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Specific a1at monoclonal antibody for detection of endometriosis

A monoclonal antibody and detection kit technology, applied in the field of detection and/or detection of endometriosis, can solve the problems of delaying the timing of doctor's inspection, insufficient sensitivity of endometriosis, and low patient acceptance

Active Publication Date: 2016-10-26
TAIPEI MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because patients often ignore the symptoms of endometriosis, thus delaying the timing of doctor's examination, resulting in complicated conditions
Although several methods are currently available to test for endometriosis, some tests have low patient acceptance or are not sensitive enough to detect certain types of endometriosis
For example, although laparoscopy is quite reliable for detecting and diagnosing endometriosis, the acceptance of this invasive detection method by patients is not high
Other methods, such as vaginal ultrasound or nuclear resonance imaging (MRI), can only detect fibroids or chocolate cysts larger than 2 cm, and their sensitivity Not yet strong enough to detect endometriosis
Furthermore, although a serum biomarker, CA125, is known to detect endometriosis (Barbieri R.L., Fertil. Steril. 45:767-769, 1986), its detection sensitivity is only 15%

Method used

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  • Specific a1at monoclonal antibody for detection of endometriosis
  • Specific a1at monoclonal antibody for detection of endometriosis
  • Specific a1at monoclonal antibody for detection of endometriosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Preparation of Antigen

[0059] The samples collected in this embodiment were women with endometriosis, and their serum samples were collected after informed consent. Serum samples were precipitated with 4 volumes of ice-cold acetone (containing 10% (w / v) trichloroacetic acid (TCA)). The mixture was placed at -20°C for 90 minutes, then centrifuged at 15,000 xg for 20 minutes at 4°C to collect the precipitate. The precipitate was then washed with ice-cold acetone, and centrifuged again at a speed of 15,000×g for 20 minutes. Remove the supernatant and wash with rehydration buffer (7M urea, 4% CHAPS ([3-(3-cholaminopropyl) dimethylamino]-1-propanesulfonate), 2M Thiourea (thiourea), 0.002% bromophenol blue (bromophenol blue) and 65mM dithioerythritol (DTE) dissolved the precipitate.

[0060] The protein contained in the above-mentioned rehydration buffer was treated with PNGase F to remove the N-glycan chains of the protein, and then the digested protein was id...

Embodiment 2

[0062] Example 2 Production of monoclonal antibodies

[0063] 2.1 Immunize animals with type A1-trypsin and measure the antibody titer

[0064] Mice were immunized with alpha 1-trypsin (A1AT, purchased from Sigma Inc), and a dose of 30 μg / animal was administered 2 to 6 times during a period of about 4 weeks. After the 2 booster doses, weekly blood samples were collected from the mice and the serum was immediately centrifuged. The separated serum was serially diluted, and then the antibody titer was measured by Western Blot.

[0065] 2.2 Preparation of antibody-producing cells

[0066] Select the animal of Example 2.1 with the desired antibody titer (i.e., in the western blotting method, the dilution concentration of the serum sample is 1:5,000, showing a positive reaction) for fusion Preparation from spleen cells, regional lymph nodes of immunized animals The antibody-producing cells were produced, and the antibody-producing cells were fused with the myeloma FO cell line ac...

Embodiment 3

[0073] Example 3 Detection of endometriosis using the monoclonal antibody of Example 2

[0074] The specificity of monoclonal antibodies 2A7 and 2C8 in Example 2 was analyzed by immunoblotting method.

[0075] In this example, serum samples were obtained from 235 females who were informed and provided samples with their consent. These subjects were divided into five groups. Among them, the control group consisted of 48 pregnant women without endometriosis; the Endo(+)GnRh(-)I / II group consisted of 89 women with mild (or early) pelvic intrauterine Composed of women with ectopic membranes who did not receive gonadotropin-releasing hormone (GnRh) therapy; Endo(+)GnRh(-)III / IV group consisted of 16 women with severe pelvic endometriosis women who did not receive GnRH therapy; the adenomyosis group, which consisted of 23 women with adenomyosis; and finally, the Endo(+)GnRh(+) group, which consisted of 59 women with Consisting of women with pelvic endometriosis who had been treat...

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Abstract

Provided herein are monoclonal antibodies with a high binding affinity to isoforms of alpha 1-antitrypsin (A1AT), hybridoma cells producing the same, and their uses in diagnosing and / or detecting endometriosis from a serum sample of a subject suspicious of having endometriosis or a subject under health examination.

Description

technical field [0001] This disclosure is in the technical field of testing and / or detecting endometriosis. In particular, the disclosure refers to a monoclonal antibody that recognizes alpha1-antitrypsin (A1AT), a fusion tumor that produces the monoclonal antibody, and the detection of the monoclonal antibody in serum samples from individuals and and / or use to detect endometriosis. Background technique [0002] Endometriosis is a disease characterized by the appearance of endometrial tissue in areas outside the uterus, for example, glands and stromal cells that should grow inside the uterus, instead grow outside the uterus while still retaining the normal uterine The same physiological form of the membrane. [0003] Endometriosis is not easy to detect, and the existence of endometriosis is often discovered and confirmed by actual examination of the patient's abdominal cavity until the patient has symptoms of pain or fever during menstruation. Because patients often ignor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28
Inventor 杨维中王惠钧陈水聪吕肯奋彭明棋
Owner TAIPEI MEDICAL UNIV
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