Development of an Immunoassay for the Detection of Amyloid Beta 1‐42 and Its Application in Urine Samples
Anurak Wongta, Surat Hongsibsong, Somporn Chantara, Mookda Pattarawarapan, Ratana Sapbamrer, Korawan Sringarm, Zhenlin Xu, Hong Wang
Abstract
Amyloid beta peptides (A β 1‐42) have been found to be associated with the cause of Alzheimer’s disease (AD) and dementia. Currently, methods for detecting A β 1‐42 are complicated and expensive. The present study is aimed at developing an indirect competitive enzyme‐linked immunosorbent assay (ic‐ELISA) to detect A β 1‐42 by using a polyclonal antibody from alpaca, an application used in urine samples. The serum was collected from the alpaca after immunizing it with A β 1‐42 at 500 μ g/injection 5 times. The ic‐ELISA was developed and showed a half‐maximal inhibitory concentration (IC 50 ) of 103.20 ng/ml. The limit of detection (LOD) was 0.39 ng/100 μ l. The cross‐reactivity was tested with A β 1‐40 and 8 synthesized peptides that had sequence similarities to parts of A β 1‐42. The cross‐reactivity of A β 1‐40 and peptide 1 (DAEFRHDSGYE) was 55% and 69.4%, respectively. The ic‐ELISA was applied to analyze A β 1‐42 in the urine and precipitated protein urine samples. This method can be used for detecting a normal level of total soluble A β (approximately 1 ng in 5 mg of precipitated urine protein) and can be used for detecting the early stages of AD. It is considered to be an easy and inexpensive method for monitoring and diagnosing AD.