Novel crytococcus neoformans GXM (glucuronoxylomannan) antigen immunodetection kit as well as preparation method and application thereof

A cryptococcus neoformans detection kit technology, applied in the field of immune detection, can solve the problems of large limitations, poor sensitivity, and low positive detection rate

Inactive Publication Date: 2016-06-08
DYNAMIKER BIOTECH TIANJIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 1) Indian ink staining method: This method has poor sensitivity, low positive detection rate, and large limitations;
[0007] 2) Culture methods such as blood culture or cerebrospinal fluid culture: this method has poor sensitivity and the positive detection rate is not high;
There are no immunological detection products for the clinical detection of Cryptococcus neoformans in my country

Method used

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  • Novel crytococcus neoformans GXM (glucuronoxylomannan) antigen immunodetection kit as well as preparation method and application thereof
  • Novel crytococcus neoformans GXM (glucuronoxylomannan) antigen immunodetection kit as well as preparation method and application thereof
  • Novel crytococcus neoformans GXM (glucuronoxylomannan) antigen immunodetection kit as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] The preparation of embodiment 1 anti-GXM polyclonal antibody

[0102] 1. Immunization of animals

[0103] Mix equal volumes of GXM antigen and complete Freund's adjuvant to an appropriate volume. After full emulsification, New Zealand big-eared rabbits were injected subcutaneously at multiple points, and the immune dose of each rabbit was controlled at 0.01-1 mg. Ear blood was collected 3 days before immunization, and serum was separated as a negative control. After the initial immunization, immunize once every 2 weeks, and the method is the same as the first time.

[0104] 2. Obtaining polyclonal antibodies

[0105] 1) Titer determination: During the immunization process, blood was collected every few days to measure the titer once after immunization, and the number of immunizations was not less than 3 times.

[0106] 2) Separation of antiserum: When the serum titer reaches the highest level, a large amount of blood is collected by carotid artery bleeding. After the...

Embodiment 2

[0117] The detection of embodiment 2 anti-GXM polyclonal antibodies

[0118] 1. SDS-PAGE electrophoresis detection

[0119] The anti-GXM polyclonal antibody prepared in Example 1 was subjected to SDS-PAGE electrophoresis, and the resulting gel was stained with Coomassie brilliant blue. See the experimental results figure 1 (The pAb swimming lane is the anti-GXM polyclonal antibody prepared in Example 1, and the M swimming lane is the protein Marker). As can be seen from the figure, there are clear and obvious bands in the 25kD and 50kD molecular weight regions, which are respectively the hydrogen chain and the heavy chain of the antibody protein, and there are no bands of foreign proteins, which illustrate the anti-GXM polyclonal antibody prepared in Example 1. The purity is very high.

[0120] 2. Potency determination

[0121] Antibody titers were determined by indirect ELISA. The enzyme-labeled secondary antibody used was horseradish peroxidase-labeled goat anti-rabbit ...

Embodiment 3

[0122] Example 3 Preparation of Cryptococcus neoformans capsular polysaccharide antigen immunoassay kit

[0123] 1. Preparation of enzyme-labeled carrier

[0124] ① Dilute GXM to 25ng / 100μL with coating buffer to obtain GXM coating solution and add it to the wells of the microplate, add 200μL of coating solution to each well, and place the microplate at 2-8°C for 8 hours;

[0125] ②Add blocking solution to the wells of the microplate, add 200 μL of blocking solution to each well, and place at 37°C for 30 minutes to block;

[0126] ③ Discard the blocking solution and place it at a constant temperature of 37°C for 30 minutes to obtain the GXM-coated enzyme-labeled carrier;

[0127] The coating buffer is a 0.01mol / L PBS buffer solution with a pH of 7.0-7.4;

[0128] The preparation of the blocking solution: 0.01mol / L PBS buffer solution containing 3% skimmed milk powder with a pH of 7.0-7.4;

[0129] 2. Preparation of GXM standard

[0130] Dilute GXM antigen with sample dilue...

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Abstract

The invention belongs to the technical field of immunodetection and relates to a novel crytococcus neoformans GXM (glucuronoxylomannan) antigen immunodetection kit. The kit comprises an enzyme-labelled carrier coated with GXM, an anti-GXM polyclonal enzyme-labelled antibody and a GXM standard substance, wherein GXM is obtained from a novel crytococcus neoformans culture solution through extraction and purification; according to the kit, an enzyme-labelled plate is coated with GXM firstly, then a to-be-detected sample or a standard antigen and a coating antigen are subjected to competitive binding with limited antibody binding sites, then an enzyme and a substrate have a chromogenic reaction, and the concentration value of a to-be-detected antigen is calculated according to the detection result. The detection kit has better sensitivity, specificity, repeatability and stability, is higher in recovery rate of target compounds, can provide more accurate and reliable detection results, is convenient and easy to use and operate and provides an effective tool for clinical detection of GXM.

Description

technical field [0001] The invention relates to the technical field of immunoassay, in particular to a Cryptococcus neoformans capsular polysaccharide (Glucuronoxylomannan, GXM) antigen immunoassay kit and its preparation method and application. Background technique [0002] Cryptococcus neoformans (Crytococcus Neoformans) is an important opportunistic pathogen that often infects immunocompromised or immunocompromised patients, leading to deep fungal infections, mainly central nervous system infections, with a high mortality rate. A large series of epidemiological studies conducted by the US Centers for Disease Control and Prevention (CDC) in 1992-1993 showed that the annual incidence of deep fungal infection was 178.3 / million. Among them, cryptococcosis was 65.5 / million, accounting for about 36.7%. [0003] In recent years, due to the long-term and extensive application of broad-spectrum antibacterial drugs, adrenocortical hormones, tumor chemotherapy, radiotherapy and imm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56911
Inventor 彭洁史东东李宁粟艳周泽奇
Owner DYNAMIKER BIOTECH TIANJIN
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