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Insulin immunogen, preparation method thereof, insulin antibody, and kit

An insulin and immunogen technology, used in insulin antibodies and kits, their preparation, and the field of insulin immunogens, can solve problems such as low antibody titers, and achieve the effects of high titers, reduced biological activity, and strong immune responses

Inactive Publication Date: 2018-11-30
SHENZHEN NEW INDS BIOMEDICAL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The main purpose of the present invention is to provide a kind of insulin immunogen, its preparation method, insulin antibody and kit, to solve the problem of low titer of antibody produced by insulin immunogen in the prior art

Method used

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  • Insulin immunogen, preparation method thereof, insulin antibody, and kit
  • Insulin immunogen, preparation method thereof, insulin antibody, and kit
  • Insulin immunogen, preparation method thereof, insulin antibody, and kit

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preparation example Construction

[0059] In the second typical embodiment of the present application, a preparation method of insulin immunogen is provided, the preparation method comprising: under the action of a cross-linking agent, intramolecular and / or intermolecular cross-linking of multiple insulin monomers Linked to obtain insulin cross-linked body, insulin cross-linked body contains one or more cross-linked units shown in formula (1):

[0060]

[0061] Wherein, ① and ② are independently selected from intramolecular and / or intermolecular chain A or chain B, and R1 is R2 and R3 are empty, or R1 and R3 are each independently selected from Or -S-, R2 is selected from C2~C74 straight chain or branched chain alkylene, or part of -CH in C2~C74 straight chain or branched chain alkylene 2 - arbitrarily replaced by -O-, -S-, -NH-, -COO-, -CO-, Arylene is substituted by heteroarylene or cyclohydrocarbon; or R1 and R3 are each independently selected from Or -S-, R2 is selected from an arylene group or ...

Embodiment 1

[0105] Example 1: Preparation and detection of polyclonal antibody to insulin (EDC method self-crosslinking)

[0106] 1. Principle of immunogen preparation

[0107] EDC is used as an activating reagent for the carboxyl group in the synthesis of amides. After the two carboxyl groups (Asn, Thr) at the C-terminal are activated during the self-crosslinking process of insulin, they can be randomly combined with the amino group (N-terminal Gly / Phe, Lys) on another insulin molecule. ) for coupling to form an insulin cross-linked body. The reaction process is shown in the following formula (11):

[0108]

[0109] 2. Preparation of immunogen: Weigh three parts of 20mg insulin (sigma, product number: I3536) and dissolve in 2ml MES (pH5.0), then add 0.5mg, 1.32mg, 5mg EDC and 12mg EDC (sigma, product number: 03449), stirred at room temperature for 2h, SDS-PAGE results showed that the insulin cross-linked body was dispersed, and the size of the insulin cross-linked body was concentra...

Embodiment 2

[0122] Embodiment 2: Insulin (Bis(NHS)PEG 2 ) Preparation and detection of polyclonal antibody

[0123] 1. The principle of immunogen preparation

[0124] Bis(NHS)PEG 2 Be the water-soluble polyethylene glycol (PEG) shown in formula (12):

[0125]

[0126] The PEG spacer arm increases the solubility of the cross-linked protein, the reactive group N-hydroxysuccinimide ester (same as bifunctional), the reaction is directed to the amino group on the surface of the protein, and the reactive groups at both ends can form amides with the amino group on different insulin molecules key, spacer arm length 2 PEG. Its cross-linking reaction product with insulin monomer contains a cross-linking unit shown in the following formula (13):

[0127]

[0128] 2. Immunogen preparation

[0129] Weigh three portions of 20mg insulin (sigma, product number: I3536) and dissolve in 2ml 0.1M NaHCO 3 (pH8.3), add 13.8mg Bis(NHS)PEG predissolved in 0.2ml DMSO 2 (Mw=400.5), stir gently at room...

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Abstract

The invention provides an insulin immunogen, a preparation method thereof, an insulin antibody, and a kit. The insulin immunogen is an insulin crosslinked body, is formed through intramolecular crosslinking and / or intermolecular crosslinking of a plurality of insulin monomers, and contains one or a plurality of crosslinked monomers disclosed in the invention, wherein 1, and 2 are independently selected from A chain or B chain, R1 is used for representing a group disclosed in the invention, and R2 and R3 are blank, or R1 and R3 are independently selected from a group disclosed in the inventionor -S-, R2 is selected from C2-C74 straight chain or branched chain alkylene, C2-C74 straight chain or branched chain alkylene with a part of -CH2- randomly substituted by -0-, -S-, -NH, -COO-, -CO-,arylidene, heteto arylidene, cycloalkylene, or groups disclosed in the invention, or R1 and R3 are independently selected from a group disclosed in the invention or -S-, and R2 is selected from arylidene or substituted arylidene, wherein in the substituted arylidene, the -H on the substituted arylidene is substituted by -OH, halogen elements, or alkane. The insulin immunogen can be used for obtaining high potency insulin antibody.

Description

technical field [0001] The invention relates to the field of insulin antibody detection, in particular to an insulin immunogen, a preparation method thereof, an insulin antibody and a kit. Background technique [0002] Insulin is transcribed from the DNA of the insulin gene region on the broken arm of the 11th chromosome of the human body to mRNA, and the mRNA moves from the nucleus to the endoplasmic reticulum of the cytoplasm, and is translated into preproinsulin (Preproinsulin) composed of 105 amino acid residues. Proinsulin undergoes proteolysis to remove the propeptide to generate a long peptide chain consisting of 86 amino acids --- Proinsulin. Proinsulin enters the Golgi apparatus along with the microvesicles in the cytoplasm, connects with disulfide bonds, and through the action of proteolytic enzymes, the chains connected by three arginines 31, 32, and 60 are cut off, and the broken chains generate C-peptide which has no effect , forming the α chain (also known as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/62C07K16/26G01N33/74G01N33/543
CPCC07K14/62C07K16/26G01N33/54326G01N33/74G01N2333/62
Inventor 饶微杜凯刘蕾高莉肖国华罗春
Owner SHENZHEN NEW INDS BIOMEDICAL ENG
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