Development and Application of Colloidal Gold Test Strips for the Rapid Detection of Canine Brucellosis

Abstract

Brucellosis is a global problem, with the causative agent being the genus Brucella. B. canis can cause undulant fever in dogs, which is a zoonotic disease that can spread not only among dogs but also to humans. This poses a public health threat to society. In this study, a rapid and straightforward immune colloidal gold test strip was developed for the diagnosis of canine brucellosis through the detection of anti-LPS antibodies in serum samples. Rabbit anti-canine IgG conjugated with colloidal gold was employed as the colloidal gold-labeled antibody. The extracted high-purity R-LPS was employed as the capture antigen in the test line (T-line), while goat anti-rabbit IgG was utilized as the capture antibody in the control line (C-line). The colloidal gold strip exhibited high specificity in the detection of brucellosis, with no cross-reaction observed with the common clinical canine diseases caused by Canine coronavirus (CCV), Canine distemper virus (CDV), and Canine parvovirus (CPV). In comparison to the commercial iELISA kit, the sensitivity and specificity of the colloidal gold test strip were found to be 95.23% and 98.76%, respectively. The diagnostic coincidence rate was 98.47%. The findings of this study indicate that colloidal gold test strips may be employed as a straightforward, expeditious, sensitive, and specific diagnostic instrument for the identification of canine brucellosis, particularly in resource-limited regions.

Keywords: canine brucellosis; colloidal gold test strips; rapid detection.

Figure 1

The schematic diagram of the colloidal gold test strip (A) and the final product of the colloidal gold test strip package (B). The test strip consisted of three pads (sample pad, conjugate pad, and absorbent pad), a nitrocellulose membrane, and a polystyrene backing. The conjugate pads contained gold-labeled rabbit anti-canine IgG. There were two lines on the nitrocellulose membrane: the test line (T-line) and the control line (C-line). The T-line contains R-LPS and the C-line contains goat anti-rabbit IgG antibody.

Figure 2

The purity of R-LPS was identified. R-LPS silver staining (A); schematic representation of Coomassie Brilliant blue R250 staining (B); identification of the R-LPS by western blotting using rabbit anti-Brucella antibody (C). Lane 1 represents the aqueous phase following hot phenol–water extraction. Lane 2 depicts the phenol phase after proteinase K treatment. Lane 3 is the aqueous phase treated with proteinase K. Lane 4 is the phenol phase after hot phenol–water extraction.

Figure 3

Identification of colloidal gold solution. The colloidal gold solution exhibited a wine-red coloration (A). Transmission electron microscopy revealed that the gold particles were predominantly within the range of from 18 to 25 nanometers, exhibiting a relatively uniform morphology (B). The peak at 519 nanometers observed in the OD400–700 nm scanning further substantiated that the colloidal gold particles exhibited a relatively small diameter, thereby confirming the high degree of specificity (C).

Figure 4

Optimal labeling conditions for colloidal gold. (A) The optimal pH for labeling colloidal gold solutions; (B) the optimal amount of antibody for labeling colloidal gold solutions.

Figure 5

Optimal antigen coating and serum dilution for colloidal gold test strips; (A) 1 mg/mL, 2 mg/mL, 3 mg/mL, or 4 mg/mL R-LPS was coated on NC membrane and positive serum was used for detection. (B) The positive serum was diluted 1:5, 1:2, and 1:1 to test the optimal dilution concentration. (C) The negative serum was tested at dilutions of 1:20, 1:10, 1:5, and 1:1, and none of them was detected as positive.

Figure 6

Evaluation of the specificity of colloidal gold test strips. Positive serum samples from clinical dogs infected with Canine coronavirus (CCV), Canine distemper virus (CDV), and Canine parvo virus (CPV) were tested negative in test strips.