DESCRIPTION

AFP (alpha-fetoprotein) is a major plasma protein found in the fetus while plasma AFP level is decreased rapidly after birth. AFP is a vital marker of the hepatocyte lineage. High AFP concentrations is related with tumor cell growth. Detection of AFP in plasma is important in diagnosis of hepatocellular carcinoma (HCC), stomach cancer and germ cell cancers. Altered levels of both fetal and maternal AFP are associated with hypothyroidism, autoimmune disorders, and heart defects. Measurement of AFP in maternal blood or amniotic fluid of pregnant women is used in screening developmental abnormalities such as aneuploidy. Serum AFP level is elevated in people with developmental birth defects and neurologic disorders.

PRINCIPLE OF THE ASSAY

Sandwich ELISA structure (Detection antibody labeled with HRP) A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody labeled with HRP also binds to the analyte. TMB acts as the HRP substrate and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns solution yellow. The color intensity is proportional to the quantity of bound protein which is measurable at 450 nm with the correction wavelength set at 630 nm.

PRECISION

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 20 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 24 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	20	2,844.3	215.7	7.6
2	20	613.7	31.4	5.1
3	20	142.4	7.4	5.2

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	24	2,713.7	220.4	8.1
2	24	554.7	41.0	7.4
3	24	143.5	13.5	9.4

RECOVERY

The recovery of mouse AFP spiked to three different levels throughout the range of the assay in mouse serum was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:250	84	78-92
	1:500	85	75-104
Cell culture supernatant	1:3,200	110	104-114

LINEARITY

To assess the linearity of the assay, samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The mouse serum was initially diluted 1:32. The cell culture superntant was initially diluted 1:200. )

		Mouse serum	Cell culture supernatant
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	89	95
	Range (%)	87-90	94-96
1:8	Average% of Expected	94	109
	Range (%)	87-100	103-115
1:16	Average% of Expected	98	115
	Range (%)	93-102	108-123

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant mouse AFP produced at Sunlong Medical™.