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Multistage enlarging fluoresconce immune analysis method

A fluorescence immunoassay, fluorescence analyzer technology, applied in fluorescence/phosphorescence, material excitation analysis, material inspection, etc., can solve the problems of scattered light reducing signal/noise ratio, low sensitivity, failure to achieve A-B multi-stage amplification, etc. , to solve the space obstacle and ensure the effect of multi-level amplification

Inactive Publication Date: 2006-06-28
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Fluorescence immunoassay technique (FIA) has attracted more attention in the field of ultra-micro immunoassay due to its non-radioactivity, instability of enzyme reagents and limitation of validity period; however, there are two problems in FIA: - it is produced in the liquid phase The scattered light reduces the signal / noise ratio; the second is that the fluorescent signal detected directly is much lower than the ionization amplification of radioimmunoassay (RIA) and the enzyme substrate amplification of enzyme-linked immunoassay (ELISA), resulting in low sensitivity of the technical analysis
[0007] The above method does not significantly change the structure between A-B, but only increases the length between the primary antibody and B, so the multi-stage amplification between A-B cannot be realized, and the sensitivity of the analysis is not ideal enough

Method used

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  • Multistage enlarging fluoresconce immune analysis method
  • Multistage enlarging fluoresconce immune analysis method
  • Multistage enlarging fluoresconce immune analysis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Serum HBsAg Determination

[0080] Weigh the amount of reagents and samples added according to Table 1:

[0081] test group

[0082] PcAb-MD, HBsAg, and B-McAb were incubated at 37°C for 2 hours, centrifuged at 8000 rpm for 30 minutes, added 200 μL of washing solution, and washed three times.

[0083] Incubate A and B-R-PE for 40 minutes, centrifuge, wash three times, combine the two and add activation buffer, incubate at 37°C for 30 minutes, wash three times, add 100 μL of activation buffer, and measure the fluorescence intensity.

[0084] Measurement results: R-PE λex 542nm; λem 572nm, the data given by the instrument is the light intensity value of the emitted wave (wavelength 572nm), and three parallel samples were measured for each sample. The results are shown in Table 2.

[0085]

[0086] Conventional ELISA experiment control ratio ≥ 2.1 can be diagnosed as positive (that is, the test subject has been infected by hepatitis B viru...

Embodiment 2

[0087] Embodiment 2. detect hepatitis B e antibody (anti-HBe)

[0088] Implementation Principle Marking

[0089]

[0090] Take HBeAg150ug / 10ul, PSMD (1.3×10 7 Each / ml) 1ml is prepared by double activation of EDC and Sulfo-NHS to prepare HBeAg-coupled PSMD (anti-HBs-coupled PSMD); the method is the same as above (coupled PcAb method in steps and conditions)

[0091] B-labeled goat anti-human IgG

[0092] Use 150 μg goat anti-human IgG body, 20 times the molecular ratio of Sulfo-NHS-LC-B labeling method as before (steps and conditions ②) label Mc anti-HBs method.

[0093] The B-labeled R-PE method and A are shared with the corresponding reagents for measuring HBsAg in the above (3) in the steps and conditions; the determination method is also the same. Results: The standard quality control serum is diluted 10 times, and the conventional ELISA method is negative. This method is diluted 10 times 3 times are still positive.

Embodiment 3

[0094] Example 3. Detection of hepatitis B virus core antibody (anti-HBc).

[0095] Principle and basic method are identical with embodiment 2.

[0096] ●Label 1.3×10 with 150 mg of hepatitis B core antigen (HBcAg) 4 PSMD,

[0097] ● B labeled goat anti-human IgG, B labeled R-PE and A are the same as those in Example 2 above. The detection method is the same as in Example 2, and the results show that: the standard quality control serum is diluted 10 times, and the conventional ELISA method is negative; this method is still positive when diluted 103 times.

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Abstract

A fluorescent immune analysis method of multistage amplification applies R ¿C PE containing 16 carbon long ¿C chain succinamides ¿C biotin label to lengthen distance between R - PE molecule and A and distance between R¿C PE and PSMD for eliminating í

Description

technical field [0001] The invention belongs to the technical field of fluorescence immunoassay. technical background [0002] Fluorescence immunoassay technique (FIA) has attracted more attention in the field of ultra-micro immunoassay due to its non-radioactivity, instability of enzyme reagents and limitation of validity period; however, there are two problems in FIA: - it is produced in the liquid phase The scattered light reduces the signal / noise ratio; the second is that the fluorescent signal detected directly is much lower than the ionization amplification of radioimmunoassay (RIA) and the enzyme substrate amplification of enzyme-linked immunoassay (ELISA), resulting in low sensitivity of the technical analysis . The former can be solved by using time-resolved, fluorescent microbead technology and flow cytometry (Flow Cytometry). Improved, but still lower than RIA and ELISA (immunofluorescence and related staining techniques, edited by W.Knapp., etc., Elsevier / North...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N21/64
Inventor 潘利华马世盐钮倩曲晓春
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI