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Anti-mHIN2 protein antibody, application thereof, and kit containing antibody

A kit and protein content technology, applied in the direction of anti-bacterial immunoglobulin, immunoglobulin, biological testing, etc., can solve the problems of antibody existence and achieve high sensitivity and wide detection range

Active Publication Date: 2020-04-28
CHENGDU OLYMVAX BIOPHARM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antibodies are not yet available

Method used

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  • Anti-mHIN2 protein antibody, application thereof, and kit containing antibody
  • Anti-mHIN2 protein antibody, application thereof, and kit containing antibody
  • Anti-mHIN2 protein antibody, application thereof, and kit containing antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Specific recognition of mHI N2 Preparation and Screening of Protein Mouse Monoclonal Antibody

[0081] mHI N2 Recombinant protein (1.4mg / mL) was mixed with adjuvant CFA and AD11.15 respectively to prepare immunogen, that is, recombinant protein was mixed with CFA at a volume ratio of 5:6 as immunogen A, and then recombinant protein was mixed with AD11.15 Mixed at a volume ratio of 1:1 as immunogen B. Immunogen A is the primary immunization and immunogen B is the booster immunization. Three mice were immunized intramuscularly, and the tail blood of the mice was drawn on the 14th day after immunization, and the antibody titer of the tail blood was evaluated by indirect ELISA method.

[0082] Use mHI N2 Recombinant protein coated ELISA plate (1 μg / mL), add 100 μL to each well, react overnight at 4°C; wash the plate 3 times with PBS solution, block with 5% milk-PBS at room temperature for 1 hr; then wash the plate once with PBS solution Finally, add mouse ta...

Embodiment 2

[0091] Example 2: Preparation of Purified Antibody

[0092] 1. Ascites preparation

[0093] Ascites was prepared from the 4 positive clones (4D11, 4H2, 5A8, 7G4) obtained above. respectively about 10 7 Each cell was injected into the peritoneal cavity of 2 Balb / C mice pre-injected with IFA adjuvant, and then about 10 days later, the ascites produced by each positive clone was extracted, and then centrifuged at 4°C and 12000rpm for 15min, and the supernatant was collected for the next step G protein purification.

[0094] 2. Purification of mouse monoclonal antibody

[0095] Take 1mL of the column material coupled with protein G and put it into an empty column, wash it with PBS solution, dilute 2mL of ascitic fluid with 8mL of PBS and put it on the column, then put the flow-through solution on the column again; then use pH 2.7 Glycine eluent was used for elution, and one tube was collected for each 1mL eluent (100μL neutralizing solution was added in advance, and the neutra...

Embodiment 3

[0101] Example 3: Antibody Pair Screening

[0102] The four purified antibodies were labeled with HRP by sodium periodate oxidation method, and then the labeled antibodies and mHI were evaluated by ELISA N2 The binding ability of the recombinant protein, the results are shown in Table 4.

[0103] Table 4: Evaluation results of HRP-labeled antibodies

[0104]

[0105]

[0106] As can be seen from Table 4, HRP-labeled antibody and mHI N2 Compared with the antibody before labeling, the binding ability of the protein is not significantly reduced, and the next pairing experiment can be carried out (see Table 5), and the selection can be used for mHI N2 Paired antibodies for protein detection.

[0107] Table 5: Pairwise pairing experiments of antibodies

[0108]

[0109]

[0110] According to the pairing results in Table 5 and the comprehensive evaluation of the yields of the purified antibodies of these four mouse monoclonal antibodies, the pairing results of choosi...

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Abstract

The invention belongs to the technical field of biology, and provides an anti-mHIN2 protein antibody, application thereof, and a kit containing the antibody. The antibody comprises a heavy chain and alight chain, the heavy chain and the light chain respectively comprise three CDR regions, the amino acid sequences of the heavy chain CDR regions are respectively shown as SEQ ID NO: 1-3 or 7-9, andthe amino acid sequences of the light chain CDR regions are respectively shown as SEQ ID NO: 4-6 or 10-12. The anti-mHIN2 protein antibody disclosed by the invention can be specifically combined withmHIN2 protein and effectively detect the content of the mHIN2 protein, and has high sensitivity and wide detection range.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a monoclonal antibody and its application. Background technique [0002] Staphylococcus aureus α-hemolysin (Hla) is an exotoxin, which is the main pathogenic factor of Staphylococcus aureus infection, and is often caused by pathogenic Staphylococcus aureus, especially methicillin-resistant Staphylococcus aureus MRSA) produced. Iron surface determinant B subunit (IsdB) is an important outer membrane anchoring protein of Staphylococcus aureus, which plays an important role in assisting bacteria to absorb and metabolize heme iron, as well as in bacterial adhesion and colonization. wxya N2 Using molecular fusion and Escherichia coli genetic engineering expression technology, the α-hemolysin nontoxic mutant of Staphylococcus aureus (mHla H35L ) and the ferric ion surface determinant B subunit N2 active fragment (IsdB-N2) are molecularly fused and solublely expressed recombinant fusion pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12G01N33/68G01N33/577G01N33/535
CPCC07K16/1271C07K2317/565G01N33/535G01N33/577G01N33/68G01N2333/31
Inventor 曾浩纪永军邹全明杨峰杨茜蔡昌芝赵莉群张怡
Owner CHENGDU OLYMVAX BIOPHARM
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