RESUMO
A highly purified and bioactive immunoglobulin G monoclonal antibody against receptor-binding domain of SARS-CoV-2 (RBD-IgG-MAb) has been accurately quantified by amino acid determination using isotope dilution liquid chromatography-mass spectrometry. Absolute quantification of RBD-IgG-MAb was achieved by averaging 4 amino acid certified reference materials, which allows the quantitative value (66.1 ± 5.8 µg/L) to be traced to SI unit (mol). Afterwards, the RBD-IgG-MAb was employed as control and calibration compound for the development of a point-of-care testing (POCT) system based on colloidal gold lateral flow immunoassay, which aimed to rapidly and accurately detect the level of protective RBD-IgG after vaccination. Under the detection parameters, a sigmoidal curve has been plotted between signal intensity and the logarithmic concentration for quantitative detection with the limit of detection of about 0.39 µg/mL. The relative standard deviations of intra-assay and inter-assay were lower than 2.3% and 14%, and the recoveries ranged from 87 to 100%, respectively. Fingertip blood samples from 37 volunteers after vaccination were analyzed by the POCT system; results showed that levels of RBD-IgG in 33 out of 37 samples ranged from 0.45 to 2.46 µg/mL with the average level of 0.91 µg/mL. The developed POCT system has been successfully established with the quantity-traceability RBD-IgG-MAb as control and calibration compound, and the scientific contribution of this work can be promoted to other areas.
Assuntos
COVID-19 , Imunoglobulina G , Humanos , SARS-CoV-2 , COVID-19/diagnóstico , Testes Imediatos , AminoácidosRESUMO
Carbohydrate antigen 199 (CA199) is a serum biomarker which has certain value and significance in the diagnosis, prognosis, treatment, and postoperative monitoring of cancer. In this study, a lateral flow immunoassay based on europium (III) polystyrene time-resolved fluorescence microspheres (TRFM-based LFIA), integrated with a portable fluorescence reader, has been successfully establish for rapid and quantitative analysis of CA199 in human serum. Briefly, time-resolved fluorescence microspheres (TRFMs) were conjugated with antibody I (Ab1) against CA199 as detection probes, and antibody II (Ab2) was coated as capture element, and a "TRFMs-Ab1-CA199-Ab2" sandwich format would form when CA199 was detected by the TRFM-based LFIA. Under the optimal parameters, the detection limit of the TRFM-based LFIA for visible quantitation with the help of an ultraviolet light was 4.125 U/mL, which was four times lower than that of LFIA based on gold nanoparticles. Additionally, the fluorescence ratio is well linearly correlated with the CA199 concentration (0.00-66.0 U/mL) and logarithmic concentration (66.0-264.0 U/mL) for quantitative detection. Serum samples from 10 healthy people and 10 liver cancer patients were tested to confirm the performances of the point-of-care application of the TRFM-based LFIA, 20.0 U/mL of CA199 in human serum was defined as the threshold for distinguishing healthy people from liver cancer patients with an accuracy of about 60%. The establishment of TRFM-based LFIA will provide a sensitive, convenient, and efficient technical support for rapid screening of CA199 in cancer diagnosis and prognosis.
Assuntos
Neoplasias Hepáticas , Nanopartículas Metálicas , Biomarcadores Tumorais , Ouro , Humanos , Imunoensaio , Limite de Detecção , MicroesferasRESUMO
Neutralizing antibody (NAb) is a family of antibodies with special functions, which afford a degree of protection against infection and/or reduce the risk of clinically severe infection. Receptor binding domain (RBD) in the spike protein of SARS-CoV-2, a portion of the S1 subunit, can stimulate the immune system to produce NAb after infection and vaccination. The detection of NAb against SARS-CoV-2 is a simple and direct approach for evaluating a vaccine's effectiveness. In this study, a direct, rapid, and point-of-care bicolor lateral flow immunoassay (LFIA) was developed for NAb against SARS-CoV-2 detection without sample pretreatment, and which was based on the principle of NAb-mediated blockage of the interaction between RBD and angiotensin-converting enzyme 2. In the bicolor LFIA, red and blue latex microspheres (LMs) were used to locate the test and control lines, leading to avoidance of erroneous interpretations of one-colored line results. Under the optimal conditions, NAb against SARS-CoV-2 detection carried out using the bicolor LFIA could be completed within 9 min, and the visible limit of detection was about 48 ng/mL. Thirteen serum samples were analyzed, and the results showed that the NAb levels in three positive serum samples were equal to, or higher than, 736 ng/mL. The LM-based bicolor LFIA allows one-step, rapid, convenient, inexpensive, and user-friendly determination of NAb against SARS-CoV-2 in serum.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Antivirais/isolamento & purificação , COVID-19/diagnóstico , Cromatografia de Afinidade , Humanos , Látex , Microesferas , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory coronavirus 2 (SARS-CoV-2) is still raging all over the world. Hence, the rapid and sensitive screening of the suspected population is in high demand. The nucleocapsid protein (NP) of SARS-CoV-2 has been selected as an ideal marker for viral antigen detection. This study describes a lateral flow immunoassay (LFIA) based on colloidal gold nanoparticles for rapid NP antigen detection, in which sensitivity was improved through copper deposition-induced signal amplification. The detection sensitivity of the developed LFIA for NP antigen detection (using certified reference materials) under the optimized parameters was 0.01 µg/mL and was promoted by three orders of magnitude to 10 pg/mL after copper deposition signal amplification. The LFIA coupled with the copper enhancement technique has many merits such as low cost, high efficiency, and high sensitivity. It provides an effective approach to the rapid screening, diagnosis, and monitoring of the suspected population in the COVID-19 outbreak.