TWI235159B - Anti-human hemoglobin monoclonal antibodies for detecting fecal occult blood, producing hybridomas and uses thereof - Google Patents

Abstract

The invention provides monoclonal antibodies specific to human hemoglobin, producing hybridomas thereof, and the use of the antibodies in detecting fecal occult blood.

Description

1235159 A7

The present invention relates to a fusion antibody specific to a human heme and a right, a line, a single antibody, a child antibody, and its use for detecting fecal occult blood. Gastrointestinal cancer and gastrointestinal lesions often cause gastrointestinal bleeding, and the fluid is often excreted with the feces. Therefore, it is checked whether the feces each have two blood (called fecal occult blood), which can be used for early diagnosis of gastrointestinal diseases, especially: Colorectal / rectal cancer has the highest sensitivity and specificity. / 、 At present, the faecal occult blood detection method commonly used in China is chemical removal. Because the central blood matrix of heme has pseudo-peroxidase activity, it can cause some colorless compounds to show color through oxidation reaction. Among them, the most commonly used chemical method is the guaiac method: 2H2〇 + 〇2 hemoglobin + chromogen Hb + H2〇2 oxidation of guaiac oleum + guaiac wood— Oxidized Acne Lignans (Colorless) (Blue)

Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. C Please read the note on the back? Please fill in this page again for details.) Phenol Quinone The chemical specificity is poor, and it is easy to be affected by foods or medicines that can be ingested by the test subject, resulting in false positives. Such as animal hemoglobin; 5 such as & and long fresh vegetables and fruits may contain substances that cause false positives, so the testees should not eat them two or one days ago. 58055.DOC \ en-4-. This paper standard applies to China Standard (CNS) A4 specification (210 X 297 mm) 1235159 A7 ----_ B7___ ^ V. Description of the invention (2) Red meat and a variety of fruits and vegetables, causing a lot of inconvenience. The k-antigen specific binding immunoassay technology has been available for many years / Ding has been equipped (please read the precautions on the back before filling out this page) The combined color layer analysis method has developed a simple and fast method, even if any Individuals can also interpret the results with the naked eye, such as the simple tomographic test. The simple tomographic test is generally implemented by two principles: the competitive immune response and the sandwich immune response. The competitive response is the sample contact area. It is located at one end of the test piece, which is in contact with the sample to be measured. The adsorbed sample moves to the other end by capillary phenomenon, so as to wet the entire test piece. The indicator area is close to the sample contact area, and the indicator is collected in a dry form in this area. -Antibody binding body. The test area is located above the indicator area, and the area is fixed with a color reaction reagent such as the test substance-carrier antigen binding body. It can react with the antibody indicator binding body to show a naked eye. Interpreted signal. The carrier antigen can be, for example, albumin, globulin, hemocyanin, ferritin, etc. Printed response from the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs During the test, the end of the sample contact area of the test piece is in contact with the sample to be tested. The sample solution is wetted up by the capillary phenomenon, and the antibody-indicator conjugate is dissolved back. If the sample contains the test object, it will contact the test area. The test substance-carrier antigen binding on the antibody-indicator binding agent has limited competition with each other. Therefore, the higher the content of the test substance in the sample, the weaker the positive signal in the test area. From the presence or absence of the signal reaction in the test area, the strength of the signal can be determined in the sample. Whether it contains the test substance and its concentration. In the sandwich type reaction, two antibodies (single or multiple antibodies against the test antigen) are fixed, one antibody is fixed in the test area, and the other antibody is connected to the indicator. If the sample Containing the test antigen, the test antigen will first react with the antibody-indicator conjugate, then move to the test area and be fixed 58055. D〇C \ en " 5-This paper size applies Chinese national standards (CNS ) A4 size (210 X 297 mm) l235l59 A7 B7 V. Another antibody function of the invention description (3_) ^ was captured and fixed in the test area, and a signal visible to the naked eye appeared. The higher the concentration, the stronger the positive signal. Due to the specificity and convenience of immunoassay methods, there is an urgent need to develop domestic immunoassay materials and methods. SUMMARY OF THE INVENTION The purpose of the present invention is to provide human hemoglobin with Specific monoclonal antibody. Another object of the present invention is to provide a fusion tumor producing the antibody. Another object of the present invention is to further provide a method for detecting fecal occult blood, a detection reagent and a kit thereof. BRIEF DESCRIPTION OF THE DRAWINGS 1 · Immunochromatographic chromatographic test reagents, where (1) is the sample contact, which uses capillary phenomenon to move the sample dropped on the lower end of the glass fiber membrane to the nitrocellulose membrane; (2) the antibody indicator area, which is used for nitration Colloidal gold_single antibody 46.24 conjugate is adsorbed on the glass fiber membrane under the fiber membrane; for the reaction test area, the c-line sprayed on the nitrocellulose membrane is difficult to resist mouse immunoglobulin (control line) and the VT line is a single antibody 37 · 24 (test line); and ⑷ is the liquid adsorption area. During the test, if only one line (control line) appears on the reagent, it is a negative reaction; if two lines (control line and test line) are displayed, it is a positive reaction. Detailed description of the invention The present invention first provides two single-body antibodies specific for human heme, and fusion tumors 37 46 and 46 24 which produce the single-body antibodies, respectively. Several of these two fusion tumors have been deposited in Food 58055 on June 25, 1998. D〇C \ en 〇_ -D-、 * This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 Public Love) (Please read the note on the back? Matters before filling out this page)

-------- Order · --------. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1235159 A7

V. Description of the invention (4) The storage numbers of the strain preservation and research center of the Koukou Industrial Development Institute are CCRC 960096 and CCRC 960097, respectively. (Please read the precautions on the back before filling out this page) In addition, the present invention also provides a method for testing fecal occult blood using the single antibody 37 46 and / or 46 24. This method can be used to diagnose gastrointestinal lesions, such as gastric ulcer, gastric cancer, esophageal ulcer, esophageal cancer, duodenal ulcer, colorectal / rectal cancer and polyps (polip. Among them, the diagnosis of colorectal / rectal cancer is preferred. All known antibodies / Antigen-binding detection methods can be applied to implement the detection methods of the present invention, such as enzyme-linked immunosorbent assay (EUSA), sandwich analysis, competitive analysis, latex agglutination, chromatographic immunoassay, etc. The invention also provides a reagent for testing fecal occult blood using the single antibody 37 46 and 46 24, which may include a tritium sample contact area, an antibody indicator area '(3) a reaction test area, (4) a liquid adsorption area, and (5 ) Carrier, where the carrier is composed of one or more porous materials that can provide capillary phenomenon and absorb liquids, such as filter paper, nitrocellulose membrane, nylon film, glass fiber membrane, etc. # 日 示 剂 can shame such as Colored latex particles (c latex particies), metal colloidal solution (eg colloidal solution) Colloidal silica particles and dye solution are all commercial products. Another object of the present invention is to provide a kit for testing fecal occult blood using the single antibody 37 46 and 46.24. And the kit can further include conventional chemical reagents that can be used to detect fecal occult blood, such as guaiac chemistry, chemistry, aminopyrine chemistry, benzidine chemistry, or o-toluidine 58055.DOC \ en „7-', this paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1235159 A7 B7 Printed by the Consumers’ Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the invention (5 :) The following examples further illustrate the present invention, but these examples should not be considered as limiting conditions of the present invention. Example 1 Preparation of Purified Human Heme (Sigma, cat. H-7379, Murine Fusion Tumors 37.46 and 46.24) lot. 32h9329) and Freund's adjuvant (Gibco) to prepare a 1: 1 (v / v) emulsion, with the exception of using the complete adjuvant for the first immunization injection and subsequent use of Full adjuvant, this emulsion was injected subcutaneously into BALB / C mice (each injected with 100 micrograms of human hemoglobin) for 6 to 8 weeks, injected once a week for four consecutive weeks, and then strengthened every four weeks Agents (40 micrograms of human hemoglobin per injection) were administered four times, and three days before cell fusion, an additional dose was administered intravenously (without adjuvant). Three days later, the mice were sacrificed and the spleen was removed. Spleen cells were fused with murine myeloma F 0 cell line (ATCC CRL1646), and 50% polyethylene glycol 400 (PEG400) was used to 1. · 3 (spleen cells: murine myeloma F 0 cell line), After the ratios are fused, the cells are suspended in RPMI medium (HAT, 20% FCS) and diluted to 105 cells per ml, and then seeded into 96-well culture plates (0.2 ml per well) and cultured. After 7 days, the cell culture supernatant was tested with an ELISA plate that adsorbed human heme, and fusion tumors specific to human heme were selected, and then singulated by the limiting dilution method. The mouse-derived fusion cell lines 37.46 and 46.24 of a single cell were isolated by this method. The monoclonal antibodies secreted by them are IgG, kappa light chain. Its 58055. D〇C \ en-8-^ ^ (Please read the notes on the back before filling this page)

Packing ---- Order --------- Φ. This paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) 1235159 A7 B7 V. Description of the invention (8) Phosphate buffer solution , ΡΗ = 7.0), block at room temperature for 30 minutes, remove the nitrocellulose membrane and place it in a drying room with constant temperature (20 ° c) and constant humidity (24% humidity) for drying. Take a backboard with a length of 7 cm x 30 cm, and draw 4.2 cm and 4.9 cm from the top with a cutter, tear off the 4 2 cm piece of paper, and stick the NC die along the lower edge of the torn part. . Then take 2 4 cm X 30 cm filter paper (Whatman, 3 MM Chr) and stick it along the upper edge of the back plate of the torn part. The paper sheet below the back panel 4 · 9 cm was torn off, and a glass fiber membrane strip containing a colloidal gold-single antibody 46.24 conjugate mixture was placed in the middle of the unteared layer of the paper sheet in the center of the back sheet. Paste the folded 4.2 cm x 30 cm glass fiber membrane along the lower edge of the back plate. Finally, take 30 cm of 3M tape and stick it down about 3mm above the fiber membrane strip. Cut the combined reagent piece into 7 cm x 08 cm strips (as shown in Figure 1) and place it on the lower cover of the kit, and then cover the open | upper top, and dry at constant temperature and humidity for 18 hours. active. During the test, about 200 microliters of sample was placed in the sample well on the kit. The samples were human heme at different concentrations, and the dilution was 0.1% citrate buffer ρΗ, 8.0 + 0.05% triton. The results are shown in Table 2 below. Table 2 (Please read the precautions on the back before filling out this page)

Packing -------- Order --------- ^^^ 1 Printed by the Consumer Affairs Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs

a: The unit of human hemoglobin concentration is micrograms / liter. b: +++ indicates a clear line; indicates a clear line; indicates a clear line; 58055. D0C \ en -11-This paper scale applies Chinese National Standard (CNS) A4 Specifications (210 X 297 mm) 1235159 A7 B7 V. Explanation of the invention (weak lines on the wristwatch;-wireless bars on the watch. From the results in Table 2, it can be known that the degree of hemoglobin is 0.15 μg / ml. Rich (Please read the precautions on the back before filling this page)

Packing ---- Order ..-------- #. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 58055. DOC \ en 12- This paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm)

Claims (1)

Ί235159 y I 1 ^^ 8111081 patent application f text, please replace the patent scope (May 1993) 6. Scope of patent application 1. A single antibody specific to human heme is produced by fusion tumor 37.46 with the deposit number CCRC 960096. 2. A monoclonal antibody specific for human heme, which is produced by fusion tumor 46.24 with the deposit number CCRC 960097. 3. · Fusion tumor 37.46 of the production system according to item i of the single-body antibody of the patent scope, and its registration number is CCRC96〇96. 4. A fusion tumor that produces a single antibody according to the scope of the patent application No. 2 46 · 24 ′, and its registration number is CCRC 9 6 0 0 9 7. 5-A method for in vitro testing of fecal occult blood, which is characterized by contacting a single antibody against a fecal sample according to item 1 or 2 of the scope of the patent application. 6. The method according to item 5 of the scope of patent application, which is used to test fecal occult blood caused by gastrointestinal diseases. 7. The method according to item 5 or 6 of the scope of patent application 'is used to test fecal occult blood caused by colorectal / rectal cancer. 8. A reagent for detecting fecal occult blood, characterized in that it comprises at least one single-body antibody according to item 1 or 2 of the scope of patent application. 9. A kit for testing fecal occult blood, characterized in that it comprises at least one monoclonal antibody according to item 1 or 2 of the scope of patent application. 10. The kit according to item 9 of the scope of the patent application, further comprising a chemical reagent that can be used for detecting fecal occult blood. 11. The set according to item 10 of the scope of patent application, wherein the chemical reagent is selected from the guaiac chemistry method, the aminopyrine chemistry method, and the benzidine chemical method; fl China National Standard (CNS) A4 specifications (21GX297 public director) 1235159 8 8 8 8 AB c D Patent application scope method, or reagent of o-toluidine chemical method-2 This paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 mm)