A New Method for Early Screening of Gastric Cancer (G17 and CA724 Dual-Labeled Time-Resolved Fluorescence Immunoassay).

Gastric carcinoma (GC) is one of the most common malignancies in the world with the great early screening challenges. The study is aimed at establishing a new detection method for early screening GC using time-resolved fluorescence immunoassay (TRFIA) via quantitative detection of gastrin-17 (G-17) and carbohydrate antigen 724 (CA724) in serum. Time-resolved analyzer measured the fluorescence intensity. The standards of G-17/CA724 were used for drawing the standard curve, which is used to calculate the concentration of G-17 and CA724 in serum sample. The sensitivity for G-17 was 0.54 pg/mL and for CA724 was 0.28 U/mL with a wide-range analyze concentration (0.1-1000) pg/mL or U/mL. The average recoveries ranged from 100.52% to 110.30% for G-17 and 103.02% to 116.00% for CA724. All CVs of the intra- and interassay were below 10% with high specificity. There was a high Pearson coefficient between this TRFIA method and the commercially available kits (Pearson r 0.9117 for G-17 and 0.9449 for CA724). Additionally, the cutoff value was 88.41 pg/mL and 5.47 U/mL for CA724 in health subjects. This study established a TRFIA method for simultaneous detection of the concentrations of G-17 and CA724 in serum, which provide a new method for sensitive, accurate, and specific early screening of gastric cancer.

Determination of human COVID-19 total antibodies in serum using a time-resolved fluorescence immunoassay.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading rapidly around the world. Antibody detection plays an important role in the diagnosis of COVID-19. Here, we established a new time-resolved fluorescence immunoassay (TRFIA) to determine COVID-19 total antibodies. A double-antigen sandwich TRFIA was optimized and established: recombinant nucleocapsid phosphoprotein (N protein) and spike protein (S protein) of COVID-19 immobilized on 96-well plates captured human COVID-19 antibodies and then banded together with the N/S proteins labeled with europium(III) (Eu3+ ) chelates, and finally, time-resolved fluorometry was used to measure the fluorescence values. We successfully established a TRFIA method for the detection of human COVID-19 total antibodies, and the cutoff value was 2.02. There was no cross-reactivity with the negative reference of the National Reference Panel for IgM and IgG antibodies to COVID-19. The CV of the precision assay was 3.19%, and the assay could be stored stably for 15 days at 37°C. Compared with that of the colloidal gold method and chemiluminescence method, the sensitivity of the TRFIA method was higher, and the false positive/negative rate was lower. This established TRFIA has high sensitivity, accuracy, and specificity, which indicates that this method provides a new detection method for the high-throughput routine diagnosis of COVID-19.

A New Method for Detection African Swine Fever Virus: Time-resolved Fluorescence Immunoassay.

African swine fever (ASF) has severely influenced the swine industry of the whole world. Fast and accurate African swine fever virus (ASFV) antigen detection is very important for ASF prevention. This study aims to establish a new detection method for detection ASFV antigen using time-resolved fluorescence immunoassay (TRFIA) in the nose and mouth discharge. A double antibody sandwich TRFIA method was optimized and established. Recombinant P30 recombinant antigen was captured by its antibodies immobilized on 96-well plate, and then banded together with another detection antibodies labeled with Europium(III) (Eu3+) chelates, finally time-resolved analyzer measured the fluorescence intensity. The performance of this TRFIA (sensitivity, specificity and accuracy) was evaluated using the clinical samples and compared with the nucleic acid testing method. The sensitivity of this TRFIA was 0.015 ng/mL (dynamic range 0.24-500 ng/mL) with high specificity. The recovery ranged from 92.00 to 103.62 %, the inter-assay CVs ranged from 5.50 to 11.96 %, and the intra-assay CVs was between 5.20 and 10.53 %. Additionally, the cutoff value was 0.016. TRFIA took only 45 min to generate results, and its detection capability comparable to the nucleic acid detection. This study developed a TRFIA method that could be used for qualitative/quantitative detection of ASFV antigen in pigs nasal discharge, which has high sensitivity, specificity and accuracy. This TRFIA provides a new method for rapidly screening ASFV infection in pigs industry.

Determination of parvovirus antigen in the vaccine using time-resolved fluorescence immunoassay.

As a highly contagious and potentially fatal disease of dogs, canine parvovirus type 2 (CPV-2) usually causes severe myocarditis and gastroenteritis, while vaccine injection has greatly reduced the incidence of CPV-2 diseases. However, there is currently a lack of simple and effective method for quantitative detection of CPV-2 in vaccine. Therefore, this study aims to prepare an accurate method to determine the CPV-2 antigen (CPV-2-Ag) in vaccine. Here, a sandwich time-resolved fluorescence immunoassay (TRFIA) was established and optimized. Anti-CPV-2 antibodies were immobilized on 96-well plates to capture CPV-2-Ag, and then bound together with the detection antibodies labeled with Europium(III) (Eu3+ ) chelates; finally, time-resolved fluorometry was employed to measure the fluorescence intensity. Vaccination was performed to evaluate the relationship between CPV-2-Ag concentration and antibody titer. The sensitivity is 1.15 mEU/mL (LogY = 1.524 + 0.8667 × LogX, R2  = 0.9933), and the average recovery is among 91.00% to 106.39% without cross-reactions with the other canine viral antigen. The correlation between ELISA assay and this method is up to 0.9861. And, there is high correlation between the CPV-2-Ag concentration and antibody titers (R2  = 0.9234). This immunoassay established has high sensitivity, accuracy, and specificity, which indicate that this method could be suitable for quantitative detection of CPV-2-Ag in vaccine evaluation.