Detection method of lactoferrin in tears and dedicated colloidal gold detecting card thereof

A lactoferrin and detection card technology, which is applied in the direction of measuring devices, biological testing, material inspection products, etc., can solve the problems of large sample volume and unsuitable lactoferrin detection, and achieve accurate results, less tear sample volume, and high sensitivity high effect

Inactive Publication Date: 2013-10-02
无锡安迪生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Spectrophotometry and high performance liquid chromatography are easy to operate, but the amount of sample to be detected is

Method used

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  • Detection method of lactoferrin in tears and dedicated colloidal gold detecting card thereof
  • Detection method of lactoferrin in tears and dedicated colloidal gold detecting card thereof
  • Detection method of lactoferrin in tears and dedicated colloidal gold detecting card thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Preparation of Lactoferrin Monoclonal Antibody

[0024] Animal procedure: Lactoferrin was used as the antigen, and the immunization dose of 100 μg / mouse was fully emulsified with an equal volume of Freund's complete adjuvant to immunize two Balb / c mice by intraperitoneal injection at multiple points, and the same mice were injected every 2 to 3 weeks. The dose of antigen was emulsified with Freund's incomplete adjuvant and immunized 2~3 times, and the immunization was boosted once three days before fusion.

[0025] 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 competitive ELISA, and positive wells were screened. Positive wells were cloned by limiting dilution until a hybridoma cell line stably secreting anti-lactoferrin monoclonal antibody was obtained.

[0026] Cell cryopreservation and recovery: Take the monoclonal hybridoma cell line of la...

Embodiment 2

[0028] Example 2 Preparation of colloidal gold-labeled lactoferrin monoclonal antibody complex

[0029] 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 above 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.

[0030] Preparation and purification of gold-labeled antibody: use 0.1mol / L K 2 CO 3 Adjust the pH of the colloidal gold solution to the optimal pH value, add the optimal protein amount of ractopamine monoclonal antibody drop by drop while stirring evenly, and finish adding it within 5 minutes. In the liquid-balanced colloidal gold solutio...

Embodiment 3

[0031] Example 3 Assembly of Lactoferrin Colloidal Gold Rapid Detection Card

[0032] Properly dilute the gold-labeled antibody, spray it on the binding pad with a gold spray machine, and dry it at 37°C to obtain a colloidal gold film. Lactoferrin is diluted to the optimum concentration with phosphate buffer solution of 0.01mol / L pH7.4, and is sprayed on the cellulose film 6 with spray film instrument, and this is detection line 10; Concentration of goat anti-mouse IgG, this is the quality control line 11. After drying at 37°C, assemble the cellulose film, colloidal gold film, sample pad and absorbent paper on the bottom plate in sequence, and cut the sample into appropriate width with an automatic cutter. The paper strip is loaded into the test card housing.

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Abstract

The invention discloses a detection method of lactoferrin in tears and a dedicated colloidal gold detecting card thereof, and belongs to the field of medical diagnostic reagent technology. The lactoferrin rapid colloidal gold detecting card comprises a detecting card and a colloidal gold test strip, and is prepared by following steps: lactoferrins monoclonal antibodies of high sensitivity and specificity are obtained by taking lactoferrins as antigens to immunize mice; the lactoferrin colloidal gold test strip is obtained by using colloidal gold chromatography; and the colloidal gold test strip is fixed in the detecting card through a groove. The content of lactoferrin in tears is detected by using immune-colloidal gold technology, so that detecting cost is low, and detecting accuracy is high.

Description

technical field [0001] The invention relates to a method for detecting lactoferrin in tears and a special colloidal gold detection card thereof, which is a detection device for detecting lactoferrin in tears and belongs to the technical field of medical diagnostic reagents. Background technique [0002] Lactoferrin (lactoferrin, LF) is a glycosylated protein that belongs to the iron-binding protein family. It can reversibly bind two iron ions. It was first isolated from milk, so it got its name. Human lactoferrin has a molecular weight of about 80kDa, an isoelectric point of about 7.8, and is composed of 692 amino acids. It exists in various body fluids and secretions of the human body, such as tears, saliva, and milk. Lactoferrin has a broad-spectrum antibacterial function, mainly because lactoferrin can combine with iron ions, so it can competitively inhibit Gram-positive bacteria that require iron. In addition, it can also combine with many proteins and DNA to exert anti-...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577G01N33/558
Inventor 周坚王黎丽
Owner 无锡安迪生物工程有限公司
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