CN-121164642-B - Kit for microorganism detection and application thereof

CN121164642BCN 121164642 BCN121164642 BCN 121164642BCN-121164642-B

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a kit for detecting microorganisms and application thereof. The kit comprises an ELISA plate coated with a capture antibody and an HRP-labeled antibody working solution, wherein the capture antibody is a monoclonal antibody L3A-1 specifically binding to the antigen of the brucella brucei, the heavy chain amino acid sequence of the capture antibody is shown as SEQ ID NO.1, the light chain amino acid sequence of the capture antibody is shown as SEQ ID NO.2, the HRP-labeled antibody is a monoclonal antibody F4B-2 specifically binding to the antigen of the brucella, the heavy chain amino acid sequence of the capture antibody is shown as SEQ ID NO.3, and the light chain amino acid sequence of the capture antibody is shown as SEQ ID NO. 4. The kit for detecting microorganisms provided by the invention can detect the antigen content of the brucella brucei in human blood, has strong specificity and high sensitivity, overcomes the defect that the brucella brucei needs professionals and equipment, and has good application prospect.

Inventors

WANG XIN
LU XUEMEI
PAN ZIXIAN
Wang Yarou
LI DANDAN

Assignees

广州医太通生物科技有限公司

Dates

Publication Date: 20260512
Application Date: 20250926

Claims (7)

1. The kit for detecting microorganisms is characterized by comprising an ELISA plate coated with a capture antibody and an HRP-labeled antibody working solution, wherein the capture antibody and the HRP-labeled antibody are antibodies which specifically bind to the antigen of the brucella brucei; The capture antibody is a monoclonal antibody L3A-1 specifically combined with the antigen of the brucella brucei, the heavy chain amino acid sequence is shown as SEQ ID NO.1, and the light chain amino acid sequence is shown as SEQ ID NO. 2; the HRP-labeled antibody is a monoclonal antibody F4B-2 specifically binding to the antigen of the brucella brucei, the heavy chain amino acid sequence is shown as SEQ ID NO.3, and the light chain amino acid sequence is shown as SEQ ID NO. 4.
2. The kit for microbial detection according to claim 1, wherein the specific preparation method of the antibody specifically binding to the brucella brucei antigen is as follows: (1) Separating the brucella brucei from the excrement, preparing into insect powder, degreasing, cooling, ultrasonically crushing, and centrifuging to prepare brucella brucei antigen; (2) Taking the brucella brucei antigen prepared in the step (1) to immunize a mouse, and fusing spleen cells of the immunized mouse with myeloma cells to form hybridoma cells; (3) ELISA screening hybridoma secreting brucella brucei antibody, subcloning and amplifying culturing positive cell line, inoculating into mouse body, collecting ascites and purifying to obtain monoclonal antibody L3A-1 and monoclonal antibody F4B-2.
3. The kit for detecting microorganisms according to claim 1, wherein the kit further comprises an antigen standard, a washing solution, a color development solution, and a stop solution.
4. The kit for detecting microorganisms according to claim 3, wherein the antigen standard is a brucella brucei antigen, the washing solution is PBST, the color development solution is TMB color development solution, and the termination solution is sulfuric acid.
5. The kit for detecting microorganisms according to claim 4, wherein the concentration of the color development solution is 2mg/L to 4mg/L and the concentration of the termination solution is 1M to 3M.
6. Use of a kit for the non-diagnostic purpose for the detection of microorganisms according to any one of claims 1 to 5, characterized in that it is used in a method of: (1) Taking out the ELISA plate coated with the capture antibody, recovering to room temperature, washing the plate with a washing liquid and spin-drying, adding a sample to be detected and a gradient diluted antigen standard substance, incubating for 1-3h at room temperature, and washing the plate with the washing liquid and spin-drying; (2) Adding HRP-labeled antibody working solution into the system in the step (1), incubating for 1-3h at room temperature, washing the plate with washing solution, and spin-drying; (3) Adding a color development liquid into the system after the reaction in the step (2), developing color for 10-20min at room temperature in a dark place, and adding a stop solution to stop the reaction; (4) The two-wavelength detection is carried out by using an enzyme-labeled instrument, the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm is measured, and the result is calculated through a standard curve.
7. The use of a kit for the non-diagnostic purposes of a microbiological test according to claim 6, wherein the sample to be tested is blood.

Description

Kit for microorganism detection and application thereof Technical Field The invention belongs to the technical field of biological medicine, and particularly relates to a kit for detecting microorganisms and application thereof. Background The fasciola brucei is a large-scale fluke, mainly lodging in the small intestine of human body to cause fasciola brucei. The brucella zoonotic pathogen forms a transmission chain taking patients, animalcules and pigs as main infectious sources, wherein the pigs constitute a main storage host of natural epidemic places. After infection of the brucella brucei, patients may suffer from abdominal pain, diarrhea, abdominal distension, dyspepsia, anemia and edema, children may suffer from growth retardation, mental retardation and the like. Traditional brucellosis etiology diagnosis mainly relies on a direct smear method of excrement, namely, the infection of the brucellosis is confirmed by microscopic examination of eggs in the excrement, but the technical bottlenecks of poor patient compliance and low detection efficiency exist. In view of the limitations of the traditional method, the system of the invention discusses the application value of an enzyme-linked immunosorbent assay (ELISA) in serodiagnosis of the schistosomiasis brucei, aims at establishing an immunological detection method with high sensitivity and high specificity, and provides scientific basis for improving the clinical detection rate and constructing an auxiliary diagnosis system. The research has important practical significance for optimizing the prevention and control strategy of the parasitic diseases and perfecting the food-borne parasitic disease diagnosis technical system. Based on the above objects, the present invention provides a kit for microorganism detection and its application. Disclosure of Invention In order to overcome the defects in the prior art, one of the purposes of the invention is to provide a kit for detecting microorganisms. In order to achieve the above purpose, the present invention adopts the following technical scheme: the kit for detecting microorganisms comprises an ELISA plate coated with a capture antibody and an HRP-labeled antibody working solution, wherein the capture antibody and the HRP-labeled antibody are antibodies which specifically bind with the antigen of the brucella brucei; The capture antibody is a monoclonal antibody L3A-1 specifically combined with the antigen of the brucella brucei, the heavy chain amino acid sequence is shown as SEQ ID NO.1, and the light chain amino acid sequence is shown as SEQ ID NO. 2; the HRP-labeled antibody is a monoclonal antibody F4B-2 specifically binding to the antigen of the brucella brucei, the heavy chain amino acid sequence is shown as SEQ ID NO.3, and the light chain amino acid sequence is shown as SEQ ID NO. 4. Further, the specific preparation method of the antibody specifically binding to the antigen of the brucella brucei comprises the following steps: (1) Separating the brucella brucei from the excrement, preparing into insect powder, degreasing, cooling, ultrasonically crushing, and centrifuging to prepare brucella brucei antigen; (2) Taking the brucella brucei antigen prepared in the step (1) to immunize a mouse, and fusing spleen cells of the immunized mouse with myeloma cells to form hybridoma cells; (3) ELISA screening hybridoma secreting brucella brucei antibody, subcloning and amplifying culturing positive cell line, inoculating into mouse body, collecting ascites and purifying to obtain monoclonal antibody L3A-1 and monoclonal antibody F4B-2. Further, the kit also comprises an antigen standard substance, a washing liquid, a color development liquid and a stop solution. Further, the antigen standard is a brucella brucei antigen, the washing liquid is PBST, the color development liquid is TMB color development liquid, and the termination liquid is sulfuric acid. Further, the concentration of the developing solution is 2mg/L-4mg/L, and the concentration of the stopping solution is 1M-3M. The second object of the present invention is to provide an application of the kit for detecting microorganisms. According to the application of the kit for detecting microorganisms, the kit is used for detecting the antigen content of the brucella brucei in a blood sample. Further, the specific steps of the detection are as follows: (1) Taking out the ELISA plate coated with the capture antibody, recovering to room temperature, washing the plate with a washing liquid and spin-drying, adding a sample to be detected and a gradient diluted antigen standard substance, incubating for 1-3h at room temperature, and washing the plate with the washing liquid and spin-drying; (2) Adding HRP-labeled antibody working solution into the system in the step (1), incubating for 1-3h at room temperature, washing the plate with washing solution, and spin-drying; (3) Adding a color development liquid into the system after the reaction in the step (