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Mannoprotein of T. marneffei, antibody, detection reagent and kit

A mannoprotein, Marneffei technology, applied in antifungal/algae/lichen immunoglobulin, biological testing, measuring devices, etc., can solve the problem of long detection time, achieve strong product stability, large output, batch small difference effect

Active Publication Date: 2022-06-28
DYNAMIKER BIOTECH TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In summary, the T. marneffei antibody detection technology in the prior art not only has cross phenomenon with Aspergillus infected samples, but also takes a long time to detect

Method used

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  • Mannoprotein of T. marneffei, antibody, detection reagent and kit
  • Mannoprotein of T. marneffei, antibody, detection reagent and kit
  • Mannoprotein of T. marneffei, antibody, detection reagent and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] As antigen markers, the chemiluminescence indirect method was used to detect anti-specific antibodies in serum samples, and a detection kit for T. marneffei mannoprotein IgG antibody was established based on the magnetic particle chemiluminescence indirect method.

[0036] The T. marneffei mannoprotein IgG antibody detection kit includes the following components:

[0037] Specific antigens coupled with carboxyl magnetic beads: the concentration of carboxyl magnetic beads is 0.4 mg / mL, and the antigen concentration is 10 μg / mg carboxyl magnetic beads;

[0038] Acridinesulfonamide-labeled mouse anti-human IgG antibody: antibody concentration 0.2 μg / mL;

[0039] QC1 negative control: diluent (without patient serum);

[0040] QC2 positive control; diluent containing patient serum;

[0041] Diluent: 0.01M PBS (pH7.4), 0.5% BSA, 0.05% ProClin300;

[0042] The specific detection process is as follows: add 10 μL of sample, 90 μL of diluent and 30 μL of carboxyl magnetic bead...

Embodiment 2

[0048] In this example, the specific secreted antigen of T. marneffei is used to immunize mice to obtain paired monoclonal antibodies for detection. The specific operation process is as follows: immunize mice with antigens, immunize mice with lymphocytes and myeloma cells of selected mice Perform fusion to form hybridoma cells, screen positive hybridoma cells that can produce the desired monoclonal antibody by ELISA, and carry out clone amplification, and verify the antibody pairing of the cloned antibody to obtain the desired capture antibody and detection antibody .

[0049] Based on the magnetic particle chemiluminescence method, a T. marneffei antigen detection kit was established. The T. marneffei mannoprotein IgG antibody detection kit includes the following components:

[0050] Carboxyl magnetic beads-coupled capture antibody: carboxyl magnetic beads concentration 0.4 mg / mL, capture antibody concentration 10 μg / mg carboxyl magnetic beads;

[0051] Acridine ester-label...

Embodiment 3

[0062] In this example, a one-step method is used to test the influence of biotin. The specific operation steps of the one-step method are as follows: 100 μL of the human sample to be tested, 40 μL of the capture antibody labeled with 0.4 mg / mL carboxyl magnetic particles, and 1 μg / mL of acridinesulfonamide labeled detection Mix 100 μL of antibodies, incubate at 37°C for 5 min, wash once to remove unbound labels, etc., add excitation solution to generate light signal, and the luminescence intensity is positively correlated with the concentration of the analyte in the sample. The specific experimental results are shown in the following table.

[0063]

[0064]

[0065] As can be seen from the above table results, the detection time of the present invention is short, only 10min; adding biotin concentration to 640ng / mL in the sample near the critical value (I value 1.0), the negative and positive values ​​of the reference product are not changed, it can be seen that the pres...

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Abstract

The invention provides a T. marneffei mannoprotein, an antibody, a detection reagent and a kit. The antigen is T. marneffei mannoprotein, and its amino acid sequence is as shown in SEQ.ID.No.1 shown. The antigen marker of the present invention is a highly specific secreted antigen, which has no obvious cross-sequence with Aspergillus protein, and there is no false positive phenomenon in Aspergillus-infected samples through subsequent cross-validation; moreover, the antigen is a gene expression product, and the product has strong stability and is widely used. The interval difference is small, and the output is large.

Description

technical field [0001] The invention and creation belong to the field of T. marneffei detection, in particular to a T. marneffei mannoprotein and a kit prepared by using the protein. Background technique [0002] Most of the T. marneffei antibody detection techniques reported in the literature use mannoprotein MP1 as a marker for T. marneffei, but studies have shown that Aspergillus-infected samples and MP1 have crossover phenomena, indicating that Aspergillus contains proteins similar to MP1. . [0003] In the ELISA method reported in the existing literature to detect T. marneffei antibody detection technology, using MP1 as a marker, an indirect method is used to detect the antibody in the serum to be tested. The serum in the sample needs to be diluted and added to the ELISA plate, incubate and wash, add ELISA reagent, incubate and wash, then add the chromogenic substrate, incubate, terminate the reaction and read. The reading value is positively correlated with the conce...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/37C07K16/14G01N33/577G01N33/569G01N33/58G01N33/543
CPCC07K14/37C07K16/14G01N33/577G01N33/56961G01N33/585G01N33/54326G01N2333/37G01N2469/10
Inventor 刘春龙张舟张玉静柏华松
Owner DYNAMIKER BIOTECH TIANJIN