Quantitative and sensitive detection of the SARS-CoV spike protein using bispecific monoclonal antibody-based enzyme-linked immunoassay

Abstract

The severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein is known to mediate receptor interaction and immune recognition and thus it is considered as a major target for vaccine design. The spike protein plays an important role in virus entry, virus receptor interactions, and virus tropism. Sensitive diagnosis of SARS is essential for the control of the disease in humans. Recombinant SARS-CoV S1 antigen was produced and purified for the development of monoclonal and bi-specific monoclonal antibodies. The hybridomas secreting anti-S1 antibodies, F26G18 and P136.8D12, were fused respectively with the YP4 hybridoma to generate quadromas. The sandwich ELISA was formed by using F26G18 as a coating antibody and biotinylated F26G18 as a detection antibody with a detection limit of 0.037μg/ml (p<0.02). The same detection limit was found with P136.8D12 as a coating antibody and biotinylated F26G18 as a detection antibody. The sensitivity was improved (detection limit of 0.019μg/ml), however, when using bi-specific monoclonal antibody (F157) as the detection antibody. In conclusion, the method described in this study allows sensitive detection of a recombinant SARS spike protein by sandwich ELISA with bi-specific monoclonal antibody and could be used for the diagnosis of patients suspected with SARS.

Laboratory detection methods for the human coronaviruses

“…In 2013, Sunwoo and coworkers reported an assay for SARS-related coronavirus detection, which exploited anti-S1 monoclonal capture antibodies, as well as HRP-labeled bi-specific monoclonal detection antibodies to detect the S1 subunits of spike proteins by the addition of 3,3′,5,5′-Tetramethylbenzidine (TMB) chromogenic substrate. This method detected as low as 19 ng/ml in approximately 2 h [120].…”

“…Subsequently, label-free or label-based detection is carried out to analyze the captured analytes. In addition to the conventional enzyme-linked immunosorbent assay (ELISA) technique [ 120 ], electrochemical [ 121 , 122 ], electrical [ 27 , 123 ], and mass-based biosensors [ 124 ], have been developed to detect coronaviruses via their spike proteins. As illustrated in Fig.…”

Bispecific antibodies: design, therapy, perspectives

“… 133 BsAbs recognizing several epitopes of the NP antigen of SARS coronavirus (causing severe acute respiratory syndrome) and binding to horseradish peroxidase were used for immunoswab assays, with sensitivity of NP detection 10 pg/mL and high specificity. 134 …”

A tale of two specificities: bispecific antibodies for therapeutic and diagnostic applications

“…The immunoswab assay facilitated rapid detection ($45 min) [61] and was robust, potentially permitting screening of numerous infected individuals within a short period of time, and hence, assisting to contain viral infection. Furthermore, by targeting the SARS-CoV spike protein (S1) in both mAband bsAb-based assays, to identify SARS infected individuals, it was also demonstrated that the bsAb detection reagent could improve the sensitivity of the assay from 37 to 19 ng/ml [62].…”