ELISA detection method of red-tide algae toxin okadaic acid (OA)

A red tide algae toxin and detection method technology, applied in the field of ELISA detection of red tide algae toxin halicic acid OA, can solve the problems of inability to distinguish the type and structure of the toxin, complex processing process, and inaccurate data, so as to reduce false positives, High sensitivity and good effect

Inactive Publication Date: 2009-12-09
SHANGHAI OCEAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The mouse method is simple and easy to implement, but the disadvantage is that it cannot distinguish the type and structure of the toxin, which is greatly disturbed and the data is inaccurate; the HPLC method can accurately analyze the content and type of the toxin, and the detection limit can be as low as ng / g, but before the sample The processing process is complex and the instruments are expensive, requiring specialized analytical technicians; the ELISA method is much simpler than the HPLC method sample pretreatment, with good specificity and no need for purification and concentration

Method used

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  • ELISA detection method of red-tide algae toxin okadaic acid (OA)
  • ELISA detection method of red-tide algae toxin okadaic acid (OA)
  • ELISA detection method of red-tide algae toxin okadaic acid (OA)

Examples

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Effect test

Embodiment 1

[0024] Embodiment 1: raw material preparation

[0025] The materials used in this example are OA standard (purchased from Taiwan Algae Biotechnology Co., Ltd.); coating antigen OA-OVA, OA monoclonal antibody, enzyme-labeled secondary antibody HRG-goat anti-mouse IgG (purchased from Shanghai Dingguo Biotechnology Co., Ltd.); newborn bovine serum, 1% gelatin, ovalbumin (OVA), bovine serum albumin (BSA), N-hydroxysuccinimide (N-hydroxysuccinimide), N, N dicycloethane carbon di Imine (N, N-dicyclohexylcarbodiimide, DCC), N, N-dimethylformamide (N, N-dimethylformamide, DMF), polyethylene glycol (polythylene glycol-4000, PEG), Freund's complete adjuvant, Freund's incomplete adjuvant, tetramethylbenzidine (TMB), dimethyl sulfoxide (DMSO), MES buffer, etc. were products of Sigma; SP2 / 0 myeloma cells (from Shanghai Veterinary Research Institute, National Academy of Agricultural Sciences) ); the rest were domestic analytically pure.

[0026] Preparation of coating antigen OA-OVA:

[...

Embodiment 2

[0034] Example 2: Obtaining of OA indirect competition ELISA standard curve

[0035]The OA-OVA conjugate was used as the coated antigen, diluted with carbonate buffer to 1:2000, coated with 96-well microtiter plate, 100ul per well, overnight at 4°C; the residual solution was discarded and washed with phosphoric acid Wash the plate with salt buffer PBST (containing 0.05% Tween-20) 3 times, 200ul per hole, and then pat dry; seal the microplate plate with 1% gelatin, 150ul per hole, and incubate at 37°C for 2h; discard the blocking solution, wash plate, dilute the OA standard solution to 200, 100, 50, 25, 12.5, 6.25, 5, 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078ng / ml, 0ng / ml (blank control) with PBST, respectively Shake and mix an equal volume of monoclonal antibody diluted at 1:10000, that is, the final dilution of monoclonal antibody is 1:20000, add 100ul to each well, and incubate at 37°C for 1h; discard the residual solution, wash the plate, and add ELISA II Antibody, diluted ac...

Embodiment 3

[0037] Example 3: ELISA detection of red tide algae toxin halicic acid OA

[0038] a. Sample processing:

[0039] Obtaining Shellfish Extract:

[0040] Clean the shellfish samples, remove impurities such as silt on the surface, drain after shelling, and homogenize the organisms with a high-speed dispersing homogenizer. Weigh 2 parts of homogenized shellfish meat samples, each 5g, place them in clean centrifuge tubes respectively, add weakly acidic methanol solution, add a certain amount of OA standard solution to one part, and not add to the other part (i.e. negative control). Centrifuge at 3500r / min for 10min, and take the supernatant. Add methanol and n-hexane to the supernatant in turn, vortex and mix well, and let the layers stand still. Then discard the n-hexane layer, evaporate to dryness on a rotary evaporator, then add a small amount of methanol to dissolve the attachment on the wall, and then evaporate to dryness. Finally, dissolve the residue with 100ul methanol...

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Abstract

The invention provides an ELISA detection method of red-tide algae toxin okadaic acid (OA) and has the advantages of quickly, simple and sensitive detection of the OA content and low cost. The ELISA detection method of the invention comprises the following steps: (1) diluting coated antigens by using carbonate buffer solution to 1:2000, coating ELISA plates; (2) washing the plates for three times and sealing the plates; (3) dumping the blocking solution, washing the plates, separately stirring and mixing OA standard solution which is diluted to series concentrations and shellfish extract liquid with isometric anti-OA monoclonal antibody which is diluted to 1:10,000 evenly to react; (4) dumping the residual liquid, washing the plates, adding goat anti-rat IgG antibody labeled with HRP, diluting the solution to 1:5000 with each pore containing 100mu l of solution, keeping temperature for 1h at 37 DEG C; (5) dumping the residual liquid, washing the plates, adding developer and measuring the OD450 value of each pore.

Description

Technical field: [0001] The invention relates to an ELISA (enzyme-linked immunosorbent) detection method, in particular to an ELISA detection method for the red tide algae toxin soft sponge acid OA. Background technique: [0002] Diarrheal shellfish poisoning (DSP) is a class of fat-soluble natural compounds produced by some species of toxic red tide algae Dinophysis and Prorocentrum. The main components are soft spongy acid (OA) and its derivative DTXs. The symptoms of poisoning caused by DSP include diarrhea, nausea, vomiting, and pain in the lower abdomen, which usually occur within a few hours after consumption, or as short as 30 minutes in severe cases, and there is no effective drug treatment. [0003] Diarrheal shellfish toxin poisoning incidents often occur in the South my country Sea. Diarrheal shellfish toxin has become a major obstacle to fishery economic development and public health. Therefore, it is very important to establish a fast and accurate DSP toxin scre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
Inventor 何培民柳俊秀胡乐琴王权李春霞
Owner SHANGHAI OCEAN UNIV
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