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Detection strip for qualitative detection of acinetobacter baumannii specific antibodies in human serum

A technology for qualitative detection of Acinetobacter baumannii, applied in the fields of bioengineering and immunology, can solve the problems of difficult to meet rapid identification, false positives, long time, etc., and achieves low preparation cost, strong antigenicity, and strong capture power. Effect

Active Publication Date: 2020-01-21
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]At present, the method for detecting the pathogen in the respiratory tract is mainly based on the traditional method, that is, the separation and identification method. Difficult to meet the needs of rapid identification; the PCR technology developed in recent years is a fast, sensitive, and specific technology, but at present this technology still relies on the pre-enrichment step of the traditional method, and the enrichment solution often There are also PCR inhibitors, which affect the effect of amplification, and false positives are also a prominent shortcoming of this method.
At the same time, this technology also requires professional testing equipment, which is not suitable for bedside testing

Method used

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  • Detection strip for qualitative detection of acinetobacter baumannii specific antibodies in human serum
  • Detection strip for qualitative detection of acinetobacter baumannii specific antibodies in human serum
  • Detection strip for qualitative detection of acinetobacter baumannii specific antibodies in human serum

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Experimental program
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Effect test

Embodiment 1

[0055] The preparation of embodiment 1 Acinetobacter baumannii fusion antigen (Fhue+OmpA+Pilf):

[0056] 1.1) Cloning of Acinetobacter baumannii FhuOmpPil fusion gene

[0057] Obtain the peptides with the most abundant antigenic epitopes in the extracellular domains of the surface proteins Fhue, OmpA and Pilf of Acinetobacter baumannii (the accession numbers in the NCBI protein database are KMV27515, AJF83030 and AJF80497 respectively) and find their gene coding sequences , and optimize its gene coding sequence, and connect the three gene sequences with two coding sequences of flexible connecting peptides (ggsggsggsggs) to form a fusion gene. At the same time, the entire gene sequence was chemically synthesized after introducing the restriction site NdeI at the 5' end of the fusion gene, the termination signal TAA and the restriction site BamHI at the 3' end, which was denoted as FhuOmpPil. The full sequence of the gene and the encoded amino acid sequence are shown in the seq...

Embodiment 2

[0073] Embodiment 2 prepares the latex microsphere marker of mouse anti-human IgG monoclonal antibody

[0074] 2.1) Activation of latex microspheres

[0075] Take 1 mL of a solution of red carboxylated polystyrene latex microspheres (100 nm) with a concentration of 10%, and add 9 mL of 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution (0.1mol / LMES, pH8.5) After mixing; use MES buffer to prepare 10mg / mL N-hydroxysuccinimide (NHS) and 10mg / mL 1-(3-dimethylaminopropyl)-3-ethyl 1-(3- Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride hydrochloride (EDC) solution;

[0076] Add 1mL of NHS solution and 1mL of EDC solution to the polystyrene latex microsphere (100nm) solution in turn, mix slowly at room temperature for 30 minutes, centrifuge at 19000g for 20 minutes after incubation, remove the supernatant, and use 10mL of borax buffer (0.1 mol / LNa 2 B 4 o 7 , pH8.5) resuspension, oscillation, ultrasonic treatment (ultrasonic breaker product model: YJ92-IIDN, power 50...

Embodiment 3

[0079] The preparation of embodiment 3 binding pads:

[0080] The polyester fiber membrane was cut to a size of 4 cm×0.8 cm / strip, and 64 μL of the latex microsphere marker prepared in Example 2 was added dropwise to the cut membrane. After spraying, dry at 37° C. for 12 hours in an environment with a relative humidity of 20%. Store in airtight and dry place at 25°C.

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Abstract

The invention relates to a detection strip for the qualitative detection of acinetobacter baumannii specific antibodies in human serum. The detection strip is composed of a sample pad, a binding pad,a nitrocellulose film, a water absorbent pad and a PVC plate. The binding pad is sprayed with a mouse anti-human IgG monoclonal antibody coupled with color latex microspheres, and the nitrocellulose film is coated with a detection line of an acinetobacter baumannii fusion antigen and a quality control line of a sheep anti-mouse IgG polyclonal antibody. When an added sample contains the acinetobacter baumannii antibody, firstly, the acinetobacter baumannii antibody and the latex-mouse anti-human IgG monoclonal antibody form a compound, the compound is captured when migrating to the detection line coated the acinetobacter baumannii fusion antigen under capillary action, and the detection line shows the corresponding color, so that whether the sample contains the acinetobacter baumannii antibody or not is detected. The detection strip has the advantages of rapidness, simplicity and convenience, high sensitivity, good specificity and low false positive, definite results can be obtained within 10 minutes, and the detection strip can be effectively used in the auxiliary diagnosis of acinetobacter baumannii infection.

Description

technical field [0001] The invention belongs to the fields of bioengineering and immunology, and specifically relates to a detection strip for qualitatively detecting the specific antibody of Acinetobacter baumannii in human serum and a preparation method thereof. Background technique [0002] Acinetobacter baumannii (Acinetobacter baumannii, Ab) is a non-fermenting Gram-negative bacillus. This bacterium is an important pathogenic bacterium of nosocomial infection, mainly causing respiratory tract infection, and can also cause bacteremia, urinary tract infection, secondary meningitis, surgical site infection, ventilator-associated pneumonia, etc. The rate of resistance to commonly used antibiotics is increasing year by year, and has caused serious concern for clinicians and microbiologists. Domestic data show that A.baumannii accounts for more than 70% of clinically isolated Acinetobacter. The drug resistance rate of A. baumannii to the third and fourth generation cephalos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/21C12N15/31G01N33/543G01N33/569G01N33/577
CPCC07K14/212G01N33/54313G01N33/54306G01N33/56911G01N33/577C07K2319/00G01N2333/212Y02A50/30
Inventor 杨波胡征王毅
Owner HUBEI UNIV OF TECH
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