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Preparation method of beta-casein monoclonal antibody, beta-casein colloidal gold test strip and preparation method thereof

A monoclonal antibody and detection test paper technology, applied in the biological field, can solve the problems of poor casein specificity, complicated operation, and need pretreatment, and achieve the effects of good specificity, simple operation and high resolution

Inactive Publication Date: 2014-06-18
长沙安迪生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The Chinese patent application with application number 200810007526.7 "A Method for Detection of Casein Content in Milk" discloses a method for detecting casein content by adding a casein precipitant to milk, filtering and removing casein in milk, and removing casein by measurement. The difference between the protein content before and after casein is used to calculate the casein content in milk. This method is complicated to operate and is not suitable for on-site operation; the Chinese patent application publication number is 200810146950.X "A method for detecting β-casein content in milk" A method for detecting the content of casein in milk by capillary electrophoresis is developed. This method needs to process the milk sample. The minimum detection line of this method is 0.15ng, the minimum detection concentration is 0.005mg / mL, and the accuracy reaches 90%. Above, this method needs pretreatment, and needs special instrument and equipment, is not suitable for field operation; αs1-casein and κ-casein serum antibodies, kits, and test strip preparation methods, the method is poorly specific for casein, and the validity of the test results is low

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  • Preparation method of beta-casein monoclonal antibody, beta-casein colloidal gold test strip and preparation method thereof
  • Preparation method of beta-casein monoclonal antibody, beta-casein colloidal gold test strip and preparation method thereof

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Embodiment 1

[0039] The preparation of embodiment 1β-casein monoclonal antibody

[0040] Animal procedure: use β-casein as antigen, immunize 100 μg / mouse with an equal volume of complete Freund's adjuvant and fully emulsify it, then immunize 2 Balb / c mice by intraperitoneal multi-point injection, and then use again every 2 to 3 weeks The same dose of antigen was emulsified with Freund's incomplete adjuvant and immunized 2-3 times, and boosted once three days before fusion.

[0041] Cell fusion and cloning: Splenocytes from immune Balb / c mice were fused with myeloma cell SP2 / 0 at a ratio of 5:1, and the cell supernatant was measured by indirect competition ELISA to screen positive wells. Positive wells were cloned by limiting dilution until a hybridoma cell line stably secreting anti-β-casein monoclonal antibody was obtained.

[0042] Cell cryopreservation and recovery: Take the monoclonal hybridoma cell line in the logarithmic growth phase and make 5×10 6 cells / mL of cell suspension, ali...

Embodiment 2

[0044] Example 2 Preparation of colloidal gold-labeled β-casein monoclonal antibody complex

[0045] Preparation of colloidal gold particles: Take 1mL of 1% chloroauric acid solution, add 99mL of ultrapure water to form a chloroauric acid solution with a final concentration of 0.01%, after heating and boiling, take 1.6mL of 1% trisodium citrate and quickly add it to the boiled In the chloroauric acid solution, continue to heat until the solution turns from light yellow to blue-black and finally to bright red. After the color is stable, continue to heat for 5 minutes, cool at room temperature, and add water to the original volume.

[0046] Preparation and purification of gold-labeled antibody: use 0.1mol / L K 2 CO 3 Adjust the pH of the colloidal gold solution to 7.0-8.5, and add 5-20 μg of β-casein monoclonal antibody per milliliter of gold solution while stirring evenly, and add all the β-casein monoclonal antibodies within 5 minutes. Polyethylene glycol (PEG MW20000), after...

Embodiment 3

[0047] The assembly of embodiment 3β-casein colloidal gold detection test strips

[0048]Properly dilute the gold-labeled antibody, spray it on the binding pad with a gold spray machine, and dry it at 37°C. Dilute β-casein with 0.01mol / L pH7.4 phosphate buffer solution to 1.5-2mg / mL, and spray it on the cellulose film with a film sprayer, which is the detection line; sprayed at a distance of 5 mm above the detection line 2mg / mL goat anti-mouse antibody, this is the quality control line. After drying at 37°C, assemble the cellulose membrane, gold standard binding pad, sample pad, and water-absorbing pad on the liner in sequence, and cut it into a suitable width with an automatic cutter to form a β-casein colloidal gold test strip.

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Abstract

The invention relates to the technical field of biology and provides a preparation method of a beta-casein monoclonal antibody, a beta-casein colloidal gold test strip and a preparation method thereof. The technical scheme is as follows: immunizing a BALB / c mouse by taking beta-casein as an antigen, taking mouse splenocytes with best immune effects to perform fusion with myeloma cells SP2 / 0, taking beta-casein, alpha s1-casein and K-casein as detection antigens, detecting supernatant of hybridoma cells by enzyme-linked immunosorbent assay, screening, retaining positive holes, further cloning by using a limiting dilution method to obtain a hybridoma cell strain which can stably secrete the anti-beta-casein monoclonal antibody, and then performing large-scale preparation and purification of the obtained monoclonal antibody. After the beta-casein monoclonal antibody obtained by the method is labeled by colloidal gold, the beta-casein monoclonal antibody can be adsorbed on a gold standard conjugate pad to prepare the colloidal gold test strip. The beta-casein colloidal gold test strip disclosed by the invention can realize fast specific detection and high resolution and shorten detection time when used for judging whether the casein content in raw milk achieves a standard or not.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method of a β-casein monoclonal antibody, a β-casein colloidal gold detection test strip and a preparation method thereof. Background technique [0002] Casein refers to a general term for a large class of protein in milk, accounting for 76-86% of milk protein. Casein is not a single protein, but consists of four types: α-, β-, γ-, and κ-, each of which has variants with different relative molecular masses and isoelectric points. Among them, α-casein accounts for about 75%, β-casein accounts for about 22%, γ-casein accounts for about 3%, and κ-casein only accounts for a small amount. [0003] At present, there is no national standard or industry standard for the detection method of casein content in China. The Chinese patent application with application number 200810007526.7 "A Method for Detection of Casein Content in Milk" discloses a method for detecting casein con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18G01N33/68G01N33/531
Inventor 周坚王黎丽
Owner 长沙安迪生物科技有限公司
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