Experimental strains, animals and serum
The Klebsiella pneumoniae strain KpY-1 was isolated in 2018 from a goat in Shandong Province and was identified by PCR and sequencing. The titer of Klebsiella pneumoniae was determined to be 106.0 CFU/mL by Colony-Forming Units (CFU) in Luria-Bertani (LB) agar plates. Positive goat sera for Klebsiella pneumoniae, Escherichia coli, Salmonella Enteritidis, Clostridium perfringens, and Pasteurella multocida type B were collected from infected goats and stored at − 80 °C in our laboratory. The negative sera were standard goat serum products we purchased randomly (C0265, SL038, and G9023). A total of 1320 serum samples were provided by the Animal Disease Prevention and Control Center of Shandong Province. All of the samples were randomly collected from different farms of 16 cities in Shandong Province.
Four one-year-old Laiwu black goats were provided by Shandong Laiwu Black Goat Seed Farm. After the experiments were completed, the goats were healthy and released.
Preparation of the Klebsiella pneumoniae goat polyclonal antibody
The four Laiwu black goats were immunized with the prepared Klebsiella pneumoniae white oil adjuvant antigen (4.0 × 109 CFU/goat) by jugular intramuscular injection for five times with immunization interval of 7 days. The serum was collected after 1 week from the last immunization and prepared for further qualification.
The collected serum was continuously diluted with normal saline and added equivalent the inactivated Klebsiella pneumoniae as antigen. The mixture was adequately reacted for 5–10 min. Then, the highest dilution in which agglutination occurs is the serum titer . The Klebsiella pneumoniae goat polyclonal antibody was feasible when the serum agglutinated and the titers were higher than 8 log2.
Inactivated Klebsiella pneumoniae culture (2.3 × 1010 CFU/mL, 20 μL) was added to a slide and fixed with 10% formaldehyde for 15 min at room temperature . After rinsing the slide with PBS three times, nonfat dry milk (5%, 20 μL) was loaded and incubated at 37 °C for 30 min. Following complete rinse with PBS for three times, the Klebsiella pneumoniae goat polyclonal antibody (1: 200 diluted, 20 μL) that was mentioned above was loaded and incubated at 37 °C for 30 min. After the extra primary antibody was rinsed off, FITC-labeled goat anti-rabbit IgG (1: 400 diluted, 20 μL, Beyotime Biotechnology, China) was loaded and incubated at 37 °C for 30 min . Finally, the slide was washed with PBS (3 times, 1 min/time) and the positive fluorescence was observed under a fluorescence microscope (Olympus BX51, China). The culture solution without Klebsiella pneumoniae served as control.
Establishment of the indirect ELISA
A square titration in 96-well ELISA microplates (BIOFIL, medium binding) was implemented to optimize the conditions for detection according to a classical indirect ELISA protocol . Firstly, the inactivated Klebsiella pneumoniae culture (2.3 × 1010 CFU/mL) was serially diluted from 1:100 to 1:6400. The bacteria solution (100 μL/well) were coated with different buffer (deionized water, phosphate buffer, or carbonate buffer) at 37 °C for 1 h or 4 °C for 12 h. The wells were blocked with different buffers (0.5% BSA, 1% BSA, 1.5% BSA, or 2% BSA, 250 μL/well) at 37 °C for 1 h and or 4 °C for 12 h. The Klebsiella pneumoniae goat polyclonal antibody was serially two-fold diluted from 1:100 to 1:102400 by using different buffers (0.5% BSA, 1.0% BSA, or 1.5% BSA). The positive and negative sera were diluted to 1:100 and served as controls (100 μL/well). Then, the plates were incubated at 37 °C for various time scale (30 min, 45 min, 60 min, or 75 min). HRP-conjugated Affinipure Rabbit Anti-Goat IgG (100 μL/well, Proteintech, USA) was optimized with different dilutions (1:1000, 1:5000, 1:10000 or 1:15000) by 1% BSA and different incubation times (30 min, 45 min, 60 min or 75 min) at 37 °C. Finally, the plates were reacted with 50 μL substrate for various time scales (20 min, 30 min, 40 min) and were measured with a microplate spectrophotometer (XMark, Bio-Rad) at wavelengths of 450 nm (OD450). The variant was single during the execution of the entire ELISA protocol and all of the samples were tested in technical triplicates. The positive value (P) was approximately 1.0, the negative value (N) was below 0.4, and thereby the maximum ratio between P and N (P/N) was no less than 2.1, which were considered to be the optimal reaction conditions .
Determination of the cut-off value
The OD450 values of 48 goat negative serum samples were determined by the ELISA method, and the experiment was repeated 3 times. The mean and standard deviation of the OD450 values were calculated. The OD450 average value plus the standard deviation of 3 times were used as the cut-off value to determine whether a serum sample was positive or negative by the ELISA.
Sensitivity analysis, also known as the lower detection limit, was estimated by end-point titration and was defined as the maximum dilution of the sample detected just above the cut-off value. Therefore, Klebsiella pneumoniae positive sera was serially diluted from 1: 100 to 1: 1600 and was conducted by the indirect ELISA procedures. Each dilution was tested in triplicate and the Klebsiella pneumoniae negative serum samples served as controls.
The ELISA method described above was used to simultaneously detect the OD values of goat-derived sera positive for Klebsiella pneumoniae, Escherichia coli, Salmonella, Clostridium perfringens, and Pasteurella, verifying whether the Klebsiella pneumoniae goat polyclonal antibody cross-reacts with serum positive for other pathogens. The goat-derived sera without antibodies against the above mentioned pathogens served as the negative control.
To evaluate the reproducibility of ELISA, intra- and inter-assays variation analyses were conducted by the indirect ELISA. Intra-assay was performed using six different ELISA plates of the same batch, inter-assay was performed using ELISA plates to detect samples from different batches. The coefficients of variation (CVs) were calculated by dividing the standard deviation of the OD value of each tested sample by its mean and multiplying the result by 100 to evaluate the reproducibility of the ELISA.
Field sample application
The established indirect ELISA assay was applied to determine Klebsiella pneumoniae infection from 1320 clinical veterinary serum samples that were collected from different goat farms in Shandong province from 2018 to 2019.