Materials and reagents
Quantum dot fluorescent microspheres were purchased from Invitrogen Corp (Carlsbad, CA, USA). A Rose Bengal plate test (RBPT) was obtained from the China Institute of Veterinary Drug Control. Bovine serum albumin (BSA), tween-20, polyvinyl pyrrolidone (PVP), sodium azide, tris base (TB), 2-(4-Morpholino) ethanesulfonic acid(MES), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA) . The test strip materials, including nitrocellulose (NC) membranes (Millipore Hiflow-95) and glass cellulose membranes (Product number 8951), were purchased from Shanghai Jiening Biotechnology Co., Ltd (Shanghai, China).
The BioDot XYZ dispensing platform (BioDot, Richmond, CA, USA) was used to dispense reagents to nitrocellulose membrane, conjugate pad and an automatic cutter (BioDot, Richmond, CA, USA) was used to cut the strips. A fluorescent strip reader JN615 was purchased from Shanghai Jie Ning Biological Co., Ltd (Shanghai, China). A 365-nm hand held UV lamp (American Precision Co., Ltd., USA) was used to test strips.
Samples and biological materials
Bovine Brucella negative and positive standard sera were purchased from the China Institute of Veterinary Drug Control. Positive sera of Y.enterocolitica O:9(2 goat serum), E.coli O:157(4 bovine serum), Salmonella Dublin (2 sheep serum) and 50 healthy negative bovine and sheep (30 bovine serum, 20 sheep serum) were preserved in the Chinese Academy of Inspection and Quarantine. A total of 150 clinical serum samples (68 bovine serum, 44 sheep and 38 goat serum) were kindly provided by the Animal Husbandry Bureau of Ningxia Hui Autonomous Region (Table7). Brucella OMP22 and OMP28 and monoclonal antibodies of OMP22 were prepared by our laboratory.
Preparation of QDFM conjugates protein
The protein is coupled to QDFM through carboxyl activation. Transfer the commercial QDFM solution (100 μL) into a centrifuge tube and prepare OMP22-QDFM using EDC and NHS as cross-linking agents. The mixture solution was dissolved in MES buffer to produce a final concentration of 0.5 mg/mL EDC and 0.2 mg/mL NHS. The solution was vortexed for 30 minutes and then reacted at 37℃for 15 minutes. Then, 100 μL of OMP22 (0.1 mg/mL) was added and the mixture was reacted for 2-4 hours under slow stirring at room temperature. 50 μL of 10% BSA was added and the mixture was incubated at room temperature for 30 minutes. The resulting QDFM conjugate was washed 3 times by centrifugation at 8000 g for 20 minutes. The QDFM-OMP22 conjugate was resuspended in 1 mL of 20 mM Trise solution (TB, pH 8.5) containing 0.5% BSA, 2% sucrose, 0.2% Tween-20, Triton 405-X and kept at 4 ℃ until use .
Assembly of the QDFM test strip
The test strip is composed of four parts: sample pad, conjugate pad, nitrocellulose membrane and absorbent pad. The sample pad is soaked in 20 mM TB (pH 8.5) buffer containing 5% sucrose, 0.5% BSA, 0.01% PVP-40, 2% Tween-20 and 0.02% NaN3. And then dried at 70℃ for 2 hours and stored at room temperature. Paste the test strip components on the PVC backplane in turn and overlap the two components by 2-mm to ensure that the test sample solution can migrate to the entire assembled test strip. In our present study, QDFM labeled Brucella OMP22 was dispensed onto the conjugate pad and then the pad was dried at 37°C overnight and stored at 4°C. 0.03 mL of OMP28 (1.5, 1, 0.75 mg/mL) and 0.03 mL of McAb OMP22 (1, 0.75, 0.5 mg/mL) were dispensed onto the nitrocellulose membrane as test and control lines, respectively, and the strip was dried at 37°C for 2 h. Finally, the whole assembled strip was cut into a 5-mm width and 80-mm length using a BIO-DOT strip cutting machine (Fig. 3). Eight brucellosis positive serum samples with different concentrations were tested to determine the NC membrane coating concentrations. The corresponding concentrations of the samples were 0.169 ng/μL, 0.666 ng/μL, 1.35 ng/μL, 2.11 ng/μL, 3.06 ng/μL, 27.06 ng/μL, 45.2 ng/μL, 64.2 ng/μL, respectively.
According to the three detection antibody analysis protocols described by Sotnikov et al our research plan is similar to the author's second protocol. The extramembrane protein OMP22 binds to the antibody in the serum sample and is captured by OMP28 on the detection line to form an OMP22-Ab-OMP28 immune complex .
Sensitivity, threshold, specificity, feasibility and repeatability test
The standard cure was established with serial 2-fold dilutions of Brucella positive serum from 1:4 to 1:1024 and ICST was used determine the limit of detection.
50 healthy bovine and sheep serum samples were tested as negative controls to determine the threshold of results. The 365 nm handheld UV lamp is used to initially observe the results of the test strip, and then use a fluorescence reader to record the ratio of the test line signal to the control line signal (HT/HC). Calculate the HT/HC threshold of ICST by the following formula: Threshold = average ± 3 × standard deviation.
ICST was used to detect Yersinia enteritidis O: 9, E. coli O: 157 and Salmonella Dublin positive serum samples and the results were recorded with a fluorescence reader. According to the size of the threshold to determine the specificity of the test strips.
In order to evaluate the feasibility of the test strip for detecting brucellosis antibodies, 150 clinical serum samples of brucellosis were collected from the Animal Husbandry Bureau of Ningxia Hui Autonomous Region. All samples were pretreated with 0.01 M Tris-HCl (pH 9.5) buffer containing 0.9% NaCl and 0.05% Tween-20 for 15 minutes. 150 clinical samples were tested using ICST and the coefficients of the test results were compared with commercial RBPT. Serial 2-fold dilution of bovine Brucella positive standard sera from 1:2 to 1:256 were used in order to determine the sensitivity of RBPT .
The repeatability of ICST was tested by 11 serially diluted standard brucellosis positive serum samples concentrations ranging from 50 ng/mL to 1 ng/mL and 1 negative serum. Each sample was detected for three times to calculated coefficient of variation (CV). The CV was calculated by dividing the mean of three measurements by the standard deviation to determine the repeatability.