title: Chemiluminescence reaction kinetics-resolved multianalyte immunoassay strategy using a bispecific monoclonal antibody as the unique recognition reagent.
abstract: The multianalyte immunoassay (MIA) has attracted increasing attention due to its high sample throughput, short assay time, low sample consumption, and reduced overall cost. However, up to now, the reported MIA methods commonly require multiple antibodies since each antibody can recognize only one antigen. Herein, a novel bispecific monoclonal antibody (BsMcAb) that could bind methyl parathion and imidacloprid simultaneously was produced by a hybrid hybridomas strategy. A chemiluminescence (CL) reaction kinetics-resolved strategy was designed for MIA of methyl parathion and imidacloprid using the BsMcAb as the unique recognition reagent. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were adopted as the signal probes to tag the haptens of the two pesticides due to their very different CL kinetic characteristics. After competitive immunoreactions, the HRP-tagged methyl parathion hapten and the ALP-tagged imidacloprid hapten were simultaneously bound to the BsMcAb since there were two different antigen-binding sites in it. Then, two CL reactions were simultaneously triggered by adding the CL coreactants, and the signals for methyl parathion and imidacloprid detections were collected at 0.6 and 1000 s, respectively. The linear ranges for methyl parathion and imidacloprid were both 1.0-500 ng/mL, with detection limits of 0.33 ng/mL (S/N = 3). The proposed method was successfully used to detect pesticides spiked in ginseng and American ginseng with acceptable recoveries of 80-118%. This proof-of-principle work demonstrated the feasibility of MIA using only one antibody.
authors.affiliation: Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University , Chongqing 400716, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Ouyang
authors.forename: Limin
authors.initials: L
authors.lastname: Wang
authors.forename: Shijia
authors.initials: S
authors.lastname: Yang
authors.forename: Wenwen
authors.initials: W
authors.lastname: Wang
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.forename: Fengquan
authors.initials: F
authors.lastname: Liu
authors.forename: Zhifeng
authors.initials: Z
authors.lastname: Fu
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D018033:Antibodies, Bispecific / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D005260:Female; D006735:Horseradish Peroxidase / Q000378:metabolism; D006825:Hybridomas; D007093:Imidazoles / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D007114:Immunization; D007118:Immunoassay / Q000379:methods*; D007306:Insecticides / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D057230:Limit of Detection; D008163:Luminescent Measurements / Q000379:methods*; D008743:Methyl Parathion / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D000073943:Neonicotinoids; D009574:Nitro Compounds / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D005894:Panax / Q000737:chemistry*; D010575:Pesticides / Q000032:analysis*
chemical_list: D018033:Antibodies, Bispecific; D007093:Imidazoles; D007306:Insecticides; D000073943:Neonicotinoids; D009574:Nitro Compounds; D010575:Pesticides; C082359:imidacloprid; D008743:Methyl Parathion; D006735:Horseradish Peroxidase; D000469:Alkaline Phosphatase