1.
Introduction
Streptococcusagalactiaeis a common
animal and animal disease, endangering a variety of important animal bacteria [1], in China, its main harm to tilapia aquaculture.
According to the World Food and Agriculture Organization (FAO) statistics, in
2008 China tilapia farming amounted to 706,585 tons, accounting for 50% of the world’s
total output, tilapia became a truly "Waterfowl" [2,3]. According to statistics, after 1980, freshwater
fish, marine fish in the Streptococcus infection in a number of countries
have been reported in the outbreak of Streptococcus aureusin the world began widespread [4,5]. To date, the epidemic area of the disease has
covered freshwater fish and marine fish in 15 countries from six continents in
Europe, Africa, Australia, Asia and North Africa, South Africa [6]. Since 2008, China's southern tilapia farming area
began a wide range of outbreak of Streptococcusagalactiaedisease, the mortality rate
as high as 50% to 70%, 2009 Guangdong Province, breeding areas 7 to 11 months 4
months of StreptococcusagalactiaeInfection
caused more than 10,000 tons of tilapia died; in 2011, Guangxi breeding areas 5
months to 6 months in the absence of Streptococcusagalactiaeinfection caused more than
7,000 tons of tilapia deaths, economic losses of up to 8,000 million [7-11]. In recent years, Guangxi, Guangdong and other
regions due to the harm of tilapia without Streptococcus has had a lot to reduce the tilapia
farming, which caused the Chinese tilapia industry hit.
Streptococcusagalactiaeis seriously
harmful and highly contagious. At present, there is no commercial vaccine
against the prevention and treatment of tilapia streptococcal disease in China.
It can only rely on strict production management and antibiotic treatment.
Therefore, the establishment of rapid and effective disease Early diagnosis
methods and monitoring of the epidemic, timely warning and forecasting are
still the key technical problems to be solved at present. In order to provide
effective diagnostic and monitoring techniques for the production of tilapia-free
Streptococcusagalactiaein
production and to control the harm of tilapia without Streptococcusagalactiae in a timely
manner, we have previously established an immunogold gold for the detection of
tilapia-free StreptococcusagalactiaeRapid
diagnosis method [12], this study we use the
preparation of monoclonal antibody and colloidal gold technology to establish a
detection of tilapia anti-Streptococcusmutansantibody detection method, that
is, tilapia anti-Streptococcus
free antibody colloid gold infiltration Filtration, the establishment of the
law will provide a new method for on-site monitoring of tilapia-free Streptococcusmutans.
2. Materials and Methods
2.1. Materials and Reagents
S. agalactiaeand other
encounter bacteria, experimental animals and reagents. [12].
All chemicals were of grade, and used as received.
2.2.
Preparation of Monoclonal
Antibodies Against Tilapia Immunoglobulin IgM
The
preparationprocedureofmonoclonal antibodies against tilapia
immunoglobulin IgMis shown (Figure 1) below:
A
monoclonal antibody against tilapia immunoglobulin IgM was prepared by
conventional methods: tilapia was immunized with inactivated Streptococcusagalactiaeand
then immunized tilapia immunoglobulin IgM was extracted, precipitated with
saturated ammonium sulfate and purified by Protein-A column[12,13]. The purified tilapia immunoglobulin IgM was
used to immunize Balb / C mice. After three times of immunization and potency,
the mice were sacrificed with high titer of mouse spleen cells. A monoclonal
antibody hybridoma cell line with specificity for tilapia immunoglobulin IgM
was selected by semi-solid culture. Anti-tilapia immunoglobulin IgM monoclonal
antibody was prepared by conventional mouse ascites method.
2.3. Preparation of Colloidal Gold Suspension
[12,14] prepared
by the Frens method of Nano-colloidal gold, prepared by the colloidal gold
solution in the UV-visible scanning, identification of the preparation of
colloidal gold quality and size.
2.4. Preparation of the Colloidal Gold-Mab Conjugate
Anti-tilapia
immunoglobulin IgM monoclonal antibody labeled with prepared colloidal gold:
Determine the optimum pH and minimum protein content by a series of optimized
tests. Take 10 mL of colloidal gold solution, add the optimal amount of K2CO3,
add a 2K10 monoclonal antibody to the
magnetic stirrer while stirring, and add 20% 2C10
monoclonal antibody on the basis of optimization. Stir for 15 min, add the
newly prepared 10% BSA 1 mL, continue stirring for 15 min. Dispense,
centrifugation, 12000 rpm, 4°C
centrifugation 1 h. After centrifugation, remove the centrifuge tube, carefully
discard the supernatant, add 1% BSA PB buffer, wash, and then centrifuged once.
Finally, discard the supernatant, add the suspension, according to every 1 mL
of gold alloy gold solution by adding 100 μL
heavy suspension, that is concentrated 10 times. The activity of the labeled
antibody was examined by ELIA or direct spotting. Colloidal gold labeled
monoclonal antibody placed 4°Cpreservation,
cannot be frozen.
2.5.
Optimization of
the Optimum Amount of Coating and Gold Standard Solution
[12,15],
take nitrocellulose membrane (NC film), perforation, diameter 3 mm, inactivated
Streptococcusagalactiae,
bacteria concentration adjusted to 1.5 × 109-1.5
× 104 CFU, Hole, 2 μL
per hole. After drying at room temperature, wash with PBST 3 times and dry.
With 5% BSA, closed for 15 min; then PBST washed 3 times, dry, put 4°C save. Test, add positive negative serum 2μL / hole, 1 min, with PBST wash, and then add 2 μL / well labeled monoclonal antibody, 1 min, with
PSBT washing, positive that color, negative color. After optimization, the
labeled monoclonal antibody solution diluted with different times to detect,
optimize the amount.
2.6. Immuno-Colloidal Gold Infiltration Test Plate Assembly and Testing
The detection mechanism of immuno-colloidal gold infiltration test
plate is shown in (Figure 2)
below.
According
to the above optimal method in the NC film spot treatment, while the anti-mouse
IgG anti-mouse point, as the control point. The treated NC film is loaded into
2.5cm × 3cm. When assembling, the absorbent
paper is loaded on the bottom of the round plate, and then the NC film is
attached and the plate can be closed. A package consists mainly of a plate, a
bottle of gold standard liquid, a bottle of washing liquid composition.
Detection,
the spot to be added to the serum 2μL, serum
quickly diafied in the past, and then add the washing solution, the filtrate
after diafiltration, add the gold standard solution, the gold standard solution
after dosing and then add the washing liquid. 5min if positive, there are two
spots, if only a negative spot; blank control group does not appear spots, the
results sentenced to invalid.
2.7.
Sensitivity of
the Immune Colloidal Gold Filtration Assay
Take
a positive serum for serial dilution, with the establishment of colloidal gold
infiltration detection method to detect the final can detect the dilution. The
same positive serum was precipitated by saturated ammonium sulfate
precipitation method. The immunoglobulin was extracted and its protein content
was measured. Then, serial dilutions were carried out. The final dilution was
detected by the established colloidal gold infiltration test.
2.8.
Specificity of
the Immune Colloidal Gold Filtration Assay
The
strains of Pseudomonas aeruginosa and Pseudomonas aeruginosa were tested as
specific test strains, the immunoassay was used to detect the specificity of
immune colloid gold infiltration by cross-reaction with immunized serum of
various bacteria.
2.9.
Colloidal Gold
Diafiltration Assay (stability
of the immune colloidal gold filtration assay)
The
prepared colloidal gold leachate test strip was placed at 4°C and the test was taken weekly.
2.10.
Comparison of
Colloidal Gold Infiltration Detection with ELISA Method
The
sera of tilapia were collected and the OD values of these sera were measured by
ELISA. A total of 20 samples were tested by ELISA. The results were compared
with the colloidal gold infiltration method. The results were compared with the
methods of medical statistics [16], including
sensitivity, specificity, total consistency, etc. Related indicators.
3.
Results and Discussion
3.1.
Production of
mAbAgainst Tilapia Immunoglobulin IgM
A
number of hybridoma cell lines secreting anti-tilapia immunoglobulin
IgM-specific monoclonal antibody were obtained by monoclonal antibody
preparation. After identification of the biological characteristics of the
antibody secreted by these hybridoma cell lines, two high titer hybridoma cell
lines were obtained, named 2C10 and
4G6, respectively. The preparation of
anti-tilapia immunoglobulin IgM-specific monoclonal antibody lays the
foundation for further establishment of standardized detection methods.
In
addition, Wang Weifang et al.[17] immunized with
Bovine Serum Albumin (BSA) to identify the pathogens, and use rabbit
anti-turbot (Scophthalmus
maximus) multi-labeled colloidal gold, in the use of colloidal gold
immunochromatographic detection of fish antibody reported, the specific
anti-BSA antibody was detected in the sera of the turbot, and the sensitivity
was similar to that of the ELISA method. Our method is to prepare this
monoclonal antibody with colloidal gold by preparing anti-tilapia
immunoglobulin IgM-specific monoclonal antibody. Because the production of
monoclonal antibodies with hybridoma cells is stable and adaptable to scale and
standardization, it can provide a better basis for the popularization and
application of immunogolds.
3.2.
Immuno-Colloidal
Gold Infiltration Test
The
positive and negative sera were detected by the established immunogold gold
infiltration method according to the method described in 3.6 The results are
shown in (Figure 3).
The
result is positive appearance of two spots, negative only a spot, blank control
spots do not appear, sentenced to invalid. Immune colloidal gold infiltration
method compared with the conventional ELISA method, this method is very fast
detection, in a few minutes to determine the test results, but also on-site
testing, this field of rapid detection method is important for the producers.
The application value and urgent needs.
The
result is positive appearance of two spots, negative only a spot, blank control
spots do not appear, sentenced to invalid. Immune colloidal gold infiltration
method compared with the conventional ELISA method, this method is very fast
detection, in a few minutes to determine the test results, but also on-site
testing, this field of rapid detection method is important for the producers.
The application value and urgent needs.
3.3.
Sensitivity,
Specificity and Stability of the Immune Colloidal Gold Filtration Assay
The
results showed that the highest dilution ratio of serum was 256 times. Protein,
immune filtration method can detect the dilution factor is 64 times. The amount
of immunoglobulin extracted with the same amount of serum was white 0.8 mg. The
minimum detection limit of immune filtration was 1.2 ×
10-6 g / mL, which indicated that the
sensitivity of the antibody was similar to that of ELISA. Immunity colloidal
gold infiltration method and the commonly used ELISA method is also similar to
the detection principle, the difference is only the antibody markers are
different, ELISA method is to use enzyme labeled antibody, and immunogold gold
infiltration method is to use colloidal gold labeled antibody, so its Detection
of antibody sensitivity and ELISA method is similar, but the ELISA method
requires a microplate reader to read the data, and immune colloidal gold
infiltration method does not need to detect the instrument, according to the
reaction color to directly determine the test results. As the method is simple,
the producers do not need to have the expertise to use, only need a simple test
training can be used, the development and application of this technology will
be able to complex laboratory and professional testing process directly into a
simple scene Detection. As tilapia lactobacilli disease in the epidemic season
is usually a trend, the incidence of acute, so rapid on-site diagnostic
monitoring, for the early control of the disease to win valuable time.
Therefore, the establishment of immune colloidal gold infiltration method will
provide a strong technical support for the monitoring and control of Streptococcusagalactiae.
The immune
spheric gold permeation test plate was prepared by using N-Streptococcus, Streptococcusdavidianus, Aeromonashydrophila,
Pseudomonas
aeruginosa and the like as coated bacteria. The results showed that
the sera of Aeromonashydrophilaand
Pseudomonas
fluorescensdid not react with Streptococcusagalactiaeand did not react with Streptococcuslactis sera.
But found that, in turn, with dolphin Streptococcus serum and Streptococcusagalactiaedetection
when there is a certain response. Indicating that the establishment of the
immune colloidal gold infiltration method has been well tested for specificity,
but also needs to be further optimized to improve the specificity of detection
with dolphins.The results showed that the immunogold gold permeation test plate
could store at least 3 months at 4°C
for 4 months.
3.4.
Comparison with
the ELISA Test Results
Colloidal
gold leachate detection method and ELISA method comparison test results in (Table 1).
According
to the data in the table, refer to the method of medical statistics [16] calculated:
Specificity
= D / (B + D) × 100% = 92.8%; Sensitivity = A /
(A + C) × 100% = 83.3%; Positive Consistency = A / (A + C) × 100% = 83.3% (B +
D) × 100% = 7.6%; false negative = C / (A + C) × 100% = false negative = 16.6%.
Total
rate = (A + D) / (A + B + C + D) ×100% = 90%
The
experimental results show that the coincidence rate of the immune colloid gold
infiltration method and the conventional ELIA method is 90%, the reliability is
very high, and it has practical application value and deserves further
optimization and development.
4.
Conclusions
An
immuno-colloidal gold infiltration assay for the detection of tilapia-free Streptococcusagalactiaeantibodies
has been developed. This new method can be used for the production and clinical
monitoring of tilapia-free Streptococcusagalactiae. The results show that the
method has high sensitivity and high specificity, and it has the advantages of
fast, simple operation and field use compared with traditional detection
methods such as ELISA method. More optimization and testing work is still in
progress.
5. Acknowledgments
This work was supported by the Guangxi
Tilapia IndustrialScientists Innovative Team Grant [grant number: nycytxgxctd-08-02], theGuangxi fisheries Animal Husbandry and Veterinary Bureau ofScience [grant
number: GUIYUMUKE201528027],
and the Open Fund ofGuangxi Key Laboratory of Aquatic Genetic Breeding [grant
number: GXKL-AQUA-2014-0X (4)].
research article 
