Sequential injection-immunoassay system with a plain glass capillary reactor for the assay of hyaluronan.
A sequential injection capillary immunoassay system (SI-CI) was developed to automate the multi-step competitive bioassay procedures for hyaluronan (HA), a potential biomarker for liver and cancer diseases. Biotinylated HA-binding proteins (b-HABPs), anti-biotin conjugated with horseradish peroxidase (anti-b-HRP) and substrate 3,3',5,5' tetramethylbenzidene (TMB) were employed. An ordinary glass capillary was utilized as a bio-reactor where competitive assay took place. Standard HA was immobilized onto an unmodified glass capillary through simple physical adsorption. This glass capillary is much lower cost compared to a fused-silica capillary, and is well suited for use as part of the tubing of a flow system. Using the proposed system, the average amount of HA in human serums from patients with liver disease and healthy subjects could be differentiated with comparable efficiency as the conventional micro-plate assay (as compared by t-test), with an improved assay time per sample (20 min vs. 5 h), and with approximately 10 times less reagent consumption (10-80 microl vs. 100-350 microl).
Kradtap Hartwell
Supaporn
S
Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand. kradtas@yahoo.com
Boonmalai
Apichart
A
Kongtawelert
Prachya
P
Grudpan
Kate
K
D000327:Adsorption; D001560:Benzidines / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D001847:Bone Diseases / Q000097:blood; D002021:Buffers; D002138:Calibration; D053801:Capillary Electrochromatography; D006801:Humans; D018960:Hyaluronan Receptors / Q000032:analysis; D006820:Hyaluronic Acid / Q000032:analysis* / Q000097:blood; D007118:Immunoassay; D007202:Indicators and Reagents; D008107:Liver Diseases / Q000097:blood; D012015:Reference Standards; D015203:Reproducibility of Results
D001560:Benzidines; D002021:Buffers; D018960:Hyaluronan Receptors; D007202:Indicators and Reagents; C021758:3,3',5,5'-tetramethylbenzidine; D001710:Biotin; D006820:Hyaluronic Acid