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).
Apparatus
The BioDot XYZ dispensing platform (BioDot, Richmond, CA, USA) was used to dispense reagents to conjugate pad, nitrocellulose membrane, and an automatic cutter was used to cut the strip. 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 strip.
Samples and biological materials
Bovine Brucella negative and positive standard sera were purchased from the China Institute of Veterinary Drug Control. Positive sera of tuberculosis, bluetongue disease, viral diarrhea, foot-and-mouth disease, bovine leukemia, peste des petits ruminants and 50 healthy negative bovine and sheep were acquired from the Chinese Academy of Inspection and Quarantine. A total of 150 clinical serum samples was kindly provided by the Animal Husbandry Bureau of Ningxia Hui Autonomous Region. Brucella OMP22 and OMP28, and monoclonal antibodies of OMP22 were prepared by our laboratory.
Preparation of QDFM-protein conjugates
Protein was coupled to the QDFMs by carboxyl activation. In brief, the protein-conjugated QDFMs were prepared using EDC and NHS as cross linkers. The surface carboxyl groups of the QDFM were bound with the amino groups of the antigen under catalysis of EDC and NHS. A commercial QDFM solution (100 μL) was pipetted into centrifuge tubes and activated with EDC and NHS. The mixture solution was dissolved in MES buffer to yield a final concentration of 0.5 mg/L EDC and 0.2 mg/mL NHS. The solution was mixed by vortex for 30 min, followed by reaction at 37°C for 15 min. Then, 100 μL of OMP22 (0.1 mg/mL) was added, and the mixture was reacted for 2–4 h at room temperature under gentle agitation. 50 μL of 10% BSA was added and the mixture was incubated for 30 min at room temperature. The resulting QDFM conjugates were washed three times by centrifugation at 8000 g for 20 min. The purified functional QDFM-OMP22 was resuspended in 1 mL of 20 mM Trise base (TB, pH 8.5) containing 0.5% BSA, 2% source, 0.2% Tween-20, and Triton 405-X and stored at 4°C until use.
Assembly of the QDFM test strip
The strip consisted of four parts: sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad. The sample pad was saturated with a 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 the pad was dried at 70°C for 2 h and stored at room temperature. The components of the test strip were sequentially laminated and pasted to a PVC backing pad with appropriate 2-mm overlap to ensure the testing sample solution could migrate through the whole 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 0.3 mL of McAb OMP22 (1, 0.75, 0.5 mg/mL) were dispense onto the nitrocellulose membraneas 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. 1). .According to the Sotnikov et al describe three schemes of analysis to detect antibodies, our research plan is similar to the author's second scheme. The out membrane protein OMP22 combine with the antibodies in serum samples and captured by OMP28 on the detection line to form an OMP22-Ab-OMP28 immune complex [44].
Sensitivity, threshold, feasibility, repeatability and specificity testing
To improve the sensitivity of the diagnostic procedure, 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. The standard cure was established with serial 2-fold dilutions of Brucella positive serum from 1:4 to 1:1024, and ICTS was used determine the limit of detection.
The test strip was applied to detect positive and negative serum samples, and the test strip was detected using a 365-nm handheld UV lamp and a Fluorescence Reader connected to a laptop. The ICTS was used to detect 50 healthy Brucella negative bovine and sheep serum samples using a Fluorescent Reader and the HT/HC values were recorded as negative controls. The ratio of the signals of the test line to that of the control line (HT/HC) provides a qualitative detection for QDFM. The HT/HC threshold values of ICTS were calculated as the following equation: threshold value = mean ± 3× standard deviation.
To evaluate the feasibility of the strip for detection of antibodies against brucellosis, and to test the reader accuracy, 150 brucellosis clinical serum samples (68 bovine serum, 82 sheep serum) were collected from the Animal Husbandry Bureau of Ningxia Hui Autonomous Region. All the samples were pretreated with 0.01 M Tris-HCl (pH 9.5) buffer containing 0.9% NaCl and 0.05% Tween-20 for 15 min. The 150 clinical samples were tested using ICTS and the coefficient of the detection results were compared with a commercial RBPT.
The repeatability of ICTS 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.