP conjugate by aspiration or decantation. The activity of the enzyme present on the surface of the well is quantitated by reaction with a suitable substrate to produce light measurable with a luminometer.
The employment of several serum references of known alpha-fetoprotein (AFP) levels permits the construction of a dose response curve of activity and concentration. From comparison to the dose response curve, an unknown specimen's activity can be correlated with AFP concentration.