阅读说明：本技术 一种定量检测鸡蛋中氟苯尼考的嵌合elisa试剂盒及其制备方法 (Chimeric ELISA kit for quantitatively detecting florfenicol in eggs and preparation method thereof ) 是由 武玉香 李建林 于 2019-09-04 设计创作，主要内容包括：本发明实施例公开了一种定量检测鸡蛋中氟苯尼考的嵌合ELISA试剂盒,所述ELISA试剂盒包括氟苯尼考单克隆抗体、氟苯尼考偶联抗原、基准液、酶标物、包被所述氟苯尼考偶联抗原的酶标板,以及用于ELISA实验的显色液、终止液和稀释液；所述基准液用于作为空白对照组的溶液；测定待测样品时,同时检测空白对照组和待测样品组的OD值,通过所述OD值计算得到所述待测样品的浓度。本发明实施例提供的定量检测鸡蛋中氟苯尼考残留的检测试剂盒,其操作简便,可批量快速准确检测,且成本低、稳定性好,需要的技术含量不高。(The embodiment of the invention discloses a chimeric ELISA kit for quantitatively detecting florfenicol in eggs, which comprises a florfenicol monoclonal antibody, a florfenicol coupled antigen, a reference solution, an enzyme label plate coating the florfenicol coupled antigen, and a color development solution, a stop solution and a diluent for an ELISA experiment; the reference solution is used as a solution of a blank control group; and when the sample to be detected is detected, detecting the OD values of the blank control group and the sample group to be detected at the same time, and calculating the concentration of the sample to be detected through the OD values. The detection kit for quantitatively detecting florfenicol residues in eggs, provided by the embodiment of the invention, is simple and convenient to operate, can be used for rapidly and accurately detecting in batches, and is low in cost, good in stability and low in required technical content.)

1. A chimeric ELISA kit for quantitatively detecting florfenicol in eggs is characterized by comprising a florfenicol monoclonal antibody, a florfenicol coupled antigen, a reference solution, an enzyme label plate coating the florfenicol coupled antigen, and a developing solution, a stopping solution and a diluent for an ELISA experiment; the reference solution is used as a solution of a blank control group;

And when a sample to be detected is detected, detecting the OD values of the blank control group added with the reference solution and the sample group to be detected at the same time, and calculating the concentration of the sample to be detected through the OD values.

2. The chimeric ELISA kit for quantitatively detecting florfenicol in eggs as claimed in claim 1 wherein,

the reference solution is an aqueous solution of boric acid, sodium hydroxide, tryptophan and ethylenediamine tetraacetic acid.

3. The chimeric ELISA kit for quantitatively detecting florfenicol in eggs as claimed in claim 1 wherein,

The monoclonal antibody is prepared from a florfenicol FF3C11 cell strain, wherein the florfenicol FF3C11 cell strain is preserved in China center for type culture Collection (CCCCCCCCCCNO: C2019152) of Wuhan university in Wuhan, China at 7/4 in 2019.

4. The chimeric ELISA kit for quantitatively detecting florfenicol in eggs as claimed in claim 1 further comprising: and the data processing device is stored with a pre-established standard curve for calculating the florfenicol content in the sample to be detected.

5. The chimeric ELISA kit for quantitatively detecting florfenicol in eggs as claimed in claim 4, wherein,

The standard curve establishment comprises the following steps: preparing florfenicol standard substance solutions with different mass concentrations by using a standard substance diluent, carrying out indirect ELISA (enzyme-linked immunosorbent assay) measurement on a florfenicol antibody diluted by using a buffer solution and the enzyme-labeled substance diluted by using the buffer solution, calculating the binding rate of the florfenicol with each concentration, and establishing IC (integrated circuit) by using the binding rate of the florfenicol as a longitudinal coordinate and the concentration of the florfenicol as a horizontal coordinate₅₀The lowest florfenicol inhibited the standard curve.

6. The chimeric ELISA kit for quantitatively detecting florfenicol in eggs as claimed in claim 1 wherein,

The enzyme labeling solution is a goat anti-mouse IgG-HRP solution.

7. A method for preparing the chimeric ELISA kit for quantitatively detecting florfenicol in eggs, which is described in claim 1, is characterized by comprising the processes of preparing a reference solution, preparing a florfenicol monoclonal antibody, preparing a color development solution, a stop solution and a diluent for ELISA experiments, and coating the florfenicol coupling antigen on an ELISA plate.

8. The method of claim 7,

The reference solution is an aqueous solution of boric acid, sodium hydroxide, tryptophan and ethylenediamine tetraacetic acid.

9. The method of claim 7,

The monoclonal antibody is prepared from a florfenicol FF3C11 cell strain, wherein the florfenicol FF3C11 cell strain is preserved in China center for type culture Collection (CCCCCCCCCCNO: C2019152) of Wuhan university in Wuhan, China at 7/4 in 2019.

10. the method of claim 7, further comprising: the method comprises the following steps of pre-establishing a standard curve for calculating the florfenicol content in a sample to be detected, wherein the establishment process comprises the following steps: preparing florfenicol standard substance solutions with different mass concentrations by using a standard substance diluent, carrying out indirect ELISA (enzyme-linked immunosorbent assay) measurement on a florfenicol antibody diluted by using a buffer solution and the enzyme-labeled substance diluted by using the buffer solution, calculating the binding rate of the florfenicol with each concentration, and establishing IC (integrated circuit) by using the binding rate of the florfenicol as a longitudinal coordinate and the concentration of the florfenicol as a horizontal coordinate₅₀The lowest florfenicol inhibited the standard curve.

Technical Field

The embodiment of the invention relates to the technical field of biological detection, and particularly relates to a chimeric ELISA kit for quantitatively detecting florfenicol in eggs and a preparation method thereof.

Background

Florfenicol is an antibiotic drug special for 3 rd generation animals of amidol, has the characteristics of broad spectrum, high efficiency, good absorption, wide in-vivo distribution, no aplastic anemia caused action and the like, and is widely used for treating bacterial diseases of livestock and poultry as a substitute drug of chloramphenicol. Florfenicol has embryotoxicity and drug resistance, and is remained in livestock and poultry products due to a large amount of application in livestock and poultry production, thereby harming the health of human bodies. At present, a kit for detecting florfenicol usually needs a set of series standard products, so that the workload is increased, and the requirements on operation are extremely high for linear compounding of a standard curve; the kit with the 96T specification is prepared by the series of standard products, so that the number of samples required to be detected is large, the operation of the kit is complex, the stability of a detection result is poor, the detection precision is poor, and the time required for detection is long.

Disclosure of Invention

Therefore, the embodiment of the invention provides a chimeric ELISA kit for quantitatively detecting florfenicol in eggs and a preparation method thereof, and aims to solve the problems that the kit in the prior art is troublesome to operate and poor in stability of detection results.

In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:

A chimeric ELISA kit for quantitatively detecting florfenicol in eggs comprises a florfenicol monoclonal antibody, a florfenicol coupled antigen, a reference solution, an enzyme label plate coating the florfenicol coupled antigen, and a developing solution, a stop solution and a diluent for an ELISA experiment; the reference solution is used as a solution of a blank control group;

And when a sample to be detected is detected, detecting the OD values of the blank control group added with the reference solution and the sample group to be detected at the same time, and calculating the concentration of the sample to be detected through the OD values.

preferably, the reference solution is an aqueous solution of boric acid, sodium hydroxide, tryptophan and ethylenediamine tetraacetic acid.

Preferably, the monoclonal antibody is prepared from a florfenicol FF3C11 cell strain, wherein the florfenicol FF3C11 cell strain is deposited in China Center for Type Culture Collection (CCTCC) at Wuhan university, Wuhan, China in 7 and 4 days in 2019, and the deposit number is CTCC NO: C2019152.

The embodiment of the invention also comprises the following steps: and the data processing device is stored with a pre-established standard curve for calculating the florfenicol content in the sample to be detected.

preferably, the standard curve establishment includes: preparing florfenicol standard substance solutions with different mass concentrations by using a standard substance diluent, carrying out indirect ELISA (enzyme-linked immunosorbent assay) measurement on a florfenicol antibody diluted by using a buffer solution and the enzyme-labeled substance diluted by using the buffer solution, calculating the binding rate of the florfenicol with each concentration, and establishing IC (integrated circuit) by using the binding rate of the florfenicol as a longitudinal coordinate and the concentration of the florfenicol as a horizontal coordinate₅₀The lowest florfenicol inhibited the standard curve.

Preferably, the enzyme labeling solution is a goat anti-mouse IgG-HRP solution.

The embodiment of the invention also provides a method for preparing the chimeric ELISA kit for quantitatively detecting florfenicol in eggs, which is characterized by comprising the steps of preparing a reference solution, preparing a florfenicol monoclonal antibody, preparing a developing solution, a stopping solution and a diluent for an ELISA experiment and coating the florfenicol coupling antigen on an ELISA plate.

Preferably, the reference solution is an aqueous solution of boric acid, sodium hydroxide, tryptophan and ethylenediamine tetraacetic acid.

Preferably, the monoclonal antibody is prepared from a florfenicol FF3C11 cell strain, wherein the florfenicol FF3C11 cell strain is deposited in China Center for Type Culture Collection (CCTCC) at Wuhan university, Wuhan, China in 7 and 4 days in 2019, and the deposit number is CTCC NO: C2019152.

The embodiment of the invention also comprises the following steps: the method comprises the following steps of pre-establishing a standard curve for calculating the florfenicol content in a sample to be detected, wherein the establishment process comprises the following steps: preparing florfenicol standard substance solutions with different mass concentrations by using a standard substance diluent, carrying out indirect ELISA (enzyme-linked immunosorbent assay) measurement on a florfenicol antibody diluted by using a buffer solution and the enzyme-labeled substance diluted by using the buffer solution, calculating the binding rate of the florfenicol with each concentration, and establishing IC (integrated circuit) by using the binding rate of the florfenicol as a longitudinal coordinate and the concentration of the florfenicol as a horizontal coordinate₅₀The lowest florfenicol inhibited the standard curve.

The embodiment of the invention has the following advantages:

The kit for quantitatively detecting florfenicol residues in eggs provided by the embodiment of the invention is simple and convenient to operate, can be used for rapidly and accurately detecting in batches, is low in cost, good in stability and low in required technical content, operators do not need to contact with standard products, and the injury of the standard products to the operators is avoided.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

FIG. 1 is a diagram illustrating the identification of the purification purity of SPG monoclonal antibody provided in the example of the present invention;

FIG. 2 is a software interface diagram of the calculation of a chimeric ELISA kit according to an embodiment of the present invention using a pre-established standard curve;

FIG. 3 is a pre-established standard curve diagram of a chimeric ELISA kit according to an embodiment of the present invention.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

EXAMPLE 1 preparation of florfenicol monoclonal antibody

1. Cell resuscitation

After the hybridoma cell strain 3C11 is quickly taken out from a liquid nitrogen tank, the florfenicol FF3C11 cell strain is preserved in the China center for type culture Collection (CTCC NO): C2019152. rapidly melting in 37 deg.C water, centrifuging for 3 min at 1000 rpm after completely melting, wiping with alcohol, sterilizing, placing on sterile workbench, removing supernatant, suspending the cells in the tube with 600ul HT medium, transferring to 24-well culture plate, and placing in 37 deg.C CO₂Culturing in an incubator.

2. Preparation of ascites

The florfenicol monoclonal antibody is prepared by an in-vivo ascites induction method of animals, desensitization is carried out according to the dosage of 0.5ml of mineral oil/mouse, cells which are 80% full are injected into the abdominal cavity of the small mouse 7 to 10 days after desensitization, the cells are injected according to the quantity of 100 ten thousand, a diluent is sterile physiological saline, and ascites is taken 7 to 10 days.

And (3) purifying the florfenicol monoclonal antibody: ascites is purified by an SPG column method, 2 batches of the purified florfenicol monoclonal antibody are subjected to antibody purity identification according to the conventional operation, as shown in figure 1, a lane 1 is a batch 2016FF23, a lane 2 is a batch 2016FF21, clear bands of 25KD and 55KD are observed by SDS-PAGE electrophoresis, and the clear bands are respectively a light chain and a reconnection of the antibody, and no impurity band exists, which indicates that the purity of the antibodies of the two batches is more than 95%.