Rapid heat-mediated method for enzyme-linked immunosorbent assay procedure

An enzyme-linked immunosorbent assay (ELISA), a fast technology, used in analytical materials, biochemical equipment and methods, and microbial determination/inspection. It can solve problems such as long-term incubation, shedding of biomolecules, and low sensitivity, saving time and time. , reduce time-consuming, simple effect

Inactive Publication Date: 2006-05-10
COUNCIL OF SCI & IND RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The traditional ELISA procedure is carried out by immobilization of antigen or antibody on polystyrene microtiter plate by adsorption, which has the following disadvantages: (i) ELISA values ​​are usually not consistent in different wells and plates (Kricka, L.J.) et al., 1980; Hermann (Hermann, J.E.) and Collins (Collins, M.F.), 1976); (ii) requires long incubation; (iii) during the washing procedure, some biomolecules are detached resulting in no reproducible results (Engvall, E. and Perlmann, P., 1971); and (iv) generally give lower sensitivity (Kemeny, 1997)
This ELISA step step too sensitive

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1 Activation of polycarbonate and polystyrene surfaces

[0084] Add 50 μl methanol-dissolved 6.0 μmol of 1-fluoro-2-nitro-4-azidobenzene (FNAB) per well to the small wells of a PCR plate (polycarbonate plate, Greiner, Germany), and completely in the dark dry. The wells coated with FNAB were then placed in a UV Stratalinker 2400 (Stratagene  , USA) in the instrument with 365nm UV radiation for 7.5 minutes, or in bright sunlight for 15 minutes. Similarly, polystyrene pores were activated with 50 μl of methanol-dissolved 10 μmol of FNAB, irradiated at 365 nm for 10 minutes, or kept in sunlight for 15 minutes.

[0085] The wells were washed several times with methanol to remove unbound linkers and dried at room temperature. These activated pores can be used for immobilization of antigen or antibody in the step of the present invention.

Embodiment 2

[0086] Example 2 Determination of Goat Anti-Human IgG Concentration in order to Prepare Solid Phase on Activated Polycarbonate Surface

[0087] Doubling dilution series (2000-0.061g / ml) of goat anti-human IgG (dissolved in 0.1M carbonic acid / dicarbonate buffer, pH 9.6) was added to three parallel wells of activated and untreated polycarbonate plates. wells (90 μl / well for each dilution). The plates were incubated in a thermal cycler at 50°C for 1 hour. It was then washed six times with washing buffer (0.05% Tween 20 containing 0.01M PBS) and incubated with 2% BSA solution (100 μl / well) at 37° C. for 1 hour to complete the blocking step. After washing, 90 µl of a human IgG solution at a concentration of 250 ng / ml (dissolved in 0.01 M PBS, pH 7.4) was added to each well, and the plates were incubated at 37°C for 3 hours. The plates were washed again six times, and 90 μl of 1 / 5000 (v / v) goat anti-human IgG-peroxidase conjugate solution (dissolved in 0.01 M PBS, pH 7.4) was adde...

Embodiment 3

[0088] Example 3 Optimization of Goat Anti-Human IgG Solidification Temperature on Activation and Untreated Polycarbonate Plates (Table 1)

[0089] Add goat-anti-human IgG solution (250ng / ml solution, 90μl / well) to three parallel wells of 8 activated polycarbonate plates, and heat them in a thermal cycler at 35°C, 40°C, and 45°C respectively. , 50°C, 55°C, 60°C, 65°C and 70°C for 60 minutes. Goat-anti-human IgG was similarly immobilized on untreated polycarbonate plates. The remaining steps of the ELISA were then carried out on these solid phases in the conventional ELISA method described in Example 2.

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Abstract

This invention relates to a rapid and efficient method for carrying out enzyme-linked immunosorbent assay for detection of minute quantities of biomolecules such as antigen, antibody etc. This invention particularly relates to heat-mediated immobilization of antigen or antibody on to the activated surface followed by performing subsequent steps of ELISA by controlled temperature. The invented procedure has reduced the total time required for ELISA to around 3 h. The invented ELISA procedure is rapid, economical, reproducible and simple. The invented procedure is useful for carrying out ELISA required in clinical diagnostics, molecular biology, agriculture, food technology, environmental science, etc. The invented ELISA method is simple, time saving and obviates the time consuming procedure. This method has the potential for automation.

Description

field of invention [0001] The present invention relates to a rapid and efficient method for enzyme-linked immunosorbent assay for the determination of trace biomolecules such as antigens and antibodies. The present invention particularly relates to the heat-mediated immobilization of antigens or antibodies on said activated surface, followed by subsequent ELISA steps by controlling the temperature. The invented procedure reduces the total time required for the ELISA to around 3 hours. The invented ELISA procedure is fast, economical, reproducible and simple. [0002] The invented procedure is quite useful in ELISA which needs to be performed in clinical diagnosis, molecular biology, agriculture, food engineering, environmental science and the like. The invented ELISA method is simple, time-saving and reduces time-consuming steps. This approach has the potential to be automated. Background of the invention [0003] Enzyme-linked immunosorbent assay (ELISA) is an extremely...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543C12Q1/68
CPCG01N33/543G01N33/54393
Inventor 普拉蒂普·纳哈尔乌特帕尔·博拉
Owner COUNCIL OF SCI & IND RES
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