CN112760294A

CN112760294A - Canine type I adenovirus monoclonal antibody/polyclonal antibody, double-antibody sandwich ELISA kit and application

Info

Publication number: CN112760294A
Application number: CN202011331361.6A
Authority: CN
Inventors: 朱言柱; 廉士珍; 孙杰; 张蕾; 邓效禹; 胡博; 闫喜军; 徐金凤
Original assignee: Institute Special Animal and Plant Sciences CAAS
Current assignee: Institute Special Animal and Plant Sciences CAAS
Priority date: 2020-11-24
Filing date: 2020-11-24
Publication date: 2021-05-07

Abstract

The invention discloses a canine adenovirus I monoclonal antibody/polyclonal antibody, a double-antibody sandwich ELISA kit and application. According to the invention, the mouse and the rabbit are respectively immunized by using the CAdV-1 as immunogen, and finally 1 strain of specific monoclonal antibody is obtained and the specific rabbit polyclonal antibody is prepared; further adopting the monoclonal antibody, the enzyme-labeled polyclonal antibody and the optimal reaction condition of screening to establish a double-antibody sandwich ELISA detection method. The double-antibody sandwich ELISA detection method established by the invention has better repeatability, sensitivity and specificity in batches and among batches. Compared with the RT-PCR method, the sensitivity of the double-antibody sandwich ELISA method is 93.75%, the specificity is 90.9%, and the coincidence rate is 92.86%, which shows that the CAdV-1 antigen double-antibody sandwich ELISA detection method established by the invention has good sensitivity, specificity and repeatability, and can be used for the clinical detection of CAdV-1.

Description

Canine type I adenovirus monoclonal antibody/polyclonal antibody, double-antibody sandwich ELISA kit and application

Technical Field

The invention relates to a canine adenovirus antibody, in particular to a canine adenovirus I monoclonal antibody/polyclonal antibody and a canine adenovirus I double-antibody sandwich ELISA detection kit constructed by the same, belonging to the field of ELISA of canine adenovirus I.

Background

Canine adenovirus (CAdV) is a double-stranded DNA virus, including type I and type II. Canine adenovirus type I (Canine adenovirus type 1, CAdV-1) mainly causes acute septicemic hepatitis (Canine) and encephalitis (bear and fox). The CAdV-1 is widely distributed all over the world, has strong pathogenicity and wide infection spectrum, and can cause lethal infection to wild carnivorous mammals (wolfs, brown bears, black bears, otters and the like) besides dogs and foxes.

CAdV can infect across species, not only can infect pet dogs, but also can infect wild animals such as wolves, suburbs, skunks, foxes, bears, lions, and the like. Infection with CAdV-1 has potential threat to domestic dogs and wild animals. Therefore, establishing an efficient and rapid detection method is a key measure for preventing and timely discovering epidemic situations.

Charaizizhu, et al, 1983, isolated CAdV-1 from dogs with infectious hepatitis for the first time in China, and established a systematic laboratory diagnostic method for CAdV-1 in 1990, confirming the ubiquitous presence of CAdV-1 in the Chinese canine industry (Charaizizhu, et al, 1984; Charaizhu, et al, 1990). The first fox source in China, CAdV-1, was isolated in 1989 by Dian Shihong et al, and CAdV-2, was isolated in bronchitis-affected dogs by Van quan et al, 1999; Dian Shi hong et al, 1990). In the following years, CAdV-1 was also detected in wild mammals such as Teddy bear, panda, tiger, etc. The CAdV-1 has strong infectivity and high lethality rate, and is one of important epidemic diseases influencing the development of the animal breeding industry of China (Fanqing water, etc. 2005; Liqin, etc. 2000; Liu Da Fei, etc. 2010). In recent years, although large-scale CAdV-1 outbreak does not occur in China, the situation of high infection rate and high outbreak rate which are continuously seen in most foreign countries is frequent, and the prevention and monitoring strength of CAdV-1 is still further enhanced.

Serum neutralization test (SN) is one of the traditional methods for detecting virus serum neutralizing antibodies, and SN has the advantages of strong sensitivity, good specificity, good stability and the like and is accepted by scholars at home and abroad (Loeffen, 2012; Whettone, 1988). SN has been used as a gold standard for CAdV serum antibody detection to assess the immune efficacy of CAdV vaccines and to monitor neutralizing antibody titers in the serum of immunized animals. Jianglili and the like establish a trace serum neutralization test aiming at the CAdV-1, and further optimize the detection effect of the traditional SN (Jianglili and the like, 2011). Due to the long detection period and the complicated operation of the SN method, the SN method is gradually replaced by other methods in large-scale epidemiological investigation.

The ELISA technique is to use a microplate made of an organic material as a solid carrier, to which proteins (antigens or antibodies) are coated, to which reaction substances are sequentially added, using the property that the organic material (e.g., polystyrene) can effectively adsorb substances such as proteins, and to which unbound components must be eluted each time, and finally, to perform color development and result determination through the interaction between a color developing solution and peroxidase (Engvall et al, 1971). ELISA detection methods play an important role in serological investigation and clinical diagnosis of CAdV-1. Zhang Haidan and Marigong et al established an early spot ELISA detection method for the detection of CIHV (Marigong et al, 1992; Zhang Haidan et al, 1991); zhenghaifa and Jianglili establish an indirect ELISA detection method for fox encephalitis (Jianglili et al, 2008; Zhenghaifa et al, 1992); the method establishes an indirect ELISA detection method aiming at the CAdV-2 by utilizing the purified CAdV-2 holovirus as an envelope antigen, such as Puyanhua and the like, has high sensitivity and specificity on the CAdV-2, but has no reported detection effect on the CAdV-1 (Puyanhua and the like, 2010); walker et al established an indirect ELISA detection method for CAdV based on the previous population using purified CAdV-1 and CAdV-2 cytotoxins as envelope antigens at the same time (Walker et al, 2016). In the traditional ELISA method using whole viruses as coating antigens, most of virus particles are obtained by ultracentrifugation, the concentration and purity of the viruses are low, and the sensitivity and specificity of the ELISA detection method are influenced to a certain extent.

Since the establishment of hybridoma technology in 1915, monoclonal antibodies (mabs) against viral structural proteins, bacteria, cytokines, and the like have been emerging. In 2015, the total amount of antibody drug trades in each country exceeds 850 billion dollars. The hybridoma technology can combine the advantages of B lymphocytes and myeloma cells, and the produced hybridoma cells have the amphiphilic function, can synthesize antibody cells and secrete specific antibodies, and can be proliferated indefinitely in vitro. With this technique, a large amount of antibody can be produced using only a small amount of antigen, and the concentration is high. Has obvious effect on the prevention and the detection of infectious diseases and has incomparable advantages compared with other methods. Furthermore, monoclonal antibodies do not require excessive antigen purity, and low purity antigens can produce highly specific antibodies.

Since the double antibody sandwich ELISA method uses two antibodies, the detection accuracy is higher. However, the establishment of the double-antibody sandwich ELISA method for detecting canine adenovirus I requires the preparation of canine adenovirus I monoclonal antibodies and polyclonal antibodies with strong specificity.

Disclosure of Invention

One of the purposes of the invention is to provide a specific canine adenovirus type I monoclonal antibody and a specific canine adenovirus type I polyclonal antibody;

the invention also aims to provide a double-antibody sandwich ELISA detection kit for detecting canine adenovirus I.

The above object of the present invention is achieved by the following technical solutions:

the invention firstly provides a hybridoma cell strain secreting canine I-type adenovirus antibody, which is named as CAdV-1-F1301-01A, and the microorganism preservation number of the hybridoma cell strain is CGMCC NO. 19661; preservation time: in 2020, 4 and 8 days, the preservation unit is: china general microbiological culture Collection center.

The invention further provides a specific canine adenovirus I monoclonal antibody, which is secreted by a hybridoma cell strain with the microorganism preservation number of CGMCC NO. 19661.

The invention further provides a canine adenovirus I polyclonal antibody, and the preparation method comprises the following steps: preparing a canine I type adenovirus polyclonal antibody by immunizing rabbits with canine I type adenovirus immunogen; more preferably, the preparation method comprises the following steps: after fully emulsifying the canine adenovirus I immunogen with Freund's complete adjuvant, healthy and clean-grade white rabbits were first immunized, and both immunization No. 2 and No. 3 were fully emulsified with Freund's incomplete adjuvant and immunogen, with the immunization dose of 500 μ g/rabbit, and the immunization interval of 14 days after subcutaneous multi-point injection on the back. And 3, collecting blood from the ear vein of the experimental rabbit 10 days after the immunization is finished, and measuring the serum titer to obtain the specific canine I-type adenovirus polyclonal antibody.

The canine I-type adenovirus monoclonal antibody and the canine I-type adenovirus polyclonal antibody provided by the invention can be applied to detecting canine I-type adenovirus, for example, the canine I-type adenovirus in a sample is detected by applying the canine I-type adenovirus monoclonal antibody by Western blot, immunofluorescence or ELISA detection methods and the like; or preparing an enzyme-labeled antibody by using HRP to mark the canine I adenovirus polyclonal antibody, and establishing the canine I adenovirus double-antibody sandwich ELISA detection method by using the obtained canine I adenovirus monoclonal antibody combined enzyme-labeled polyclonal antibody.

The coating of the canine adenovirus I monoclonal antibody on the ELISA plate is a crucial step in double antibody sandwich ELISA. When the coating concentration of the antibody is too low, the surface of the solid carrier cannot be completely covered, so that the amount of the antigen adsorbed by the captured antibody is insufficient, and false negative may occur; when too high a concentration of antibody coating is used, interaction between antibody molecules is easily caused. These antibodies are easily washed away during washing, affecting the sensitivity of the assay. If the concentration is too high, non-specific reaction is enhanced, resulting in too high a positive result, which affects the sensitivity of measurement. Therefore, screening the coating concentration of the antibody is very important for the established ELISA detection method.

The invention further provides a CAdV-1 antigen detection ELISA kit, which comprises: a primary antibody, an enzyme-labeled secondary antibody, an antibody diluent, a washing solution, a confining solution and a developing solution; wherein the primary antibody is a monoclonal antibody secreted by a hybridoma cell strain with the microorganism preservation number of CGMCC NO.19661, and the antibody is used as a coating antibody; the second antibody is a canine adenovirus type I polyclonal antibody, and the canine adenovirus type I polyclonal antibody is prepared by immunizing rabbits by using a CAdV-1 antigen as an immunogen; the enzyme label is marked by HRP.

The antibody diluent is preferably 0.5% BSA solution, the washing solution is preferably PBST buffer solution, the blocking solution is preferably 5% skim milk solution, the developing solution is preferably TMB substrate developing solution, and the stop solution is preferably 2mol/l concentrated sulfuric acid solution.

The invention further provides a method for detecting the CAdV-1 antigen in a sample by using the CAdV-1 antigen detection ELISA kit, which comprises the following steps:

(1) coating an enzyme label plate with the monoclonal antibody, standing overnight at 4 ℃, washing the plate for 3 times by PBST, adding 5% skim milk, and sealing for 2 h; (2) adding the antigen to be detected, incubating for 1h at 37 ℃, and washing the plate for 3 times by PBST; (3) adding diluted enzyme-labeled polyclonal antibody, and incubating for 1h at 37 ℃; (4) developing with TMB developing solution for 15min. After color development is stopped, detecting an OD value at 450nm by using an enzyme-labeling instrument; 30 CAdV-1 negative samples were tested, and the OD450 values of the 30 samples were recorded, by calculating the Mean value to be 0.269 and the standard deviation to be 0.032, and calculating the cut-off value for positive and negative samples (Mean +3SD) to be 0.366 according to the formula.

The invention discovers that the mouse source is obtained by a large number of screening testsThe optimal coating concentration of the CAdV-1 monoclonal antibody is 2.15 mu g/ml, and the optimal dilution multiple of the HRP-labeled rabbit-derived polyclonal antibody is 1: 2000, coating overnight at 4 deg.C, and selecting 5% skimmed milk as sealing liquid, wherein the optimal sealing time is 1 h. The optimal incubation time for the antigen is 1 h; the acting time of the enzyme-labeled antibody is 1 h; the positive-negative cutoff value of the sample was 0.366. The double-antibody sandwich ELISA method established by the parameters has relatively good repeatability no matter in batches or between batches, and can detect 100 TCIDs at minimum₅₀The virus of (1). When a plurality of viruses are used for detecting the method, only the CAdV-1 shows a positive result, which shows that the detection method has good specificity. Compared with the RT-PCR method, the sensitivity of the method is 93.75%, the specificity is 90.9%, and the coincidence rate is 92.86%.

According to the invention, a mouse and a rabbit are respectively immunized by using CAdV-1 as an immunogen, and a specific mouse-derived CAdV-1 monoclonal antibody and a specific rabbit-derived polyclonal antibody are respectively prepared; the obtained antibody is identified by Western blot, and the type of the monoclonal antibody is identified, so that 1 monoclonal antibody of IgG1 type and rabbit polyclonal antibody are obtained. The invention further utilizes HRP to mark rabbit source polyclonal antibody to prepare enzyme-labeled antibody. The obtained mouse-derived CAdV-1 monoclonal antibody and the enzyme-labeled antibody are adopted to establish a double-antibody sandwich ELISA detection method, and the optimal reaction conditions are searched and screened. The double-antibody sandwich ELISA detection method established by the invention has better repeatability, sensitivity and specificity in batches and among batches. Compared with the RT-PCR method, the sensitivity of the method is 93.75%, the specificity is 90.9%, and the coincidence rate is 92.86%, which shows that the CAdV-1 antigen double-antibody sandwich ELISA detection method established by the invention has good sensitivity, specificity and repeatability, and can be used for the clinical detection of CAdV-1.

Drawings

FIG. 1 CAdV-1 antigen; m, Marker; 1, BSA (0.5mg/ml), 2, CAdV-1(7mg/ml), 3, CAdV-1(7 mg/ml).

FIG. 2 Western blot identification of monoclonal and polyclonal antibodies.

FIG. 3 shows the screening results of the optimal coating conditions.

Figure 4 best blocking solution selection results.

FIG. 5 best block time screening results.

FIG. 6 determination of optimal incubation time.

Figure 7 optimal incubation time screening results for antibodies.

FIG. 8 Western blot analysis of monoclonal antibody results.

FIG. 9 results of immunofluorescence assays.

FIG. 10 results of tissue immunofluorescence assays.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

1. Experimental materials and statistical analysis methods

1.1 serum, strains, cells and vectors

Cell: MDCK cells are stored in the laboratory.

Strain: the CAdV-1F1301 strain, CDV and CPV antigens were stored in the inventors' laboratory.

Sample preparation: the inventor stores 70 CAdV-1 positive samples and negative samples in the laboratory.

1.2 Primary reagents and instruments

Fast pfu high-fidelity DNA polymerase and T₄DNA ligase (lnvitrogen, Inc.); His-Bind Purification Kit (Thermo Fisher Co.); HiTrap rProtein A FF: GE Healthcare 17-5079-01, Goat anti-Dog IgG (AbCom), 0.45 μm and 0.22 μm filters (MILLIPORE), IMDM medium, IMDM complete medium (15% serum), cell culture plates (Nest), 50ml and 15ml centrifuge tubes (Nest) and cell culture flasks (Corning).

37 ℃ constant temperature cell culture box (Thermo), gel imaging system (UK UVP), 96-well plate washer (Thermo), ImageQuant300 gel imager (GE), MDF-U32V-80 ℃ refrigerator (Thermo), ELISA plate with medium binding force (COSTAR corporation), protein electrophoresis apparatus (Bio-Rad), PCR apparatus type 070-, JY92-2D type sonicator (Ningbo Xinzhi Bio Inc.), high speed centrifuge (Beckman), Milli-Q II pure water instrument (Millipore), multifunctional microplate reader (Bio-Tek) for bacterial cell wall disruption.

Coupling buffer: 20mM sodium phosphate buffer, pH 7.0.

High salt coupling buffer (IgG 1): 20mM sodium phosphate buffer, 3M NaCl, pH7.0, pH 3.4.

Elution buffer: 0.1M sodium citrate buffer.

Neutralizing liquid: 1M Tris-HCl buffer, pH 9.0.

0.01M PBS buffer, pH 7.4.

1.3 statistical analysis

Data are expressed as mean ± standard deviation, with the same letter representing no difference, 1 letter different case representing a significant difference (P <0.05), and 2 letter different case representing a very significant difference (P < 0.01).

EXAMPLE 1 preparation of Canine type I adenovirus monoclonal/polyclonal antibodies

Test method 1

1.1 preparation of monoclonal antibodies

(1) CAdV-1 inactivated antigen immunization program

The CAdV-1 virus was concentrated using an ultrafiltration tube and the concentration was checked by SDS-PAGE. SPF-grade BALB/c female mice were used as test animals, and the primary immunization dose was 60. mu.g/mouse. Four consecutive immunizations, booster immunizations, identical doses each, were 30. mu.g/mouse.

(2) Establishment of Indirect ELISA method

To determine the serum titer of the immunized mice, indirect ELISA methods were established using a CAdV-1 plate. Plates were plated with 2. mu.g/ml CAdV-1 overnight at 4 ℃. Skim milk with a final concentration of 2% was used as the blocking solution, and the optimal blocking temperature was 37 ℃ for 2 h. Serum was diluted in a 2-fold gradient starting at 200 fold, and the blank control and negative control were PBS and negative serum diluted 200 fold, respectively, and incubated at 37 ℃ for 1 h. Adding TMB color development liquid for color development, and detecting an absorbance value (OD) value by an enzyme-labeling instrument at 450 nm.

(3) Cell fusion assay

SP2/0 cells were grown well, and the cells were transferred from the flask to a 50ml centrifuge tube using a pipette. The immunized mice were bled from the eyeballs and, after bleeding was completed, the mice were killed by pulling the neck and soaked in 75% alcohol for 5min. The cell sieve, plunger and a small amount of serum free IMDM were placed in a dish. The spleen of the mouse was removed and crushed on the cell sieve with a syringe, and the movement was carefully and gently performed to sufficiently crush the spleen. The obtained spleen cells were transferred to a centrifuge tube containing SP2/0 cells and centrifuged at 1500rpm/min for 5min. The thymus of the mice was removed and crushed with the inner core of the syringe. The resulting thymocytes were transferred to a 15ml centrifuge tube. After the supernatant was decanted, serum-free IMDM was pipetted into the centrifuge tube, the cells were resuspended and pipetted evenly, and centrifuged (1500r/min, 5 min). The supernatant was decanted, the cell resuspended at the bottom of the tube was tapped, the tube was placed in warm water at 37 deg.C, 1ml of PEG (completed within 1 min) was added slowly, and the tube was allowed to stand in warm water for 1 min. After completion of the standing, 2ml of serum-free IMDM was added to the centrifuge tube (completed within 2min, and attention was paid to slow movement), and finally 8ml of serum-free IMDM was added slowly (completed within 2 min). Centrifuge at 1000rpm/min for 5min. The supernatant was discarded, 10ml of serum was added, the cells were resuspended with a pipette and pipetted evenly and poured into the treated thymocytes. Adding 25ml of sterile semisolid culture medium into a centrifuge tube, uniformly mixing the cells and the culture medium in the centrifuge tube, and uniformly subpackaging the mixture into 30 cell culture dishes. The cell culture dishes were transferred to a wet box and incubated in an incubator.

(4) Screening for Positive hybridoma cells

And (3) picking the monoclonals on the semisolid culture medium to a 96-well culture plate, coating the plate with 'CAdV-1', and primarily screening the selected clones by using an ELISA method, wherein the result shows that 45 hybridoma cell strains are positive. The 14 positive cell lines were selected again by the above method, and the results showed that 5 of them were positive. Subclass identification shows that 3 IgG positive hybridoma cell strains are found in 5 positive cell strains. And (3) carrying out mass culture and cryopreservation on of the IgG positive hybridoma cell strains, wherein the results show that 1 cell strain is successfully cryopreserved.

(5) Preparation of ascites

1) The temperature of the constant temperature water bath box is adjusted to 37-40 ℃.

2) Taking out the required cell strain frozen in the liquid nitrogen tank, and immediately placing the cell strain in warm water at 37-40 ℃ to quickly shake until the liquid is completely dissolved.

3) And transferring the thawed cell freezing solution to a centrifuge tube, adding 5ml of complete culture medium into the centrifuge tube, and uniformly blowing and beating the mixture, wherein the action needs to be gentle.

4) The cell suspension in the centrifuge tube was centrifuged (1000rpm/min, 5min) and the supernatant was discarded.

5) And (3) resuspending the cell sediment at the bottom of the tube by using a complete culture solution, uniformly blowing, transferring the cell sediment into a cell culture dish, and culturing the cell sediment in a constant-temperature incubator at 37 ℃.

6) Cells in logarithmic growth phase were taken, washed with PBS and quantified: the amount of cells injected per mouse was 5X 10⁵-9×10⁵One per ml.

7) Paraffin oil was injected intraperitoneally 7 days earlier, and 500. mu.l was injected intraperitoneally into each Balb/c mouse.

8) Injecting the cell suspension into abdominal cavity of mouse, 1ml each, ensuring cell amount to 5 × 10⁵-9×10⁵One per ml.

9) The ascites of the mice were collected 7 days after the hybridoma cells were injected into the abdominal cavities of the mice.

10) The collected ascites fluid was centrifuged (5000rpm/min, 10min), and the supernatant was collected and stored at-20 ℃.

(6) Purification of antibodies

1) Sample pretreatment: diluted 1:3 with the corresponding coupling buffer, centrifuged at 4 ℃ (12000rpm/min, 10min) and the fat, disrupted cells and small particulate matter are filtered off using a 0.22 μm filter.

2) Balancing: the column was equilibrated with the corresponding 10 column volumes of coupling buffer, with a fixed flow rate of 1 ml/min.

3) Loading: the sample was injected into the column from the upper port at a fixed flow rate of 1ml/min and the effluent was collected.

4) And (3) column washing: 5 column volumes of coupling buffer were passed through the column at a fixed flow rate of 1 ml/min.

5) And (3) elution: the antibody was eluted at a fixed flow rate of 1ml/min and collected in an EP tube. The pH was immediately adjusted to 7.0 with 1M Tris-HCl buffer pH 9.0.

6) Balancing: the column pH was equilibrated to neutral using 10 column volumes of coupling buffer at a fixed flow rate of 1 ml/min.

And (3) dialysis: the antibody was dialyzed overnight against 0.01M PBS buffer and the solution was changed 3 times.

(7) Western blot identification

CAdV-1 infected MDCK cells were collected, mixed well with 6 Xprotein loading buffer using Total protein extraction kit (doctor Deg), boiled at 95 ℃ for 5min, and centrifuged. And (3) carrying out SDS-PAGE electrophoresis on the sample, transferring the protein onto a PVDF membrane after the electrophoresis is finished, blocking the PVDF membrane by using 3% BSA, and incubating for 2h at 37 ℃. The purified antibody and CAdV-1 standard positive serum (1: 500) were used as primary antibody, and the incubation conditions were 4 ℃ for 12 h. Goat anti-Dog IgG (HRP) (1: 5000 dilution) was used as secondary antibody at 37 ℃ for 1 h. And dripping ECL color developing solution, and detecting the strip by adopting a chemiluminescence imager.

(8) Subclass measurement method

The subclass of the antibody generated by the hybridoma cell is identified by adopting a monoclonal antibody subclass identification kit (IgG1\ IgG2a \ IgG2b \ IgG3\ IgM \ IgA) (Beijing Boolong immune technology Co., Ltd., BF 06001).

(9) Ascites titer determination

Add 100. mu.l of CAdV-1 antigen to each well and coat overnight at 4 ℃. Unbound antibody was washed away with PBST, 3% BSA was added, and incubated for 2h at 37 ℃ in an incubator. After PBST was washed to remove excess blocking solution, 100. mu.l of hybridoma supernatant was added to each well and incubated at 37 ℃ for 1 hour in a constant temperature incubator. PBST 3 times washing. Goat anti-Dog IgG (HRP) (1: 3000 dilution) was used as secondary antibody, 0.1ml per well, and incubated at 37 ℃ for 1 h. PBST washing 3 times; after 50. mu.l of TMB developing solution was added to each well and reacted for 10 minutes, OD (dual wavelength 450, 630) was measured and data was recorded.

1.2 preparation of polyclonal antibodies

(1) Immunizing animals

The test animals selected from New Zealand white rabbits of healthy and clean grade, all of which are 3 months old (3 rabbits are purchased from Changchun Dada animal center). The primary immune is fully emulsified with Freund complete adjuvant, the 2-immune and the 3-immune are fully emulsified with Freund incomplete adjuvant, the immune dose is 500 mu g/mouse, and the immune interval is 14d by subcutaneous multi-point injection on the back. The ear vein of the experimental rabbits was sampled 10 days after the end of the 3-immunization and the serum titer was measured. Comparing the titer of each group of serum, selecting the serum of the experimental rabbit with high titer, and carrying out subsequent experiments.

(2) Determination of polyclonal antibody titer

The polyclonal antibody titer was detected by indirect ELISA. CAdV-1 was added to the ELISA reaction plate with coating solution, 50. mu.l per well, and coated overnight at 4 ℃. After the coating solution is discarded, the plate is washed by PBST for 5 times continuously, the interval of each time is 5min, the reaction plate is tapped until no obvious water drop exists, the skim milk powder and the PBST are used for preparing a 5% sealing solution, 200 mu l of the sealing solution is added into each hole, and the sealing is carried out in a constant temperature incubator at 37 ℃ for 1 h. Incubating the primary antibody: before incubation of the primary antibody, the plate is washed by PBST for 5 times continuously and dried, rabbit source polyclonal antiserum (the dilution range is 1: 1000-1: 128000) is diluted by 5% skim milk to be used as the primary antibody, 50 mu l of diluted primary antibody is added into each hole, the primary antibody is incubated for 30min in a constant temperature incubator at 37 ℃, and the non-immune serum is adopted as a negative control. Wash plate 5 times and pat dry, remove excess PBST, dilute goat anti-rabbit IgG to 1: 5000, 50. mu.l of each well, adding into a 96-well plate, and reacting in a 37 ℃ incubator for 30 min. And (3) washing the plate by PBST for 5 times continuously, slightly patting the plate to remove redundant PBST, adding 50 mu l of TMB color development solution into each hole, and reacting for 10min in a dark place at room temperature. Add 25 μ l H₂SO₄(2mol/L) color development was stopped. OD was measured at 450nm using a microplate reader.

(3) Rabbit-derived polyclonal antibody (HRP) labeling

An HRP labeling kit (BF 06095-1000, Beijing Boolong immuno-technologies, Inc.) is adopted. The antibody was adjusted to a concentration of 2mg/ml, and 1/10 volumes of the labeling buffer were added and mixed well to avoid the formation of air bubbles. And (3) adding equal mass of horseradish peroxidase into the substance to be marked, fully and uniformly mixing to avoid generating bubbles, and reacting for 3 hours at room temperature in a dark place. An appropriate amount of the reaction termination solution was added to the labeled reaction tube at a ratio of 1. mu.l of the reaction termination solution per 10. mu.l of the reaction solution. Mixing well, standing at room temperature for 1 h. Adding marker preserving fluid with the same volume, mixing well, and preserving at-20 deg.C.

2. Test results

2.1 preparation of the CAdV-1 antigen

After CAdV-1 infection of MDCK, continuous passage and mass culture are carried out. The virus solution was concentrated by ultrafiltration tube to obtain 7mg/ml protein, as shown in FIG. 1, which was used for immunization.

2.2 serum titers

As shown in Table 1, the mice with the highest titer of CAdV-1-4# were selected for cell fusion experiments based on serum titers.

TABLE 1 results of serum titer detection in immunized mice

2.3 cell line screening results

14 positive cell lines were coated with CAdV-1 and screened again by ELISA to find 5 positive cell lines (Table 2).

TABLE 2 screening results of cell lines

2.4 subclass measurement results

Subclass identification was performed on 5 positive cell lines, of which 1 line was an IgG1 type hybridoma cell line (Table 3).

TABLE 3 identification of cell line subtypes

2.5 Western blot identification of monoclonal and polyclonal antibodies

The hybridoma cell strain with the cell number 5 is selected to carry out the experiment. Both the mouse-derived monoclonal antibody and the rabbit-derived polyclonal antibody can generate specific bands with the CAdV-1 infected cells collected at 0h, 12h and 36h (figure 2).

Test example 1 establishment of detection method for detecting canine adenovirus type I by double antibody sandwich ELISA and optimization of detection conditions

Establishment of CAdV-1 double antibody sandwich ELISA detection method

The monoclonal antibody was coated on an ELISA plate overnight at 4 ℃. PBST wash plate 3 times. Adding 5% skimmed milk, and sealing for 2 hr. 30 portions of the CAdV-1 negative sample to be detected were added and incubated at 37 ℃ for 1 h. PBST wash plate 3 times. Diluted enzyme-labeled polyclonal antibody is added, and incubation is carried out for 1h at 37 ℃. Developing with TMB developing solution for 15min. After the color development is stopped, an enzyme-linked immunosorbent assay (ELISA) instrument is used for detecting the OD value at 450nm

After the experimental conditions are optimized, 30 CAdV-1 negative samples are detected, and 30 sample OD (optical density) are recorded₄₅₀The cut-off value of (a) was calculated to be 0.366 by calculating the Mean value to be 0.269 and the standard deviation to be 0.032 and calculating the cut-off value of a positive and negative sample (Mean +3SD) according to the formula.

1.2 optimization of reaction conditions for CAdV-1 double antibody sandwich ELISA

(1) Optimum concentration screening of coated antibody and enzyme-labeled antibody

Monoclonal antibodies were diluted to 7 concentrations of 17. mu.g/ml, 8.6. mu.g/ml, 4.3. mu.g/ml, 2.15. mu.g/ml, 1.075. mu.g/ml, 0.5375. mu.g/ml, 0.26. mu.g/ml using a matrix titration method, and 96-well plates were coated with 1: 500,1: 1000,1: 2000,1: 4000,1: enzyme-labeled antibody was diluted at a dilution of 8000. Different gradients of monoclonal antibodies were coated on the elisa plate overnight at 4 ℃. PBST wash plate 3 times. Adding 5% skimmed milk, and sealing for 2 hr. Antigen was added and incubated at 37 ℃ for 1 h. PBST wash plate 3 times. Enzyme-labeled secondary antibodies of different dilutions were added and incubated at 37 ℃ for 1 h. Developing with TMB developing solution for 15min. After the color development is stopped, an enzyme-labeling instrument is adopted to detect the OD value at 450nm, and the optimal dilution of the coating antibody and the enzyme-labeled antibody is determined.

(2) Screening for optimum coating time

Coating the ELISA plate according to the optimal antibody coating concentration determined by the method (1). The coating time is 2h, 3h, 4h and 4 ℃ overnight respectively. Antigen was added and incubated at 37 ℃ for 1 h. PBST wash plate 3 times. The optimal concentration of enzyme-labeled antibody determined according to method (1) was incubated at 37 ℃ for 1 h. Developing with TMB developing solution for 15min. After the color development is stopped, an enzyme-labeling instrument is adopted to detect the OD value at 450nm, and the optimal coating time of the antibody is determined.

(3) Selection of confining liquids

Coating the ELISA plate according to the optimal monoclonal antibody coating concentration determined by the method (1) and the optimal coating time determined by the method (2). 3% BSA, 1% gelatin, 1% casein and 5% skim milk were selected as blocking solutions and allowed to react at 37 ℃ for 2 h. Antigen was added and incubated at 37 ℃ for 1 h. PBST wash plate 3 times. The optimal concentration of enzyme-labeled antibody determined according to method (1) was incubated at 37 ℃ for 1 h. Developing with TMB developing solution for 15min. After the color development is stopped, an enzyme-labeling instrument is adopted to detect the OD value at 450nm, and the most suitable confining liquid is determined.

(4) Optimizing seal time

Coating the ELISA plate according to the optimal monoclonal antibody coating concentration determined by the method (1) and the optimal coating time determined by the method (2). According to the optimal sealing liquid determined by the method (3), 0.5h, 1h, 1.5h and 2h are selected for sealing at 37 ℃. Antigen was added and incubated at 37 ℃ for 1 h. PBST wash plate 3 times. The optimal concentration of enzyme-labeled antibody determined according to method (1) was incubated at 37 ℃ for 1 h. Developing with TMB developing solution for 15min. After the color development is stopped, an enzyme-labeling instrument is adopted to detect the OD value at 450nm, and the most suitable sealing time is determined.

(5) Determination of incubation time for antigen

Coating the ELISA plate according to the optimal monoclonal antibody coating concentration determined by the method (1) and the optimal coating time determined by the method (2). And (3) adopting the optimal sealing liquid determined by the method (3), the optimal sealing time determined by the method (4) and sealing at 37 ℃. Antigen was added and incubated at 37 ℃ for 0.5h, 1h, 1.5h and 2 h. PBST wash plate 3 times. The optimal concentration of enzyme-labeled antibody determined according to method (1) was incubated at 37 ℃ for 1 h. Developing with TMB developing solution for 15min. After the color development is stopped, an enzyme-labeling instrument is adopted to detect the OD value at 450nm, and the optimal antigen incubation time is determined.

(6) Enzyme-labeled antibody reaction conditions

Coating the ELISA plate according to the optimal monoclonal antibody coating concentration determined by the method (1) and the optimal coating time determined by the method (2). The optimal sealing liquid determined by the method (3), the optimal sealing time determined by the method (4) and sealing at 37 ℃. The optimal antigen incubation time determined according to method (5), antigen was incubated at 37 ℃. PBST wash plate 3 times. The optimal enzyme-labeled antibody concentration was determined according to method (1), and incubated at 37 ℃ for 0.5h, 1h, 1.5h, and 2 h. Developing with TMB developing solution for 15min. After the color development is stopped, an enzyme-labeling instrument is adopted to detect the OD value at 450nm, and the optimal incubation time of the enzyme-labeled antibody is determined.

(7) Determination of positive and negative cutoff values

30 CAdV-1 negative samples are detected by adopting an optimized double-antibody sandwich ELISA method, the average value (Mean) and the Standard Deviation (SD) of OD450 of the 30 samples are calculated, and the critical value of the positive and negative samples is calculated according to a formula (the positive and negative critical value is Mean +3 SD).

(8) Repeatability test

And (3) repeatability experiment between plates: and (3) detecting 10 parts of positive samples and 2 parts of negative samples by using the same batch of the purified enzyme label plate coated by the CAdV-1 monoclonal antibody under optimized experimental conditions, performing 3 parallel controls on each sample, and analyzing the obtained data to obtain the intra-batch difference value. In-plate repeatability experiments: coating the ELISA plate with CadV-1 monoclonal antibodies purified in different batches, detecting 10 parts of positive samples and 2 parts of negative samples under optimized experimental conditions, performing 3 parallel controls on each sample, and performing statistical analysis on the obtained data to obtain the batch difference value. Coefficient of Variation (CV) × (standard deviation/average) × 100%.

(9) Sensitivity test

Diluting the CAdV-1F1301 strain by 10 times according to the screened ELISA reaction conditions, setting 3 times for each dilution, setting negative and positive controls for ELISA detection, and determining the maximum dilution times of the detectable CAdV-1 positive samples according to the detection results so as to compare the sensitivity of the two detection methods.

(10) Specificity test

And (3) detecting Canine Distemper Virus (CDV) and canine parvovirus (MEV) by adopting an optimized double-antibody sandwich ELISA method. 3 replicates were set for each sample, with CAdV-1 as the positive control and PBS as the negative control. The OD value was measured at 450nm using a microplate reader. And (4) judging whether the detected sample is positive or negative by comparing the OD450 value of the sample with the critical value.

(11) Comparative test of double-antibody sandwich ELISA detection method and RT-PCR

Respectively adopting RT-PCR and optimized CAdV-1 double-antibody sandwich ELISA methods to detect 70 samples stored in the laboratory, and calculating the specificity, coincidence rate and sensitivity of the detection results of the CAdV-1 double-antibody sandwich ELISA method and the RT-PCR method.

2. Test results

2.1 optimal dilution concentrations of Capture antibody and enzyme-labeled antibody

The optimal coating concentration of the mouse-derived CAdV-1 monoclonal antibody is 2.15 mu g/ml, and the dilution multiple of the HRP-labeled rabbit-derived polyclonal antibody is 1: 2000, the P/N value at this time was 4.219 (Table 4).

TABLE 4 optimal working concentration of antibody

2.2 determination of optimal coating conditions

Through experiments, as shown in the following table 5, compared with the ELISA plates coated for 4h, 3h and 2h, the P/N value of the ELISA plate detection sample coated overnight at 4 ℃ is higher. Therefore, the coating conditions were determined to be 4 ℃ overnight (table 5, fig. 3).

TABLE 5 optimal coating conditions

2.3 determination of optimal blocking fluid and optimal blocking time

Comparing the blocking effect of 5% skim milk, 3% BSA, 1% casein and 1% gelatin in the same blocking time, the result shows that the P/N value of the detected sample is the largest and reaches 10.49 when the sample is blocked by 5% skim milk. Therefore, 5% skim milk showed the best blocking effect (table 6, fig. 4), with the best blocking time being 1h (table 7, fig. 5).

TABLE 6 selection of optimal blocking solution

TABLE 7 optimal blocking time screening

2.4 determination of incubation time of samples

As shown in table 8 and fig. 6, the optimal incubation time for the antigen was 1 h.

TABLE 8 determination of optimal incubation time

2.5 optimal action time screening of enzyme-labeled antibodies

As shown in Table 9 and FIG. 7, the P/N value increased and then decreased within 0.5h-2h as the incubation time of the enzyme-labeled antibody increased. At 1h, the P/N value reached the highest. Therefore, the optimal incubation time for the enzyme-labeled antibody was 1h (Table 9, FIG. 7).

TABLE 9 optimal time of action screening for antibodies

2.6 determination of Positive and negative cutoff values

2.7 repeatability test

2.7.1 results of the batch-to-batch repeatability experiment

5 ELISA plates of different batches are taken, CAdV-1 is added, 3 holes of negative control and blank control are set simultaneously, and the result shows that the batch variation coefficient is lower than 10 percent and the batch repeatability is better (Table 10).

TABLE 10 repeatability results Table between lots

2.7.2 results of the in-batch reproducibility experiment

5 ELISA plates of the same batch are taken, CAdV-1 is added, and a blank control and a negative control are arranged at the same time. As shown in table 11, the coefficient of variation was less than 5% within a batch, with better batch repeatability.

TABLE 11 results of the in-batch repeatability experiments

2.8 sensitivity test

Setting TCID50 to 10⁸According to 10, CAdV-1 of^-1，10^-2，10^-3，10^-4，10^-5，10^-6， 10^-7Diluting, testing the sensitivity of the established double-antibody sandwich ELISA detection method, and the result shows that the highest dilution multiple is 10^-6100 viruses of TCID50, the results are shown in Table 12.

TABLE 12 sensitivity of the double antibody sandwich ELISA detection method

2.9 specificity test

The result is verified to show that only CAdV-1 is positive, which shows the good specificity of the method, and the result is shown in the table (Table 13).

TABLE 13 specificity of the double antibody sandwich ELISA detection method

2.10 percent coincidence of double-antibody sandwich ELISA result and RT-PCR result

In order to determine the accuracy of the double-antibody sandwich detection method, the RT-PCR method is used for verifying the double-antibody sandwich ELISA method. The results showed that of the 70 samples, 47 positive samples and 23 negative samples were detected by the double antibody sandwich ELISA method, while 48 positive samples and 22 negative samples were detected by RT-PCR. 47 copies of both were detected, 22 copies of negative, 93.75% sensitivity, 90.9% specificity, and 92.86% agreement (Table 14). Embodies the better accuracy of the method and can be used for detecting clinical samples.

TABLE 14 coincidence rate of double antibody sandwich ELISA results with RT-PCR results

Test example 3 application test of monoclonal antibody

Western blot detection

1. Collecting protein samples

2. Electrophoresis

(1) SDS-PAGE gel formulation

(2) Sample processing

An appropriate amount of concentrated SDS-PAGE protein loading buffer was added to the collected protein samples. Heating at 100 deg.C or boiling water bath for 3-5 min to fully denature protein.

(3) Sample loading and electrophoresis

And cooling to room temperature, and directly loading the protein sample into the SDS-PAGE gel sample loading hole. Typically, the electrophoresis is stopped when bromophenol blue reaches near the bottom end of the gel.

3. Rotary film

PVDF membrane is selected. The membrane was transferred by a semi-dry transfer apparatus from Bio-Rad at 24V for 15min.

4. Sealing of

And immediately placing the protein membrane into a prepared TBST after the membrane is transferred, and rinsing for 1-2 minutes to wash off the membrane transfer liquid on the membrane. Adding 5% skimmed milk powder sealing solution, and sealing at 4 deg.C overnight.

5. Primary antibody incubation

Antibodies were raised according to 1: 100 dilution primary antibody. TBST wash for 5-10 minutes. The total number of washes was 3.

6. Incubation with secondary antibody

Goat anti-mouse secondary antibody, as 1: diluted 2000, incubated 1 hour at room temperature and washed 5-10 minutes with TBST.

7. Protein Detection (Detection of proteins)

ECL developing solution is added dropwise, a chemiluminescence apparatus is adopted for developing, protein is detected, and the detection result is shown in figure 8.

II, immunofluorescence

1. Preparing a cell plate and a cell slide:

1) the cell density is about 90-95%, the cells are digested by preheated pancreatin, then the cells are resuspended in a complete culture medium, and the cells are fully blown to form a single cell suspension and counted.

2) Taking a 12-hole cell culture plate, placing a climbing piece in each hole, dripping a few drops of culture medium at the position where the climbing piece is to be placed in each hole according to the size of the climbing piece, then placing the climbing piece on the liquid drop, and compressing the climbing piece to enable the climbing piece and a culture dish to be adhered together by the tension of the culture medium, so that the climbing piece is prevented from floating when a cell suspension is added, and a double-layer cell patch is caused. According to the needs, the cells with proper density are planted in the 12-hole plate culture plate.

3) After 24h, the cell density is observed and judged according to the growth speed of the cells, and cell immunofluorescence is carried out when the cell density is about 90 percent.

2. Fixation and immunofluorescence of cells

1) The medium was aspirated off and the cells were washed 3 times for 5min each time, typically with PBS.

2) To the well was added 1ml of 4% paraformaldehyde, and cell fixation was performed at room temperature for 20 minutes.

3) Paraformaldehyde was aspirated and washed 3 times with PBS for 5min each.

4) 0.5% Triton X-100 (in PBS) was added to the wells and allowed to permeate for 20min at room temperature in order to allow the cells to permeate.

5) Triton X-100 was removed and washed 3 times with PBS for 5min each.

6) Blocking was performed with 10% goat serum (PBS) or 5% BSA homologous to the secondary antibody for 2 hours (selected blocking was consistent with antibody dilutions during subsequent manipulations). No washing with PBS was required after blocking.

7) The blocking solution is aspirated, a sufficient amount of primary antibody with an appropriate concentration is added dropwise to each well (the concentration of the antibody can be recommended according to the antibody specification for the first time, and the appropriate concentration of the antibody can be groped in subsequent experiments), and the mixture is incubated overnight in a wet box at 4 ℃.

8) Primary antibody was aspirated and washed 3 times with PBS for 5min each.

9) Sufficient secondary antibody with appropriate concentration is added dropwise into the wells, and incubated at 37 ℃ for 1 hour at room temperature in the dark. Note that the secondary antibody is labeled with fluorescein, so the procedure is performed in the dark as much as possible.

10) The secondary antibody was aspirated and rinsed 3 times with PBS for 5min each.

11) Dropping DAPI or Hoechst on the glass slide to counterstain the cell nucleus, which is generally blue fluorescence; incubating for 5-10min in dark.

12) The cells were washed gently with PBS 3 times for 5min each, and excess DAPI was washed off.

13) When the climbing sheet is taken, the climbing sheet is tightly combined with the bottom of the culture dish, the tension is high, the needle tip of the syringe needle can be hooked with a small hook towards the back, and the climbing sheet is hooked slightly and taken out by a small forceps.

14) And (3) absorbing the liquid on the slide by using absorbent paper, sealing the slide by using a sealing liquid containing an anti-fluorescence quencher, attaching the slide to a polylysine glass slide in a reverse mode, observing and collecting images under a fluorescence microscope, and selecting an excitation light source corresponding to the antibody.

The results are shown in FIG. 9.

Tissue immunofluorescence assay

1. Wet paper towels are spread at the bottom of a glass slide box to form a humidifying box, the glass slide with the slices is taken out by a freezing section instrument and put into a humidifying box (6 slices are put at each side), and the glass slides in the humidifying box do not contact with each other.

2. When the slides were wet and not dry in the chamber, PBS was applied to the sections (no overflow slide).

3. The diluted primary antibody was centrifuged at 13500 g for 2min at 4 ℃ in a microcentrifuge (40-50. mu.l antibody per slide should cover the section).

4. Using a Pasteur pipette connected to a pump, the PBS on the slide was aspirated off at one end of the section and the antibody was added from the other end, the humidified box was covered, and incubated at room temperature for 1 h.

5. The slides were washed 3 times with PBS (5 min/time), fresh PBS buffer was added from one end of the section, and old buffer was aspirated from the other end.

6. The diluted secondary antibody was centrifuged at 13500 g for 2min at 4 ℃ using a microcentrifuge (40-50. mu.l antibody per slide can be added).

7. The secondary antibody was added to the slides and incubated in a humidified chamber at room temperature for 1h, and the slides were washed 3 times with PBS (5 min/time).

8. Place the coverslip on a paper towel and drop 1 Gelvatol into the center of the coverslip. The cover glass was placed on the cover glass (without pressure), the slide was placed on a bench top, covered with aluminum foil and left in the dark for 30min to allow Gelvatol to set.

9. Observing the result under a microscope, or storing the result in a slide box at 4 ℃ in a refrigerator.

The results are shown in FIG. 10.

Claims

1. A hybridoma cell strain secreting canine I-type adenovirus antibody is characterized in that the microorganism preservation number is CGMCC NO. 19661.

2. A canine adenovirus type I monoclonal antibody secreted by the hybridoma cell line of claim 1.

3. The canine adenovirus I polyclonal antibody is characterized in that the preparation method comprises the following steps: the rabbit is immunized by the canine I type adenovirus immunogen, and the specific canine I type adenovirus polyclonal antibody is prepared by collecting rabbit serum.

4. The polyclonal canine adenovirus type I antibody according to claim 3, which is prepared by a method comprising: fully emulsifying the canine adenovirus I immunogen with Freund complete adjuvant, then fully emulsifying healthy clean-grade white rabbits with Freund incomplete adjuvant for 2-immunization and 3-immunization, wherein the immunization dose is 500 mu g/rabbit, injecting subcutaneous multi-point on the back, and the immunization interval is 14 d; and (3) collecting blood from the ear vein of the experimental rabbit 10 days after the 3-day immunization and measuring the serum titer to obtain the canine I type adenovirus polyclonal antibody.

5. Use of the monoclonal antibody against canine adenovirus type I according to claim 2 for the preparation of a reagent for diagnosing or detecting canine adenovirus type I.

6. Use of the polyclonal canine adenovirus type I antibody according to claim 3 or 4 for the preparation of a reagent for diagnosing or detecting canine adenovirus type I.

7. A dog type I adenovirus double-antibody sandwich ELISA detection kit is characterized by comprising: a primary antibody, an enzyme-labeled secondary antibody, an antibody diluent, a washing solution, a confining solution and a developing solution; wherein the primary antibody is the canine adenovirus type I monoclonal antibody of claim 2; the secondary antibody is the canine adenovirus type I polyclonal antibody of claim 3 or 4.

8. The canine adenovirus type I double-antibody sandwich ELISA detection kit of claim 7, wherein the blocking solution is a 5% skim milk solution.

9. The dog type I adenovirus double antibody sandwich ELISA detection kit of claim 7, wherein the antibody diluent is 0.5% BSA solution, the washing solution is PBST buffer solution, the color developing solution is TMB substrate color developing solution, and the stop solution is 2mol/l concentrated sulfuric acid solution.

10. The canine type I adenovirus double-antibody sandwich ELISA detection kit according to claim 7, wherein the dilution factor of the secondary antibody is 1: 2000.