Measurement of vasoactive intestinal peptide using a competitive fluorescent microsphere immunoassay or ELISA in human blood samples.
The concentration of Vasoactive Intestinal Peptide (VIP) as measured by recycling immunoaffinity chromatography (RIC) has been reported to be elevated in the blood of patients with autism as compared with normal subjects. In this study, we have developed a "Competitive Fluorescent Microsphere Immunoassay" (cFMI) in which VIP competes with biotinylated VIP in binding to polyclonal antibodies on microspheres. The results were obtained using the Luminex100 system. We measured VIP in serum, plasma, and material eluted from dried blood spots on filter paper with both the cFMI and an ELISA procedure. We found that a purification procedure was necessary for obtaining useful results from plasma and serum, however, a preincubation step was required with the blood eluates. This newly developed cFMI was more sensitive (2.5 vs. 20.0 pg/ml), and more reproducible than the ELISA. To get accurate measurements of VIP in eluted material high sensitivity is especially important. Thus, the cFMI using the Luminex system has definite advantages over a conventional ELISA including the possibility that samples can be assayed at higher dilutions. We have determined that the VIP concentrations of serum, plasma, and dried blood spot eluate specimens as measured with the cFMI assay system were similar to those measured with ELISA. Thus, the new cFMI using Luminex system may be useful for detection of VIP in human blood samples.
Song
Eun Young
EY
LDN, National Institute of Child Health and Human Development, National Institutes of Health, Building 31 Room 2 A 25, Bethesda, MD 20892-4480, USA.
VanDunk
Cassandra
C
Kuddo
Thea
T
Nelson
Phillip G
PG
D000328:Adult; D001667:Binding, Competitive; D002021:Buffers; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D015200:Fluoroimmunoassay / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D006801:Humans; D008863:Microspheres; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014660:Vasoactive Intestinal Peptide / Q000032:analysis / Q000097:blood* / Q000592:standards
D002021:Buffers; D014660:Vasoactive Intestinal Peptide