A Modified Quantum Dot-Based Dot Blot Assay for Rapid Detection of Fish Pathogen Vibrio anguillarum

Abstract

Vibrio anguillarum, a devastating pathogen causing vibriosis among marine fish, is prevailing in worldwide fishery industries and accounts for grievous economic losses. Therefore, a rapid on-site detection and diagnostic technique for this pathogen is in urgent need. In this study, two mouse monoclonal antibodies (MAbs) against V. anguillarum, 6B3-C5 and 8G3-B5, were generated by using hybridoma technology and their isotypes were characterized. MAb 6B3-C5 was chosen as the detector antibody and conjugated with quantum dots. Based on MAb 6B3-C5 labeled with quantum dots, a modified dot blot assay was developed for the on-site determination of V. anguillarum. It was found that the method had no cross-reactivity with other than V. anguillarum bacteria. The detection limit (LOD) for V. anguillarum was 1 × 10³ CFU/ml in cultured bacterial suspension samples, which was a 100-fold higher sensitivity than the reported colloidal gold immunochromatographic test strip. When V. anguillarum was mixed with turbot tissue homogenates, the LOD was 1 × 10³ CFU/ml, suggesting that tissue homogenates did not influence the detection capabilities. Preenrichment with the tissue homogenates for 12 h could raise the LOD up to 1 × 10² CFU/ml, confirming the reliability of the method.

Introduction

Vibrio anguillarum is among the most devastating bacterial pathogens that can infect a broad range of marine fish worldwide, with the consequence of an extremely lethal septicemia named vibriosis, causing great economic loss in aquaculture industries [4]. Therefore, the development of an effective diagnostic method of Vibrio anguillarum in cultured fish and rearing water has been the research emphasis.

Traditionally, biochemical detection techniques of V. anguillarum were laborious and time-consuming [10,11]. The multiplex PCR method proved to be a useful tool [15] and immunohistochemical studies using specific MAbs have also been performed [14]. Although these methods are specific and sensitive, their operations are time-consuming and impracticable in non-laboratory conditions. Thus, it is impossible to be applied to on-site diagnosis. Lateral-flow technology methods have also been developed and extensively used because of their high sensitivity, specificity, rapidity, low cost, and applicability to large numbers of samples [7,18]. Recently, quantum dots (QDs) have gained increasing interest because of their unique optical and electrical characteristics. The features of size-tunable emission, broad absorption, intense brightness, narrow emission spectra, and exceptional resistance to photobleaching have made QDs more attractive than traditional colloidal gold or fluorescent probes for developing analytical applications [2]. The present work describes a modified dot blot method based on QD-labeled immunoassay for the rapid detection for V. anguillarum. Compared with traditional dot blot immunoassay, which uses enzymatic labeling, Cdse/Zns QDs were used to conjugate MAb in this study, which can leave out the requirement of multiple incubation steps to allow the enzyme to react with substrates, and shorten the required time for detection from hours to 15 min, with a higher sensitivity and few requirements of both equipment and technical skill at the same time. This method is also applicable to other fish pathogens for on-site rapid diagnosis of diseased fish.

 

Materials and Methods

Strains, Media, and Chemicals

The bacterial strains employed in this study are listed in Table 1. All isolates were revived from stock cultures preserved at −80℃. Vibrio strains were grown in LB broth with 2% NaCl (LB20) at 30℃; Escherichia coli strains, Staphylococcus aureus, and Aeromonas hydrophila were cultured in LB broth at 37℃; and Edwardsiella piscicida and Edwardsiella ictaluri were cultured in brain-heart infusion (BHI) broth at 30℃ for 20 h. The bacteria were centrifuged and washed three times with pre-chilled phosphate-buffered saline (PBS, 10 mmol/l, pH 7.4), inactivated with 1% (v/v) formalin for 1 h at 30℃, then washed three times again with PBS to remove the formaldehyde, and stored at −80℃ for subsequent experiments.

Table 1.Strains used in this study.

The goat anti-mouse IgG was purchased from Tiangen Biotech (China). Freund’s complete adjuvant (FCA), Freund’s incomplete adjuvant (FIA), and HAuCl4·3H2O were from Sigma (USA). Cdse/ZnS QDs were from Wuhan Jiayuan Co. Ltd.

Generation of Antibodies against V. anguillarum

MAb specific to V. anguillarum MVM425 was produced according to previously described methods [25]. Five BALB/c mice were immunized by intraperitoneal injection of 200 μl 108 CFU/ml inactivated bacteria after being mixed with equal volume of Freund’s adjuvant for three times at 2-week intervals; FCA for the first immunization, and FIA for the second and third immunizations. Indirect ELISA was performed to determine serum antibody titers on formalin-killed V. anguillarum-coated plates at 1 week after the third immunization. The dispersed spleen lymphocytes from two mice were removed and fused with myeloma cells (SP2/0-Ag-14) to produce hybridoma cells. Two hybridoma cells (designated as 6B3-C5 and 8G3-B5) were picked for characterization by ELISA test, and then injected intraperitoneally at 5 × 106 CFU per mouse for ascite production. After 1 to 2 weeks, ascite was collected, precipitated with 50% ammonium sulfate, dialyzed, and purified through a protein G agarose column.

Reactivity and Characterization of MAbs

Indirect ELISA tests were completed to appraise the reactivity and sensitivity of MAbs to the formalin-killed V. anguillarum, according to the method performed by Liu et al. [9]. The MAb isotyping was classified with a MAb isotyping kit (Sigma).

Preparation of QD-Antibody

Cdse/ZnS QDs were conjugated with anti-V. anguillarum MAb 6B3-C5 through an EDC/NHS reaction at the concentrations of 0.06 and 0.1 mmol/l, respectively. The mixture was vortexed for 10 sec after addition to the dBSA-coated QD solution (pH 7.4), followed by the addition of MAb, and then incubated for 2 h at 20℃, and stored at 4℃ for further use.

Preparation of Immunochromatographic Test Strip

The MAb-colloidal gold particle (diameter = 40 nm) conjugation and immunochromatographic test strip were produced with the method described by Liu et al. [9]. The rabbit polyclonal antibody (2 mg/ml) and goat anti-mouse IgG (1 mg/ml) were micro-sprayed with XYZ rapid test reagent dispenser HM3030 (Shanghai Goldbio Tech, China) onto a nitrocellulose membrane (NCM) for test line (T) and control line (C) separately.

Sensitivity and Specificity Assays of Modified Dot Blotting

In order to assess the cross-reactivity of the modified dot blot assay, V. anguillarum MVM425, V. anguillarum VIB72, E. piscicida EIB202, Vibrio alginolyticus, Vibrio harveyi, Vibrio vulnificus EIBVF1, E. coli DH5-α, E. coli CC118, S. aureus, and Vibrio parahaemolyticus were diluted to 1 × 106 CFU/ml with PBS. Bacterial samples (10 μl) were spotted onto the NCM, baked at 60℃ for 1 h, blocked in 5% Blotto (bovine lacto transfer technique optimizer, 5% nonfat dry milk, 0.1% Triton X-100 in PBS), then incubated with QD-MAb 6B3-C5 at a 1:100 dilution for 3 h, washed again in 0.5% Blotto, and hatched in PBS for 5 min [19]. Then, the NCM-displayed immunoreactivity specific to V. anguillarum was confirmed by 16S DNA sequencing.

The sensitivity of the modified dot blot assay was determined qualitatively. A series of diluted V. anguillarum (10 μl) of gradient concentrations from 1 × 109 to 1 × 102 CFU/ml in sample buffer were spotted onto NCM and applied to the dot blot. All of the detections were performed in triplicates.

Detection of V. anguillarum in Turbot Tissue

Healthy turbots (~30 g) were injected intraperitoneally with 100 μl of 5 × 106 CFU/ml V. anguillarum (determined by counting the bacterial colonies grown on LB20 plates after 24 h incubation). Spleen tissues from fresh turbot were dissected out, and each 0.01 g spleen tissue was homogenized in 1 ml of LB20. Samples with various concentrations of V. anguillarum cultures in turbot spleen tissue homogenate were prepared by diluting with LB20 and incubated at 30℃. All of the tissue homogenates were heat killed at 60℃ for 30 min and performed in triplicates.

 

Results and Discussion

Preparation and Characteristics of Antibodies against V. anguillarum

Based on O-antigens, V. anguillarum has as many as 23 serogroups. The different serogroups exhibit various pathogenicity and host selectivity. O1 and O2 are by far the most typical virulent serogroups in turbot [1]. In this study, V. anguillarum MVM425 with serogroup O1 was used as the immunogen in the production of MAb against V. anguillarum. Out of the 1,728 wells of culture supernatants from two fusions of spleen lymphocytes and myeloma cells after immunization of formalin-killed V. anguillarum MVM425 for three times, two hybridoma cells, 6B3-C5 and 8G3-B5, displayed a strongly positive signal in indirect ELISA. After purification, MAbs 6B3-C5 and 8G3-B5 reacted strongly with V. anguillarum MVM425, while a weaker reaction with V. anguillarum VIB72 (serogroup O2) was also observed, suggesting that these two MAbs can be specific to V. anguillarum with serogroups O1 and O2, but not with the whole bacterial cells of common bacterial pathogens of maricultured fish, such as A. hydrophila, E. coli, E. piscicida, E. ictaluri, S. aureus, V. harveyi, V. alginolyticus, V. parahaemolyticus, and V. vulnificus (Fig. 1A). The isotyping results classified the two anti-V. anguillarum MAbs as subclass IgG3.

Fig. 1.Reactivity and sensitivity of MAbs to V. anguillarum and other bacterial strains with indirect ELISA. (A) A fixed quantity of formalin-killed whole cells of V. anguillarum (108 CFU/ml) were coated to the plate, and 1:200 dilution of MAbs 6B3-C5, 8G3-B5, 5G4-G4, and 5G4-G8 was used as the primary antibody. (B) The ELISA plate was coated with 10-fold diluted solutions (108 to 101 CFU/ml) of formalin-killed whole cells of V. anguillarum MVM425, and 1:200 dilution of MAb 6B3-C5 was used as the primary antibody. The dotted lines indicated the positive control values.

In our pervious study, two MAbs (5G4-G4 and 5G4-G8) against V. anguillarum were also generated and showed specificity to V. anguillarum [28]. In this study, we showed that MAb 6B3-C5 reacted stronger than MAbs 8G3-B5, 5G4-G4, and 5G4-G8 did (Fig. 1A). The LOD of MAb 6B3-C5 to detect V. anguillarum was 1 × 104 CFU/ml (Fig. 1B). According to the results shown above, the cell line 6B3-C5 (CCTCCC201478) was selected for subsequent experiments.

Preparation of QD-Antibody

Activated by the EDC and NHS mixture, MAb 6B3-C5 IgG was linked to QDs. There was a significant decrease of the fluorescence intensity in QD-labeled MAb 6B3-C5 due to energy transfer from QDs to MAbs upon their conjugation [3,8] (Fig. 2A). The conjugation was also confirmed by gel electrophoresis (Fig. 2B). QD-antibodies displayed a lagging band because of its larger molecular weight compared with the bare QDs (Fig. 2B). These results indicated that QDs were successfully conjugated to antibodies.

Fig. 2.Characteristics of QD-antibody conjugates. (A) Fluorescence intensity of the QD-antibody conjugation products. (B) Electrophoresis of the QD-antibody conjugation products. Lane 1 and lane 2: QD-antibodies; lane 3 and lane 4: bare QDs. (C) The optimal amount of antibody labeled by QDs. Line 1: QDs; line 2: 20 μl antibody-QDs; line 3: 30 μl antibody-QDs; line 4: 40 μl antibody-QDs; line 5: 50 μl antibody-QDs; line 6: BSA.

To determine the optimal combination between QDs and antibodies, 1 mg/ml antibodies were added to 5 μl of QDs with different volumes. The amount of antibodies were 50, 40, 30, 20 μl, respectively, and then the PBS buffer was added up to 3 ml. Fluorescence spectrometry was used to measure the emission spectrum diagram of these samples (Fig. 2C). The fluorescence intensity of all samples was decreased with the decrease of antibody concentration. The fluorescence of QD conjugates with antibodies was weaker than the bare QDs. The optimal amount of antibody labeled by QDs was determined as 5 μl of QDs with 40 μl of 1 mg/ml antibodies.

Sensitivity and Specificity of Modified Dot Blotting for Bacterial Suspension Detection

The sensitivity for detection of QD-antibody was determined through modified dot blotting. Heat-killed bacterial suspensions (10 μl) of V. anguillarum at serial concentrations from 1 × 109 to 1 × 102 CFU/ml were spotted onto NCM, while PBS was used as the blank and BSA as the negative control. The results indicated that the fluorescence intensity was enhanced with the increasing of microbial cells and could be detected visually up to 1 × 103 CFU/ml (101 CFU per spot, Fig. 3A).

Fig. 3.Sensitivity and specificity evaluation of the modified dot blot assay and immunochromatographic test strip in bacterial suspension. (A) Sensitivity evaluation of the modified dot blotting. Bacterial suspensions of V. anguillarum at serial concentrations from 1 × 109 to 1 × 102 CFU/ml; PBS and BSA were used as blank and negative control, respectively. (B) Sensitivity evaluation of immunochromatographic test strip. Bacterial suspensions of V. anguillarum at serial concentrations from 1 × 109 to 1 × 102 CFU/ml; PBS and BSA were used as blank and negative control, respectively. (C) Specificity evaluation of the modified dot blotting with various bacteria. The same bacterial suspensions mentioned above were applied.

Recently, the immunochromatographic test strip was used for fish pathogenic bacteria detection [13]. In this study, the MAb 6B3-C5 was also labeled with colloidal gold and developed as an immunochromatographic test strip. When 1 × 105 CFU/ml bacteria suspension was added on the strip, the intensity of the test line was significantly weaker than the control line (Fig. 3B), suggesting 1 × 105 CFU/ml of V. anguillarum could be the visual detection limit of the immunochromatographic test strip, which is similar to the report of Guo et al. [6]. Thus, the modified dot blotting showed 100-fold more sensitivity than the immunochromatographic detection.

According to the modified dot blot method in this study, the detections of V. anguillarum with serogroups O1 and O2 produced positive results when the concentration of 1 × 106 CFU/ml was spotted onto NCM. There were no cross-reactions of the dot blotting with E. piscicida EIB202, V. alginolyticus, V. harveyi, E. coli DH5-α, E. coli CC118, and V. parahaemolyticus (Fig. 3C). The modified dot blotting showed a high specificity to V. anguillarum and proved to be an effective method for the sensitive detection of V. anguillarum.

Detection of V. anguillarum in Turbot Tissue

In order to determine the sensitivity of the modified dot blot detection in artificially infected turbots, healthy turbots were injected intraperitoneally with V. anguillarum bacterial suspension with a final concentration of 5 × 106 CFU per fish. Spleen from three turbots of each group were sampled and homogenized at 0, 1, 2, 3, 5, 6, 7, 8, 9, and 10 days post infection. The survival rate of turbots challenged with V. anguillarum decreased from 1 to 10 days post infection (Fig. 4A), which correlated with the increasing contemporaneous bacterial counts of V. anguillarum in spleen tissue (Fig. 4B). An obvious immunoreactivity was detected in the turbot homogenate samples challenged with V. anguillarum from 1 to 10 days post infection through the modified dot blot assay, while the sample at days 1 and 2 post infection showed no immunoreactivity through the immunochromatographic test strip, due to the low counts of V. anguillarum in spleen tissue (Figs. 4B and 4C). Therefore, the sensitivity of the dot blot assay was determined as 105 CFU/g of fish tissue (1 × 103 CFU/ml, 0.01 g sample homogenized with 200 μl of PBS and PBS added to 1 ml), which was 100-fold more sensitive than the immunochromatographic test strip experiment (Fig. 4C). Furthermore, after pre-enrichment of the sample for 12 h, an obvious chromogenic reaction could be observed in samples containing 1 × 102 CFU/g V. anguillarum with the modified dot blot method, while after pre-enrichment for 24 h, a positive result could be observed at the concentration of 10 CFU/g in LB20 (Fig. 4D). These results indicated that with a high performance and modified dot blot assay, a rapid and highly sensitive on-site diagnostic test was developed for the purpose of earlier determination of V. anguillarum infection in fish. A similar development strategy could also be applied to other fish pathogens.

Fig. 4.Sensitivity and specificity evaluation of the modified dot blot assay and immunochromatographic test strip in turbot tissue. (A) Survival rate of turbot infected with V. anguillarum MVM425 by i.m. injection. (B) Propagation of V. anguillarum MVM425 in turbot spleen following i.m. injection. The number of viable bacteria was shown as the mean ± SD of three samples. (C) Modified dot blot and immunochromatographic test strip tests of V. anguillarum MVM425 with turbot spleen tissue homogenates at 1 to 10 days after i.m. injection. (D) Modified dot blot and immunochromatographic test strip tests of V. anguillarum MVM425 with turbot tissue homogenates. V. anguillarum MVM425 at various concentrations (from 10 to 108 CFU/ml) were pre-enriched with turbot tissue homogenates for 0 to 24 h before being applied to the modified dot blot and immunochromatographic test strip tests; BSA and PBS were used as the blank sample.