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ELISA reagent kit for on-site detection

An on-site detection and kit technology, applied in biological testing, material inspection products, etc., can solve problems such as inconvenience in carrying and mailing, failure of kits, impact of antibody and enzyme titers, etc., to increase biological safety and increase resistance. The effect of increasing the range and seismic performance

Active Publication Date: 2013-03-20
HANGZHOU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 1. Because the kit contains various liquid reagents such as antibody solution, enzyme marker solution, sample dilution buffer (concentrate), etc., the kit needs to be refrigerated, which makes it inconvenient to carry and mail; and the kit is in use. The titers of antibodies and enzymes are susceptible to being affected by repeated rewarming, and losses and contamination are easily caused during the process of absorbing solutions for many times. Sometimes, all kits will become invalid due to power failure or refrigerator failure.
[0006] 2. Poor biological safety: the quality control samples of the commonly used plant virus detection kits are obtained by directly freeze-drying the extracts of positive or negative samples
Although the virus is easily inactivated under dry conditions at room temperature, if it is frozen and then vacuum-dried, the virus can survive for a long time
[0007] 3. ELISA is a test with very fine operation requirements. At present, the alkaline phosphatase-labeled ELISA reagent lacks a suitable indicator, and the operation process is prone to errors, especially in the TSA-ELISA (three-antibody sandwich type) kit. Detection antibodies and enzyme-labeled anti-antibodies are two colorless and trace precious reagents. If they are not marked clearly, mistakes are more likely to occur.
[0008] 4. The currently commonly used 3mol / L sodium hydroxide terminates the alkaline phosphatase reaction, and the result can only be maintained for several hours, and sodium hydroxide is corrosive

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Preparation and use of TAS-ELISA detection kit for Cymbidium mosaic virus (CyMV) on-site detection. The kit contains ①antibody detection plate, ②sample dilution reagent, ③washing reagent, ④quality control (positive / negative control), ⑤ enzyme conjugate reagents (including detection antibodies, enzyme-labeled anti-antibodies, diluents), ⑥ substrate reagents, ⑦ termination reagents, ⑧ some auxiliary equipment.

[0085] 1. The specific preparation process is as follows:

[0086] 1.1 Antibody detection plate:

[0087] Preparation of coating buffer, containing Na 2 CO 3 1.59g / L, Na 2 HCO 3 2.93g / L, NaN 3 0.2g / L to adjust the pH to 9.6. Take 50 microliters of coating antibody (rabbit anti-Jianlan mosaic virus, produced by American company Agdia) and dilute it 1:200 times with coating buffer (that is, add 9.95 mL of coating buffer per 50 μL to prepare 10 mL of use solution) , on a detachable 96-well high-affinity microtiter plate (produced in Canada), add 100 microl...

Embodiment 2

[0175] TAS-ELISA detects the preparation and use of CyMV on-site detection kit, the difference with embodiment 1 is only described below: the carrier used is a cotton ball; the detection antibody does not contain an indicator, and an indicator is added on the enzyme-labeled anti-antibody Reagent I; The indicator I used is acid chrome blue K; The indicator II is preserved in a small centrifuge tube.

[0176] 1.5.2 Preparation of temporary solid carrier for antibody: Dissolve 0.05 g of Guangming brand skimmed milk powder in 10 ml of enzyme conjugate dilution buffer → knead the absorbent cotton into small balls of 3-4 mm (mung bean size), soak for 2 hours, during each Stir once every 30 minutes → soak in washing buffer 4-6 times → control the water, spread it on a clean glass slide, dry it in a desiccator, and store it in a desiccator for later use. Thereby obtaining a temporary solid carrier of the antibody.

[0177] 1.5.3 Preparation of absorbent cotton pellets containing dete...

Embodiment 3

[0184] DAS-ELISA detection of Potato Leaf Roll Virus (Potato Leaf Roll Virus) (antibody produced by agdia company) field detection kit preparation and use. Only the difference from Example 1 is described below: 1. The enzyme conjugate reagent only contains enzyme-labeled antibody and enzyme conjugate diluent; 2. The indicator is added to the enzyme conjugate diluent, and the indicator used is methyl red.

[0185] 1. Prepare enzyme conjugate diluent: weigh Na 2 HPO 4 0.620g, KH 2 PO 4 0.100g, and NaCl 4.00g, KCl 0.10g, NaN 3 0.10g, bovine serum albumin 1.0g, polyvinylpyrrolidone K3010±1g, Tween -20 0.25±0.05g→dissolve reagent in 80ml pure water→add 1mL of 1g / L methyl red indicator, mix well→use 0.10g / mL Na 2 HPO 4 solution; or 0.02g / mL KH 2 PO 4 Adjust the pH of the solution to 7.03→concentrate to 100mL to obtain a 5-fold concentrated solution of ECI→test: take 2mL and dilute it with water to 10mL to obtain the enzyme conjugate dilution buffer (pH 7.40~7.42)→fill e...

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Abstract

The invention discloses an ELISA kit used for on-site detection. The kit comprises a kit body; an antibody detection plate, a sample dilution reagent, positive / negative contrast, a cleaning reagent, an enzyme conjugate reagent, a substrate reagent, a stopping reagent, a disposable dropper and a cover plate membrane are arranged in the kit body; the substrate reagent is composed of a substrate, a substrate diluent and an indicator II; the substrate diluent is a concentrated solution; the sample dilution reagent, the cleaning reagent, the enzyme conjugate reagent, the stopping reagent, the substrate and the indicator II are all solid. The ELISA kit has the advantages of great portability, convenient use and high safety. Furthermore, the requirement of the on-site detection can be met.

Description

technical field [0001] The invention relates to an ELISA kit for on-site detection. Background technique [0002] Enzyme-linked immunosorbent assay (ELISA) is a detection technique that combines the catalytic reaction of enzymes with the immunological reaction of antigens and antibodies. Due to the high specificity of the antigen and antibody reactions, coupled with the high catalytic efficiency of the enzyme, the result of the immune reaction is indirectly amplified, so the determination method can achieve high specificity and sensitivity. In recent years, ELISA has been widely used in the detection of viruses, bacteria, hormones, and drug residues due to its sensitivity, accuracy, good repeatability, and no radioactive contamination. [0003] However, the currently used ELISA kits need to be refrigerated and stored in the refrigerator because the reagents such as antibodies and enzyme markers are liquid, and cooling measures such as ice packs and incubators are also requi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/53
Inventor 柳爱春赵芸刘超邹礼根童朝明徐晓丹
Owner HANGZHOU ACAD OF AGRI SCI
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