Human HE4/WFDC2 Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human HE4/WFDC2 Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human HE4/WFDC2 in cell culture supernates, serum, plasma, saliva, and urine. It contains NS0-expressed recombinant human HE4/WFDC2 and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human HE4/WFDC2 showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for naturally occurring human HE4/WFDC2.

Preparation and Storage

Background: HE4/WFDC2

Human Epididymal Protein 4 (HE4), also known as WAP Four-Disulfide Core domain protein 2 (WFDC2), is a secreted 25 kDa glycosylated member of the Whey Acidic Protein (WAP) family. WAP family proteins (e.g. SLPI, Trappin-2/Elafin, WFDC5, and WFDC12) share multiple conserved cysteines and exhibit protease inhibitory and anti-microbial activities (1, 2). Mature human HE4 consists of two tandem WAP domains, while mouse and rat HE4 have an additional 54 amino acid (aa) or 48 aa region between the WAP domains, respectively (3, 4). Mature human HE4 shares approximately 60% with comparable regions of mouse and rat HE4. Alternate splicing of human HE4 generates additional isoforms in which either the first or second WAP domain is deleted and may be replaced by substitutions of 10 aa, 22 aa, or 28 aa (3). HE4 is expressed in the normal epithelium lining the male and female genital tracts (4-6), upper respiratory tract (5-7), and ducts of the salivary glands and breast (5-7). It is also variably expressed in the renal distal convoluted tubule, colon, and endometrium (5, 6, 8). HE4 is pathologically expressed in lung adenocarcinomas and cystic fibrosis lungs (7, 9, 10). Expression of the isoform which has 22 aa in place of the first WAP domain is associated with lung tumor size and invasiveness (9). Tissue and serum HE4 levels are elevated in type I and type II endometrial cancer, particularly of the endometrioid subtype (5, 8, 11, 12). HE4 elevation in the serum increases with endometrial tumor progression (8, 11). In breast cancer, tissue expression of HE4 correlates with lymph node involvement (13). Urinary HE4 levels are elevated in transitional cell cancer (14) as well as in early and late stage ovarian carcinoma (15). HE4 is not expressed in normal ovarian epithelium (5, 6), although the serum HE4 level rises during the ovulatory phase of the menstrual cycle (16). HE4 is strongly upregulated and secreted in ovarian cancer. Among ovarian cancer subtypes, serous and endometrioid tumors exhibit the most frequent and greatest magnitute of upregulation (5, 6, 17-19) with expression in clear cell and mucinous subtypes relatively sporadic and weak (5, 18). The serum elevation of HE4 is highest in advanced stages of ovarian cancer (17, 19, 20). Serum levels of the mucin glycoprotein CA125 are also elevated in ovarian cancer, and this marker exhibits comparable sensitivity but lower specificity than HE4 (19, 21, 22). The study of ovarian and endometrial cancer progression may be improved by the incorporation of both HE4 and CA125 values in the predictive computer model, risk of malignancy algorithm (ROMA) (8, 18-20, 23-25). The Quantikine® Human HE4/WFDC2 Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human HE4 in cell culture supernates, serum, plasma, saliva, and urine. It contains NS0-expressed recombinant human HE4 and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human HE4 showed linear curves that were parallel to the standard curves obtained using the Quantikine® kit standards. These results indicate that this kit can be used to determine relative mass values for natural human HE4.

Assay Procedure

Refer to the product datasheet for the complete 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.

1、Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2、Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3、Add 50 μL of Assay Diluent to each well.

4、Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5、Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6、Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7、Aspirate and wash 5 times.

8、Add 100 μL Substrate Solution to each well.

9、Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.