WO2018103630A1

WO2018103630A1 - Hybridoma cell strain c1 for secreting anti-paromomycin monoclonal antibody and use thereof

Info

Publication number: WO2018103630A1
Application number: PCT/CN2017/114598
Authority: WO
Inventors: 徐丽广; 陈燕妮; 胥传来; 匡华; 刘丽强; 宋珊珊; 吴晓玲; 郑乾坤
Original assignee: 得利斯集团有限公司; 江南大学
Priority date: 2016-12-06
Filing date: 2017-12-05
Publication date: 2018-06-14
Also published as: CN106520705B; CN106520705A

Abstract

Disclosed are a hybridoma cell strain C1 for secreting an anti-paromomycin monoclonal antibody and the use thereof, belonging to the technical field of immunochemistry. A murine monoclonal cell strain C1 has been prepared through the conventional cell fusion technologies and deposited in the China General Microbiological Culture Collection Centre, and the deposit number is CGMCC No. 12025. The monoclonal antibody secreted by the cell strain is determined by the indirect competitive enzyme-linked immunosorbent assay, and the IC50 to paromomycin and neomycin is 0.61 and 2.43 μg/L respectively. The range of the recovery rate of paromomycin and neomycin in animal foodstuff is 64.56-105.85% and 54.08-100.55%. The hybridoma cell strain C1 provides a raw material for the immunological detection of the paromomycin residue in animal foodstuff, and has a practical application value.

Description

Hybridoma cell line C1 secreting anti-paromomycin monoclonal antibody and application thereof

Technical field

The invention relates to a murine hybridoma cell line C1 and a secreted monoclonal antibody capable of recognizing paromomycin, which can be used for detecting the residue of paromomycin in food, and belongs to the technical field of immunochemistry.

Background technique

Paromomycin is a broad-spectrum antibiotic that is secreted by Streptomyces. Paromomycin is structurally very similar to neomycin, the only difference being that the 6-amino group on the neomycin structure is replaced by a hydroxyl group. The antibacterial spectrum of paromomycin is similar to kanamycin, it has a strong killing effect on amoeba, and also has antibacterial activity against some protozoa, including: leishmania, dysentery amoeba, cryptosporidium insect. Clinically, paromomycin is used to treat amebiasis, giardiasis and leishmaniasis. In the livestock industry, it is used to treat bacterial infections, including trichomoniasis in turkeys in poultry.

In the livestock industry, paromomycin is added to the feed as a feed additive, thus creating some antibiotic resistance problems. In the early years, studies have shown that low doses of paromomycin can also cause intestinal bacterial resistance and even resistance to other aminoglycoside antibiotics. Therefore, the regulation of the use of paromomycin in the aquaculture industry and the supervision of the residue of paromomycin in edible animal foods are imminent. This also requires highly sensitive, high-throughput detection technology to achieve regulation.

In food safety testing, chromatographic methods are widely used, and liquid chromatography has been used to detect paromomycin in food. However, like other aminoglycoside antibiotics, paromomycin has no UV absorption. This is because there is no chromophore in its chemical structure and the chromatographic performance in reversed phase liquid chromatography is very poor. Moreover, the chromatographic method is expensive, the pretreatment process is complicated, and the detection process takes a long time. Therefore, the detection of paromomycin in food requires a faster, higher-throughput assay instead. Enzyme-linked immunosorbent assay (ELISA) and colloidal gold chromatographic strips have accelerated the development of rapid food safety detection techniques. They are simple, fast, specific, sensitive, inexpensive, and easy to prepare before the sample. Most aminoglycoside antibiotics have corresponding ELISA methods, but there is no ELISA for paromomycin.

Summary of the invention

The object of the present invention is to develop a monoclonal antibody against paromomycin and to establish an ELISA method based thereon. This invention includes two aspects: preparation of monoclonal antibodies and their applications

Anti-paromomycin monoclonal antibodies are obtained by immunizing mice with a paromomycin immunogen. The immunogen was synthesized by the glutaraldehyde method, and the coating was synthesized by the carbodiimide method. Screening seropositive and highly inhibited mice for spleen and cell fusion Hehe. After the fusion test, the cell line with high positive and good inhibition of paromomycin was subcloned three times to obtain a pure cell strain. Ascites was prepared by injecting a cell strain into the peritoneal cavity of a mouse that had been subjected to paraffin oil, and the obtained ascites was purified by a caprylic acid-ammonium sulfate method. The recognition of the monoclonal antibody against the structural analog of paromomycin was determined, and the cross-reaction rate was calculated. A sample addition recovery experiment was performed to calculate the recovery rate.

The technical scheme of the present invention: a hybridoma cell line C1 secreting anti-paromomycin monoclonal antibody has been deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the preservation number is CGMCC No. 12025.

The anti-paromomycin monoclonal antibody was secreted by the hybridoma cell line C1 and had an IC ₅₀ of 0.61 μg/L.

The use of the anti-paromomycin monoclonal antibody is in the application of the detection of paromomycin residues in food.

The beneficial effects of the present invention: the monoclonal antibody secreted by the cell line C1 is determined by indirect competitive enzyme-linked immunosorbent assay, and the IC _{50 of} paromomycin and neomycin is 0.61 and 2.43 μg/L, respectively. The recovery rate of paromomycin and neomycin in animal foods ranges from 64.56% to 105.85% and 54.08% to 100.55%. It provides raw materials for the immunoassay of paromomycin residues in animal foods, and has practical application. value.

DRAWINGS

Figure 1. Standard curve of monoclonal antibody secreted by monoclonal cell line C1 against paromomycin and neomycin.

detailed description

The following examples of the invention are intended to be illustrative only and not to limit the scope of the invention. The invention is further illustrated by the following examples.

Example 1: Immunogen and coating original synthesis

The immunogen was coupled by the glutaraldehyde method: 31.96 mg of paromomycin was dissolved in 4 mL of a 0.01 M phosphate buffer solution, and 200 μL of a 1% aqueous glutaraldehyde solution was added and stirred for 15 min. 20 mg of bovine serum albumin was dissolved in 2 mL of 0.01 M phosphate buffer solution, reacted at 4 ° C for 1 h, and the reaction was stopped by adding an appropriate amount of sodium borohydride, and incubated at 4 ° C for 2 h. The reaction mixture was freed of dialysis to remove the free paromomycin to obtain an immunogen. The coating was originally synthesized by a carbodiimide method: 10 mg of ovalbumin was dissolved in 1 mL of a pH 4.7 0.1 M MES (morpholine ethanesulfonic acid) buffer solution, 2 mg of EDC and 1.2 mg of NHS were added, and reacted at room temperature for 2 h. 19.03 mg of paromomycin was dissolved in 1 mL of 0.05 M KH ₂ PO ₄ , and the activated protein solution was slowly added dropwise to the paromomycin solution, and reacted at room temperature for 3 hours. The mixture after completion of the reaction was dialyzed against 0.01 M PBS to obtain a pure coating.

Example 2: Mouse immunization

The immunogen was diluted with physiological saline to a suitable concentration, and then emulsified with an equal volume of Freund's adjuvant, and multi-point injection was performed on the back of 6-8 week old BALB/c mice. For the first immunization, Freund's complete adjuvant was used at a dose of 100 μg. The next four boosters were Freund's incomplete adjuvant, with an immunization dose of 50 μg and an immune interval of 21 days. After the third immunization, blood was collected from the tail vein of the mouse, and serum was detected by an indirect ELISA. High titer of screening titers after the fifth immunization A good mouse was immunized with abdominal spurt, and the spleen of the mouse was taken out for cell fusion three days later.

Example 3: Cell fusion and screening

Mouse spleen cells and tumor cells were fused under the action of PEG 1500 to form hybridoma cells. On the seventh day after the fusion, the detection was performed, and the cell line with high titer and good recognition of paromomycin was subcloned, and so on, 5 subclones were obtained to obtain a pure cell strain.

Example 4: Purification of monoclonal antibodies

After the selected cell lines were expanded, they were injected into the peritoneal cavity of mice that had been beaten with paraffin oil, and ascites could be taken from the abdominal cavity of mice after 7 to 14 days. Ascites was purified by octanoic acid-ammonium sulfate method and stored at -20 °C.

Example 5: Properties of monoclonal antibodies

The sensitivity and specificity of the antibodies were determined by indirect ELISA.

1. Crossover of monoclonal antibodies

The coated PR-EDC-OVA was diluted with 0.05 M pH 9.6 carbonate buffer, 100 μL/well, and reacted at 37 ° C for 2 h. The original coating concentration of 0.8 (μg/mL) and the monoclonal antibody concentration of 0.6 (μg/mL) were determined by the checkerboard method. The solution in the plate was decanted, dried, and washed 3 times with the washing solution PBST for 3 min each time. After pat dry, 200 μL/well blocking solution was added and reacted at 37 ° C for 2 h. Wash and dry for use. 50 μL of standard solution and 50 μL of antibody were added to each well of the plate, and the cells were incubated at 37 ° C for 30 min, washed three times, and then patted dry. Each well was added with 1.3000 dilution of secondary antibody 100 μL, and cultured at 37 ° C for 30 min and then washed 4 times. After patted dry, 100 μL of the prepared TMB chromogenic solution was added, cultured at 37 ° C for 15 min, and finally 50 μL of 2 M sulfuric acid solution was added to each well to terminate the reaction. The absorbance at 450 nm was measured with a microplate reader.

Cross-over experiments with monoclonal antibodies determined other aminoglycoside antibiotics: Kanamycin, Streptomycin, Neomycin, Gentamycin, Apramycin ( Apramycin), Amikacin, Lincomycin, Spectinomycin and Tobromycin. The cross-reaction rate is the ratio of the IC50 of the analog to the paromomycin IC50. The crossover experiment is shown in Table 1.

Table 1. Cross of C1 monoclonal antibodies

2, add recycling experiment

Paromomycin and neomycin were added to Bovine Milk, Whole Egg, and Swine Liver, respectively, and detected by monoclonal antibodies to calculate the recovery. See the table 2 for the addition of recycling experiments.

Table 2. Addition recovery experiment

Solution preparation:

Carbonate buffer (CBS): Weigh 1.59g of Na ₂ CO _{3 and} 2.93g of NaHCO ₃ , dissolve in a small amount of double distilled water, mix with double distilled water to about 800mL, adjust the pH to 9.6, add double Dilute to 1000mL with distilled water, store at 4 °C for use;

Phosphate buffer (PBS): 8.00 g NaCl, 0.2 g KCl, 0.2 g KH ₂ PO ₄ , 2.9 g Na ₂ HPO ₄ · 12H ₂ O, dissolved in 800 mL of pure water, adjusted to pH 7.2 with NaOH or HCl 7.4, constant volume to 1000mL;

PBST: PBS containing 0.05% Tween 20;

TMB color developing solution: liquid A: Na ₂ HPO ₄ · 12H ₂ O 18.43 g, citric acid 9.33 g, pure water to 100 mL; B liquid: 60 mg TMB dissolved in 100 mL of ethylene glycol. A and B liquids are mixed with a volume ratio of 1..5 to be a TMB coloring liquid, which is currently used.

The above is only the preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Fan Yiben Equivalent changes and modifications made in the content of the scope of the invention should be considered as the technical scope of the invention.

Claims

A hybridoma cell line C1 secreting anti-paromomycin monoclonal antibody has been deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee under the accession number CGMCC No. 12025.
An anti-paromomycin monoclonal antibody produced by the hybridoma cell line C1 of claim 1 having an IC 50 of 0.61 μg/L.
Use of the anti-paromomycin monoclonal antibody of claim 2 for use in the detection of paromomycin residues in food.