Nanobody specifically recognizing heavy metal cr and its application

A nanobody, heavy metal technology, applied in applications, specific peptides, viruses/phages, etc., can solve the problems of high screening difficulty, difficult to identify, and small metal ions

Active Publication Date: 2019-08-09
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, even if a suitable immunogen carrying heavy metal ions is obtained, there are still many bottlenecks in the subsequent antibody screening process, especially in the complete immunogen, the metal ions are very small, and the specific antibodies obtained during the screening process are very small, even It is difficult to identify; and specific antibodies against materials other than metal ions in the complete immunogen, such as chelating agents and carrier proteins, will account for a large proportion; therefore, the difficulty of screening is much higher than that of conventional monoclonal antibody screening methods

Method used

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  • Nanobody specifically recognizing heavy metal cr and its application
  • Nanobody specifically recognizing heavy metal cr and its application
  • Nanobody specifically recognizing heavy metal cr and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1, Preparation and Identification of Heavy Metal Complete Antigen

[0085] 1. Preparation of complete antigen

[0086] The phage antibody library display technology used by the present inventors is a screening method for solid-phase purification of antigen screening. Heavy metal ions themselves have very small molecular weights and are not immunogenic and cannot be solid-phased, and they have charges themselves, which will interact with them. The molecule has a strong irreversible reaction, so it is coupled with the carrier protein (BSA, OVA) to form a complete antigen and then screened. Here the inventors utilize a bifunctional chelating agent p-SCN-Bn-DTPA (abbreviated as DTPA), which can not only chelate metal ions but also be coupled with carrier proteins, thereby obtaining complete antigens Cr-DTPA-BSA, Cr - DTPA-OVA and metal-free antigens DTPA-BSA, DTPA-OVA.

[0087] Chelation of heavy metal ions and chelating agents:

[0088] Take a certain amount of DT...

Embodiment 2

[0098] Example 2, Screening and Enrichment of Cr-Nanobody Library

[0099] 1. Screening

[0100] There are special single-chain antibodies in animals such as camels and sharks. This kind of antibody naturally lacks light chains, so it is called heavy-chain antibodies (Heavy-Chain Antibodies, HCAbs), the smallest unit of antigen that can be bound to the variable region of the heavy chain It is called VHH antibody (Variable domain of Heavy chain of Heavy chain antibody, VHH), with a molecular weight of about 13kDa, also known as Nanobody. Because of its small molecular weight and easy penetration into tissues, it is easy to access the grooves, cracks and hidden epitopes on the target surface, and can recognize many antigen binding sites that cannot be recognized by traditional antibodies; in addition, VHH also has stable High solubility, high water solubility, easy expression, low cost, unlimited amplification, and small steric hindrance in binding to specific antigenic determi...

Embodiment 3

[0136] Example 3, monoclonal antibody positive clone screening

[0137] 1. Monoclonal preliminary screening - positive screening

[0138] Verification by indirect ELISA: ELISA plates were coated with Cr-DTPA-BSA and DTPA-BSA respectively, and the clones that were only positive for Cr-DTPA-BSA but negative for DTPA-BSA were selected as positive clones, and then analyzed Proceed to the next step of verification.

[0139] Two groups of experiments were carried out simultaneously, and Cr-DTPA-BSA and DTPA-BSA were used as antigens for detection respectively. The specific process is as follows:

[0140] (1) Antigen coating: complete antigen Cr-DTPA-BSA and metal-free antigen DTPA-BSA coating, the concentration is 2.5 μg / ml, 100 μl / well, 4 ° C for 12 hours;

[0141] (2) Wash 5 times with 1×PBST, dry the liquid in the well, then add 5% skim milk powder to seal, 200 μl / well, 37°C for 1.5h;

[0142] (3) Wash 5 times with 1×PBST, drain the liquid in the well, add the supernatant of ...

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Abstract

The invention relates to a nano antibody of specific identification heavy metal Cr and an application of the nano antibody. A nano antibody for the heavy metal Cr is disclosed, and the nano antibody has good specificity for the heavy metal Cr, and very low cross reactivity with other metals. By utilizing the nano antibody, a kit for conveniently, rapidly and accurately detecting the heavy metal Cr can be prepared.

Description

technical field [0001] The invention relates to the technical field of monoclonal antibodies, more specifically, the invention relates to nanobodies specifically recognizing heavy metal Cr and applications thereof. Background technique [0002] Heavy metals are widely distributed in the atmosphere, water, soil and organisms. Chromium (Cr) is ubiquitous in nature and widely used. Chromium pollution in food mainly comes from industrial wastewater in agricultural soil. Eating foods high in chromium can cause symptoms such as quarrel, diarrhea, digestive disorders, and even cancer. Chromium and its compounds can enter the human body through the digestive tract, respiratory tract, skin and mucous membranes, often pollute the environment in the form of solution, dust or steam, and endanger human health. Therefore, it is very important to detect the presence or absence and the amount of heavy metal chromium in the environment or in the sample. [0003] Traditional detection met...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/44C12N15/13C12N7/01G01N33/543
Inventor 姚刚王娟俞华齐姜忍忍依布拉音
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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