阅读说明：本技术 检测副溶血性弧菌的试剂盒及检测方法 (Kit for detecting vibrio parahaemolyticus and detection method ) 是由 王慧 邱红玲 王永 于 2019-11-21 设计创作，主要内容包括：本发明提供了一种检测副溶血性弧菌的试剂盒及检测方法。本发明人筛选获得一株独特的抗副溶血性弧菌的单克隆抗体,对于不同型别的副溶血性弧菌具有广谱结合/识别能力,而对于副溶血性弧菌以外的其它菌株则不发生结合/识别,特异性和敏感性俱佳。本发明还提供了含有该单克隆抗体的试剂盒以及利用该单克隆抗体进行副溶血性弧菌检测的方法。(The invention provides reagent kits for detecting vibrio parahaemolyticus and a detection method. strain of unique monoclonal antibody for resisting vibrio parahaemolyticus is obtained by screening, the monoclonal antibody has spectrum binding/identifying capability for different types of vibrio parahaemolyticus, and does not bind/identify other strains except the vibrio parahaemolyticus, and the specificity and the sensitivity are good.)

binding molecules comprising the amino acid sequence of heavy chain CDR1 shown in SEQ ID NO. 8, heavy chain CDR2 shown in SEQ ID NO. 9 and heavy chain CDR3 shown in SEQ ID NO. 10, light chain CDR1 shown in SEQ ID NO. 14, light chain CDR2 shown in SEQ ID NO. 15 and light chain CDR3 shown in SEQ ID NO. 16.

2. The binding molecule of claim 1, comprising a heavy chain variable region having the amino acid sequence shown in SEQ ID No. 2.

3. The binding molecule of claim 1, comprising a light chain variable region having the amino acid sequence set forth in SEQ ID No. 4.

4. Use of the binding molecule of any of claims 1-3 in the preparation of a reagent for detecting vibrio parahaemolyticus.

immune conjugates, said immune conjugate comprising:

the binding molecule of any of claims 1-3 to , and a detectable label attached to the binding molecule.

6. a kit for detecting vibrio parahaemolyticus comprising the binding molecule of any of claims 1-3 to or the immune conjugate of claim 5.

7. The kit of claim 6, further comprising: a capture antibody which is a polyclonal antibody against vibrio parahaemolyticus outer membrane protein K.

8. A method for detecting Vibrio parahaemolyticus by contacting the binding molecule with a test sample and detecting the presence and amount of Vibrio parahaemolyticus by detecting the binding of the binding molecule of any of claims 1-3- or the immunological conjugate of claim 5 to the test sample.

9. The method of claim 8, wherein the method comprises,

(1) capturing vibrio parahaemolyticus by using a polyclonal antibody as a capture antibody;

(2) specific detection of the binding molecule of any of claims 1-3 or the immunoconjugate of claim 5 as a detection antibody to obtain the presence and amount of vibrio parahaemolyticus.

Technical Field

The invention belongs to the field of microbial detection, and particularly relates to a kit and a detection method for detecting vibrio parahaemolyticus.

Background

Vibrio (Vibrio) is a gram-negative bacterium with short and curved thallus and flagella at the tail, and Vibrio (Vibrio) is 1 genus of Vibrionaceae, has various species of Vibrio, is distributed in cycles, and is especially common in water.

The vibrio parahaemolyticus is kinds of halophilic marine vibrio, is easy to cause food poisoning, and the pollution mainly comes from marine products, such as cuttlefish, sea fish, sea shrimp, sea crab and jellyfish, and pickled foods with high salt content, such as salted vegetables and salted meat.

At present, the detection of vibrio parahaemolyticus in food in China is mainly based on the gold standard GB/T4789 plus 2013 'food safety national standard food microbiology test for vibrio parahaemolyticus' or PCR detection method, the detection of the national standard method needs professional microorganism technical personnel, and the PCR method has high requirements on instrument and equipment and detection environment.

The ELISA method based on immunology is simple and convenient to operate, short in detection time, capable of realizing high-throughput (up to 96 samples) detection of the samples and plays a crucial role in a food-borne pathogenic bacteria detection monitoring and control system.

In view of the above, there is a need in the art to develop antibodies against Vibrio parahaemolyticus with good specificity and low cross-interference to meet the needs of food detection and the like.

Disclosure of Invention

The invention aims to provide monoclonal antibodies capable of specifically recognizing vibrio parahaemolyticus and application thereof.

binding molecules comprising the amino acid sequence of heavy chain CDR1 shown in SEQ ID NO. 8, heavy chain CDR2 shown in SEQ ID NO. 9 and heavy chain CDR3 shown in SEQ ID NO. 10, light chain CDR1 shown in SEQ ID NO. 14, light chain CDR2 shown in SEQ ID NO. 15 and light chain CDR3 shown in SEQ ID NO. 16.

The binding molecule comprises a heavy chain variable region, wherein the heavy chain variable region has an amino acid sequence shown in SEQ ID NO. 2.

The binding molecule comprises a light chain variable region, wherein the light chain variable region has an amino acid sequence shown in SEQ ID NO. 4.

In another preferred embodiment of , the binding molecule comprises a heavy chain variable region and a light chain variable region having the amino acid sequences shown in SEQ ID NO. 2 and SEQ ID NO. 4, respectively.

In another preferred embodiment, the binding molecule is a monoclonal antibody.

Nucleic acid molecules encoding the above binding molecules.

Use of the above-mentioned binding molecule in the preparation of a reagent for detecting Vibrio parahaemolyticus.

In another aspect of the invention, expression vectors are provided which contain DNA encoding the binding molecules.

In another aspect of the invention, host cells are provided, said host cells comprising said expression vector.

immune conjugates, wherein said immune conjugate comprises:

the binding molecule; and a detectable label linked to the binding molecule; preferably, the detectable label comprises: fluorescent markers, chromogenic markers; more preferably, the method comprises the following steps: and (4) biotin.

A kit for the detection of Vibrio parahaemolyticus comprising said binding molecule or said immunological conjugate.

The kit also comprises: a capture antibody which is a polyclonal antibody against vibrio parahaemolyticus outer membrane protein K; the capture antibody is immobilized on a solid phase carrier; preferably, the solid support includes (but is not limited to): test paper (such as colloidal gold test paper), microspheres, coated plates, slides or chips.

preferably, the polyclonal antibody is obtained by immunizing animal with outer membrane protein K, preferably rabbit.

A method for detecting Vibrio parahaemolyticus, wherein said binding molecule is contacted with a sample to be tested, and the presence and amount of Vibrio parahaemolyticus is detected by detecting the binding of said binding molecule or said immunological conjugate to the sample to be tested.

Preferably, the method comprises:

(1) the polyclonal antibody was used as a capture antibody ( th antibody) to capture Vibrio parahaemolyticus.

(2) Specific detection is carried out by using the binding molecule or the immune conjugate as a detection antibody (second antibody), and the existence amount of the vibrio parahaemolyticus are obtained.

In another preferred embodiment, the sample to be tested is not a sample from an animal or human, and preferably the sample to be tested comprises food, dairy, beverage or medicine.

In another preferred embodiment , the method for detecting Vibrio parahaemolyticus is a non-diagnostic method, and preferably, the method is applied to detection of bacteria-containing sites, bacteria-containing environments, articles (such as vessels) or food, dairy products, beverages or medicines.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

The present inventors have conducted extensive and intensive studies at and screened strain of hybridoma cell lines producing antibodies against Vibrio parahaemolyticus, which antibodies have unique complementarity determining regions (CDR regions) having spectrum binding/recognizing ability for different types of Vibrio parahaemolyticus, and do not bind/recognize other strains other than Vibrio parahaemolyticus, and are excellent in both specificity and sensitivity.

As used herein, the term "test sample" encompasses a variety of sample types, including various samples that require detection of a microorganism, particularly Vibrio parahaemolyticus. For example, the "sample to be tested" may be from a bacteria-containing place, a bacteria-containing environment, an article (such as a vessel) or a food, a dairy product, a beverage, or a pharmaceutical product.

As used herein, "capture antibody", "coating antibody", " antibody", and " anti-antibody" are all used interchangeably to refer to an antibody that is used to enrich Vibrio parahaemolyticus from a sample.

As used herein, "detection antibody", "second antibody", and "secondary antibody" are used interchangeably and refer to specific anti-Outer Membrane Protein K (OMPK), an antibody that recognizes or binds in coordination with a corresponding th antibody for Vibrio parahaemolyticus of interest in the present invention, the corresponding th and second antibodies are different and bind to different epitopes of the OMPK of Vibrio parahaemolyticus simultaneously.

As used herein, the term "detectable signal" refers to a signal that is linked to a second antibody and is used to indicate the specific binding of the second antibody to Vibrio parahaemolyticus.

Binding molecules

In view of the problems of the prior art, the present inventors have focused on the search for antibodies that enable the specific identification of Vibrio parahaemolyticus, avoid the cross interference of other strains (particularly closely related strains), isolate various proteins of Vibrio parahaemolyticus, prepare antibodies using them as immunogens, and examine the specificity of the obtained antibodies, and have been extensively tested and demonstrated by using the envelope protein K (OMPK) as immunogen and the specific antibodies screened therefrom.

The present invention provides binding molecules capable of binding to Vibrio parahaemolyticus. Preferably, the binding molecule of the invention exhibits a specific binding/recognition activity for vibrio parahaemolyticus.

The binding molecules of the present invention may be intact immunoglobulin molecules, which may be antigen-binding fragments, including but not limited to Fab, F (ab') 2, Fv, dAb, Fd, Complementarity Determining Region (CDR) fragments, single chain antibodies (scFv), bivalent single chain antibodies, bispecific double chain antibodies, triple chain antibodies, quadruple chain antibodies, and (poly) peptides or fragments thereof containing at least a fragment sufficient to confer specific antigen binding to vibrio parahaemolyticus.

The antigen binding properties of an antibody can be described by 3 specific regions located in the variable regions of the heavy and light chains, called Complementarity Determining Regions (CDRs), which separate the variable regions into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction.

Another aspect of the invention includes functional variants of the antibodies described herein that compete with the parent antibody for specific binding to OMPK, and that have the ability to recognize OMPK of Vibrio parahaemolyticus in close proximity to the specific antibodies provided in the examples of the invention.

The present invention also provides immunoconjugates comprising the antibodies described herein and further comprising at least other types of functional molecules including, but not limited to, detectable labels.

The invention is not particularly limited to the use of labels that are capable of binding to the binding molecules of the invention and that, when appropriately treated, accurately indicate the presence or absence and amount of the target strain in the sample to be detected, the detectable labels may include, but are not limited to, fluorescent labels, chromogenic labels, such as enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions.

The label may be directly provided on the second antibody, or the label may be provided on an anti-antibody specific to the second antibody, and the skilled person may select an appropriate label according to the kind and characteristics of the antibody to be used, for example, the label may be selected from horseradish peroxidase (HRP), Alkaline Phosphatase (AP), glucose oxidase, β -D-galactosidase, urease, catalase, or glucoamylase when enzyme labels as shown above are used, it is also necessary to use substrates that bind to the corresponding enzymes to report the presence or amount of the label by means of color development or the like.

As a specific example of , the detectable label is biotin, which serves as a revealing marker that generates a fluorescent signal upon laser excitation when bound to streptavidin-conjugated phycoerythrin (streptavidin-phycoerythrin, also known as PE-labeled streptavidin, SA-R-PE).

Detection reagent and kit

The binding molecule can be used for preparing a reagent or a kit for detecting vibrio parahaemolyticus, the kit for conveniently, quickly and accurately detecting vibrio parahaemolyticus can be prepared on the basis of the binding molecule, and in a preferred mode, polyclonal antibodies which are combined with anti-vibrio parahaemolyticus are applied simultaneously.

Therefore, the present invention provides detection kits for detecting the presence of Vibrio parahaemolyticus in a sample, the kit comprising the Vibrio parahaemolyticus-resistant binding molecule of the present invention.

As the detection methods of the present invention, an indirect ELISA method was used in which an antigen to be detected was coated on a solid phase carrier and detection was performed using the binding molecule of the present invention.

The solid phase carriers include but are not limited to: test paper or test strips, microspheres, slides, well plates (e.g., 48-well plates, 96-well plates), chips, and the like.

As a preferred mode of further of the present invention, the detection is carried out according to the principle of a double antibody sandwich method, in which antibody (polyclonal antibody against Vibrio parahaemolyticus) is immobilized on a carrier, then antibody is reacted with an antigen, and after washing, the antigen is reacted with a secondary antibody, which is a monoclonal antibody against Vibrio parahaemolyticus of the present invention (the secondary antibody carries a detectable signal or can be bound to a substance carrying a detectable signal), and finally a chemiluminescent or enzyme-linked chromogenic reaction is carried out to detect a signal.

For convenience in detection, the kit may contain, in addition to the binding molecules of the invention, other detection reagents or auxiliary reagents, such as reagents conventionally used in ELISA kits, the nature of which reagents and their formulation are well known to those skilled in the art, such as chromogenic reagents, sensitizers, hybridization solutions, antigen retrieval solutions, blocking solutions, PBS, xylene, ethanol, H, etc₂O₂Methanol solution, and the like. It will be understood by those skilled in the art that various modifications of the detection kit are encompassed by the present invention as long as the binding molecule of the present invention is utilized therein as a reagent for recognizing Vibrio parahaemolyticus.

In addition, instructions for use may be included in the kit to instruct the method of use of the reagents loaded therein.

After obtaining the binding molecule and/or the kit provided by the present invention, various immunology-related methods can be used to detect the presence or amount of vibrio parahaemolyticus in the sample, so as to determine whether the donor of the sample to be tested contains vibrio parahaemolyticus, and these methods are all included in the present invention. Preferably, the method is for the purpose of non-disease diagnosis.

preferred modes, the present invention provides methods for in vitro (non-diagnostic or therapeutic) detection of Vibrio parahaemolyticus, comprising the steps of:

(a1) coating the polyclonal antibody against the vibrio parahaemolyticus on a solid phase carrier;

(a2) adding a sample to be detected to the solid phase carrier of (a1), so that vibrio parahaemolyticus in the sample to be detected is combined with the polyclonal antibody, and a solid phase carrier with a 'vibrio parahaemolyticus-polyclonal antibody' binary complex is formed;

(a3) adding the binding molecule (carrying the detectable label) of the invention to the solid phase carrier added in (a2) to form a solid phase carrier with a 'polyclonal antibody-vibrio parahaemolyticus-binding molecule of the invention' complex, wherein the binding molecule of the invention carries detectable label;

(a4) detecting the marker in the complex of (a3), and determining the presence or absence or the amount of Vibrio parahaemolyticus in the sample to be detected.

As measurement methods, the content of Vibrio parahaemolyticus in a sample to be measured can be obtained by setting an antigen control with a known concentration, making a concentration standard curve, and then comparing the concentration standard curve.

Has the advantages that:

(1) brand-new binding molecules are provided, which are obtained by screening by taking the vibrio parahemolyticus OMPK protein as an antigen, can identify the spectrum of the vibrio parahemolyticus , have no cross reactivity to strains of related species except the vibrio parahemolyticus, and have very high specificity and sensitivity.

(2) The binding molecule can be well matched with an anti-OMPK polyclonal antibody, and is particularly suitable for double-antibody sandwich ELISA detection.

The invention is further illustrated at in conjunction with specific examples, it being understood that these examples are intended to illustrate the invention only and are not intended to limit the scope of the invention the experimental procedures, without specifying the specific conditions in the following examples, are generally in accordance with conventional conditions, such as those described in J. SammBrook et al, molecular cloning guidelines, third edition, scientific publishers, 2002, or in accordance with the manufacturer's recommendations.

Drawings

FIG. 1 is a graph showing the results of SDS-PAGE detection of the expression product obtained in example 1 after purification.

Detailed Description

The invention is further illustrated in with reference to the following examples:

the extracted vibrio parahaemolyticus and other strains used in the embodiment are purchased from China center for industrial microorganism culture collection; mice were purchased from slaick laboratory animals llc.