TWI283750B - Immunoassay method for determining glycosylated protein and test liquid thereof - Google Patents

Abstract

This invention discloses an immunoassay method for determining glycosylated protein, such as advance glycosylation end products (AGEs) and a test liquid thereof, which comprises a test solution for determining a glycosylated protein containing an indication carrier suspension liquid and antigen or antibody immobilized on the surface of the indication carrier. A test strip is used, containing a bottom plate and assembly parts disposed on the bottom plate, in which the assembly parts comprise a specimen water-absorption pad, a porous fiber membrane, an indication carrier fiber block and at least one immobilization material. After the test liquid is injected into the test strip, an immune reaction of the glycosylated protein antigen and the glycosylated protein antibody is determined by the coagulation phenomenon or accompanying absorbance change or color change. Therefore, it can be determined if the existence of AGEs before the diabetes is taking effect, which then allows medical staff to prevent the occurrence of diabetic complication or further inhibit progression of the complication at the earliest time.

Description

93108448

1283750" _ case number five, invention description (1) [Technical Field of the Invention] The present invention relates to a measuring solution for determining an antigen or antibody of final saccharification (AGEs) by immunoassay; and injecting a test strip through a measuring solution 'The resulting agglutination phenomenon or the accompanying change in absorbance or colorization to determine the final glycated protein antigen or anti-finalized glycoprotein antibody. [Prior Art] Diabetes is not terrible, most afraid of complications of diabetes, such as kidney disease High blood pressure, eye diseases, etc. At present, the most basic method for detecting diabetes is to measure the blood sugar level before and after meals to know if there is a sugar disease. When a person is diagnosed with diabetes, usually the doctor will The patient smoked 2 to detect the value of HbAlC, which was used to determine whether the patient's blood glucose control was good or not. However, neither the blood glucose test reagent nor the HbA1 C test reagent could be used to detect the occurrence of diabetic complications at an early stage. A microalbumin test, which can be used to measure kidney complications in diabetics, which is a pity A kind of micro-protein detection reagent can only be detected after the formation of kidney disease, that is to say, the micro-protein detection reagent 'must be detected after the complication occurs, and it may not be effective for the occurrence and prevention of the early symptoms. Although we are fully aware of the pathogenesis of diabetes today, for example, in the case of hyperglycemia, proteins (eg albumin) will be glycated into glycated proteins, which are then saccharified. The final glycated proteins (AGEs), when the final concentration of glycated protein in the blood rises abnormally, will cause abnormal destruction of the cells, especially for diabetics, which will further produce complications (eg kidney disease, etc.) In addition, although currently available, drugs can also be used effectively.

930313002.ptc Page 5 1283750 Amendment No. 1 93108448 V. INSTRUCTIONS (2) Control the gray sugar content of diabetics, but still can not prevent the occurrence of complications. So far, there is no detection reagent for early detection of diabetic complications. occur? Or complications will happen? The final glycated proteins (AGEs) appear in the body fluids (eg, blood) of the human body a few months before or during the onset of complications. Therefore, the development of a specific way, tincture, to quickly test to know whether the final glycation protein (AGEs) of diabetes is elevated, so that medical staff can prevent the occurrence of complications in diabetic patients at the earliest time, Or try to lower the final glycated protein (AGEs) in the blood, and then stop the complications. The most specific and sensitive of the specific methods and reagents used to test diabetes mellitus, the final glycosylated protein, is the use of immunoassay to determine the antigen or antibody of the final glycated protein (AGEs), which can be used as an early diagnostic complication. An indicator formed. It is a pity that no test reagents for determining final glycated proteins (AGEs) are available. SUMMARY OF THE INVENTION Based on the foregoing, the inventors of the present invention have intensively researched and developed immunoassay techniques and devices for determining final glycated protein (AGEs) antigens or anti-finalized glycoprotein (AGEs) antibodies, which are basically via agglutination or The accompanying absorbance change or color change is used to determine the immune response of the final glycated protein antigen or the antibody against the final glycated protein, thus completing the present invention. In another aspect, the present invention provides immunoassay techniques for determining a final glycated protein antigen (or using a final glycated protein antigen to detect a final glycosylated protein antibody) using an anti-final glycosylated protein antibody, including the use of immunoassay techniques Changes or changes in absorbance via agglutination or concomitant

1283750 _J No. 9310844S__年胄日 Silk 5, invention description (3) body). In a further part, the present invention proposes the use of an anti-finalized protein antibody to determine the final glycated protein antigen (or the use of a final glycated protein antigen to determine an antibody against a final glycosylated protein) by, for example, resistance to the final The agglutination phenomenon between the glycosylated protein antibody and the immune complex of the final glycated protein antigen (or, for example, the final glycated protein antigen and the anti-finalized protein antibody), to determine the presence of the antigen (or antibody) of the final glycated protein in the test subject Whether or not. In a further part, the present invention proposes another immunoassay reagent for determining a final glycated protein antigen (or using a final glycated protein antigen to determine an antibody against a final glycated protein) using an anti-finalized glycoprotein antibody, for example, by The change in absorbance caused by the formation of an immune complex of the final glycated protein antibody and the final glycated protein antigen (for example, a final glycated protein antigen and an anti-finalized glycoprotein antibody) to determine the antigen of the final glycated protein in the sample to be assayed (or The presence or absence of antibodies). In yet another aspect, the present invention provides an immunoassay test strip for the determination of a final glycated protein antigen (or the use of a final sugar, a protein antigen to determine an antibody against a final glycosylated protein) using an anti-final glycosylated protein antibody. The bottom plate is formed by, for example, an immune complex against a final glycated protein antibody and a final glycosylated white antigen (or a final glycated protein antigen and an anti-finalized protein antibody, etc.), and is determined by a color change caused by a control line. The presence or absence of the antigen (or antibody) of the final glycated protein. The features, objects, and advantages of the invention will be apparent from the description and appended claims. In the drawings. [Embodiment]

930313002.ptc Page 7 1283750 安—Λ_ _ Case No. 93108448 V. INSTRUCTIONS (4) DATES __ MODIFICATION The present invention utilizes several immunoassay techniques to determine the presence of antigen or antibody to the final glycated protein in the sample to be tested. Whether or not. The antibody used therein can be obtained by immunizing directly with a purified antigen (for example, a final glycated protein antigen) via rabbit or goat (for example, a plurality of antibodies), or can be immunized by a mouse to form a fusion tumor cell. Obtained (for example, · monoclonal antibody). Among the commonly used immunoassay techniques, see, for example, F uji kawa Η·, Igarashi, Η·, published in 1988, which uses high-density latex particles to make rapid latex agglutination reagents for detection. Portuguese ball

Enterotoxin Α ~E (Appl· Envir· Microb iol·, 54/10, 2345-2348, 1998) °Delanghe, JR·, Chape le, JP·, Vander schueren, SC·, at 1 990 The nephelometry method was used to determine Myoglobin (Clin·Chem., 36/9, 1675-1678, 1990). WO 88/0 8 534 (1 988) describes a so-called immunoassay device for measuring HCG and LH as a medium using an immunochromatographic membrane. U.S. Patent No. 5,238,652 (1,939) discloses the use of immunochromatography to determine non-protein antigens. However, none of the above prior art techniques mentions immunoassay techniques and devices for determining the antigen (or antibody) of the final glycated protein.

In a part of the present invention, an immunoassay reagent for determining a final glycated protein antigen (or a final glycated protein antigen using a final glycated protein antigen) using an anti-finalized glycoprotein antibody is proposed, which utilizes immunoagglutination technology. Determining a final glycated protein antigen (or antibody); including a sputum display carrier suspension; and whether agglutination occurs after contact with a sample immobilized on the surface of the display carrier to determine the final glycated protein 111 ___

a final glycated protein antibody (or antigen) via the suspension reagent and to be tested 1283750, 931flR44R 5. Invention (5) The presence or absence of an antigen (or antibody) in one embodiment, will be immobilized in suspension The blank of the display carrier in the middle is full. When the sample to be tested is fixed with a suspension of the carrier, and the carrier is reacted positively against the reaction plate, the carrier is suspended. The size of this material is called the anti-display configuration. Contains: Aggregate the same thing (example), other test tubes should be. On the other hand, after the fixed floating liquid has been fixed, the present invention is in the form of 0 · 01 microsurgery), and the lipid microsomes are the above-mentioned body immobilized by the final sugar to resist the final sugar reaction, which is the result of the experiment, such as: There is one type to be tested or one meter of the meter to be measured. The present invention, one of the gold sol or one of the structured protein anti-chemical protein anti-positive anti-for example: Quantitatively, the above-mentioned fixed product or a plurality of anti-final saccharides are used to re-use a certain amount of the final When saccharification or a variety of anti-finalized glycated eggs are mixed, the antibody will be formed into a carrier--final glycated protein-glycosylated protein antibody---~~-, and the immune complex will form a 'visually visible' within 3 - 5 minutes. There is no final sugar in the sample. The anti-finalized protein sample does not produce agglutination. The carrier is between a colored rice (Bangs Labs's particles can be: milk particles, carbon black particles, etc.) The final glycated protein type final glycated protein antigen ' can be used to determine the body; non-agglutination reaction is required. Of course, using the competition method according to the above determination method, the concentration of the final glycated egg is mixed in the test tube, and the final glycated protein antibody is present. The protein antibody is a protein that binds to the white antibody of the carrier antigen and forms a "antigen primary antibody." The reaction is a display of the antibody against the protein of the protein, which is a negative microparticle, Inc., USA gelatinous microparticles, dyed. The antibody of the present invention, if it is a negative reaction in the original or various test samples, will also be obtained, A competing white antibody or antibody followed by each or antigen is shown in 1283750 _____SM 93108448_年月日日 Revision__ V. Inventive Note (6) The carrier suspension is reacted to determine the final sputum protein antigen or antibody Existence or not. , '

The antibody of the present invention is used to determine the change of the final determination of the final sugar content, such as: after the display of the display carrier or the final saccharification of a light-absorbing contact, the final glycated egg is measured on an implementation suspension, and then a negative unknown is prepared. The sample to be tested shows the carrier suspension; A1 colorimetric tube is added with 20 μl of unknown sample by optical chromatography), and the OD value is recorded (colorimetric, recording OD, one colorimetric OD, another part) An anti-finalized protein glycosylated protein antigen, or an immunoassay reagent against an antibody against a final glycated protein antigenizing protein, which uses a light-absorbing result to determine a final glycated protein antigen or antibody, such as a carrier suspension; The display carrier is full. A multi-antibody and A3 colorimetric test are added to the 0 0, and the gas should be returned to the zero second again. The anti-body surface of the product is fixed with the above-mentioned anti-finalized glycoprotein antigen; An assay device; whether it produces an absorbance change via the reagent and the display carrier to be determined, the presence of a white antigen or antibody no.

In the case of 930313002.ptc, on page 10, one or more antibodies are immobilized in a blank using a blocking protein to occupy the standard serum, and a weak positive standard serum, and then each takes 25 microliters. Fix one or float into the prepared three colorimetric tubes Ai, A2 and add 20 μl of negative standard serum, A2 liter of weak positive standard serum, A3 colorimetric test tube; the above three colorimetric tubes are added. After the sample, the analyzer was 340 nm (nm) colorimetric (previously absorbance value) and the 240th value (absorbance value) after the sample was added. The value of the second colorimetric D value is the difference of the reaction 0 D. At this time, if the unknown i^83^ case number _ face ^ day, positive five, invention description (7) " should 0D difference is greater than the weak positive standard serum reaction 〇 D difference, is a positive reaction. If the unknown sample has a 0D difference that is less than the weak positive standard serum, 〇D ^ ΐ ' is a negative reaction. If you prepare a standard solution of known concentration, such as ^ear position / ml, unit / ml, 2 units, ml, 4 units C liter: 8 units / ml, χ 6 units, ml, can be based on the standard solution The concentration of the sample to be tested is calculated by the standard curve of the composition. The display carrier referred to in the present invention is a microparticle or an enzyme (Bangs Ub〇rat〇ries nc' USA 'Sigma, USA), and the microparticles may be latex microparticles, lipid microparticles, I ethylene glycol microparticles, NAD microparticles, carbon microparticles, Dye particles, enzyme network NADH particles, etc., the size is between 2 〇 2 micron, the colorimetric range: Ϊ? 260 nm ~ 840 nm, · different particles choose different light «曰/ It is well known. The antibody referred to in the present invention is one or more of the above-mentioned anti-finalized protein antibodies; if the final glycated protein antigen of J, = 冓 is immobilized on the display vector, the final glycation of various structural formulas, It can be used to determine whether the sample to be tested contains anti-final glycosylated protein antibodies. Of course, using the competition method, the same experimental results will be obtained. For example, according to the above-mentioned measurement method, a competitor (for example, a certain amount = a final glycosylated protein antibody or antigen) can be used for the sample to be tested, No; liquid, negative standard solution, first mixed in individual tubes, then = in these individual tubes, then added to the final glycated egg antibody or anti-/, the carrier suspension is reacted, and then absorbance by light. Difference, you can determine the final glycated egg resistance] = presence or absence. In the antibody = : part, 15 out of the use of anti-final glycated protein to obtain the final glycated protein antigen, or use the final glycated protein antigen

930313002.ptc Page 11 Inverse 3750 Case No. 93_ V. Inventive Note (8) ί"; The final immunoassay test strip of glycated protein antibody, which uses the = color layer: analysis technique to determine the final glycated protein antigen or antibody.丄 = shows the immunochromatographic test (4) 13 is a porous fiber membrane, the mountain is like a knot - the bottom plate 17. 7 is a sample absorbent pad, located in the test strip i 〇 the most advanced and with the fiber The film 13 is overlapped. 12 is a display carrier fiber, and the block is infiltrated with a blue display carrier 'and the carrier is immobilized with an antibody or an anti-display carrier fiber block 12 under the sample absorbent pad" and the porous fiber and sample The water absorbing pad U and the porous fiber membrane 13 are mutually different. Ϊ is a 5-purchase section (which may be a line or a dot-like distribution) on which a:: or the anti-caries interpretation section 14 is located on the surface of the porous fibrous membrane 13. - 2μ走^ Γ Section 1 4 front end facing the display carrier fiber block 1 2, the appropriate appropriate = behind the control section! 5 (can be line or point distribution), also located in a section of the porous HJ13 surface ; the imaging area (4) is fixed with an antibody or anti-u-end toward the absorption pad 16. The material of the fibrous membrane 13 is a fiber membrane, a nitrocellulose membrane, a polyester fiber membrane, a cellulose pancreas, or a chemical synthetic membrane ____, etc. Fig. 2 shows one of the built-in immunoassay strips. Waterproof box". In the eighth, 2 1 is the sample hole. 2 2 is the reaction zone hole. ^ 3 is a test strip containing 1 内? A three-dimensional view of the square water device box. Wherein, the sample absorbent pad u is located below the sample hole 21, and the area of the Un1 is not smaller than the sample absorbent pad 11. The interpretation section 14 and 搵:C: 1 1 β stand in the reaction zone hole 22' but are not in contact with it and can be directly observed by the naked eye. The 16 absorbent pad is located within the rearmost end of the device cartridge 2〇. Since the display contains: the stabilizing St t blue shows that the antibody or antigen is immobilized on the carrier, so the blue color on the display carrier fiber block 12 shows (4) (four) (four) sample, 930313002.ptc page 12 1283750 antibody reaction, due to blue display The anti-binding of the anti-final glyco-antigen combined with the blue line of a final sugar-colored display carrier, and the absence of blue lines should be negative or negative; otherwise, the visible blue on the carrier is visible on the upper side of the carrier.

930313002.ptc Page 13

Free flowing on the test strip 10; the bottom plate 17 is connected to the filler with a porous fiber reading section 14 and a blank of the control zone. In addition to the reaction of the test carrier fiber block containing the test object, and the test sample containing the final antimony and the full complement of the carrier protein and occupying the protein; If the body or the anti-inhibition occurs, it should be determined. The blue-colored blue-stained protein on the liquid sample of the fixed color line is anti-finalized and the anti-finalized egg is fixed against the antibody immobilized on the final sugar, so the antigen or more Through the display of the original carrier (for example, the naked eye can be seen as 11⁄4 sex, the blue color does not appear in the species or the end of the final sugar.

Case No. 9310844S V. Description of the Invention (9) The blue display carrier connects the direction of the 133 and the absorbent pad 16, and the fiber membrane 13 on the bottom plate 17 is set to have a complete white covering and fill the remaining sample hole 2 The test sample at 1 site can be moved in addition to the immobilized antibody or antigen-absorbent pad 16. If the antigen binding site of the final glycosylated protein body is to be displayed immediately with the blue color; the terminal glycosylated protein antibody or more will be completely bound to the chemical protein antigen immobilized on the final glycated egg reading segment 14, the blue 14 does not form on the line. It can be seen that 'and the immunoglobulin G antibody with the control segment 15'; therefore, the segment 14 is treated as a partial self-quality test, regardless of the positive reaction. Combining with the original to form a piece of meat to the bottom surface T film 13 inside the porous fibrous membrane box 20, the porous section 15 and blocking the egg, the carrier to be displayed will show white when the carrier is original The antibody or the chemical protein is resistant to an anti-allergic site and cannot continue to move with the final sugar interpretation segment: the blue line of the anti-mouse should be 'and the line, used to be tested, and the anti-finalized protein against blue is 1283750

Tjfe 9310844S V. INSTRUCTIONS (10) Θ__ The vector will bind to the control segment 丨5 and is a competitive method reaction. Therefore, the blue line of the interpretation section 14 is a negative reaction; the absorption pad 6 is used to absorb all the liquid at the end, so that it continues to form a capillary reaction. Of course, as in the above, as long as the segment 14 is to be interpreted. If the immobilized final glycated protein antigen is modified to immobilize one or more anti-finalized protein anti-antibody, the second Meiji assay can be performed, and if the sample to be tested contains the final glycated protein antigen, it will be immediately immobilized on the blue display carrier. An anti-finalized glycoprotein antibody or a plurality of anti-final glycosylated antibody antibodies, forming a blue display vector-anti-finalized protein antibody-final glycated protein antigen complex, wherein the complex is moved to the interpretation section At 14 o'clock, it will bind to an anti-finalized glycoprotein antibody or multiple anti-finalized glycoprotein antibodies immobilized on it to produce a blue line, which is a positive reaction; some blue display vectors continue to move and will be compared with the control segment. The antibody immobilized on the 5 (for example, anti-rabbit immunoglobulin G antibody) binds to form a blue control line. When the sample to be tested does not contain the final glycated protein antigen, the interpretation section 14 will not produce a blue line, which is a negative reaction. The porous fibrous membrane used in the present invention has a pore size ranging from 〇·1 μm to 60 μm. Between (What man, Mill ipore) The display carrier used in the present invention is a colored fine particle, a fluorescent substance or an enzyme; the fine particles of color have a size of 微米·〇1 μm to 20 μm (Bangs Between Labs In c, USA), the fine particles (Bangs Ubs IncUS A; Sigma, USA) referred to in the present invention may be latex particles, dye particles, lipid particles, gold sol particles, carbon black particles, polymeric particles, and the like. When the display carrier used in the present invention is *^ kinds of colored φά»fine particles, the result can be directly interpreted, which is called direct display carrier; when the display carrier used in the present invention is an enzyme, it needs to be

1283750 Case No. 93108448 月 正 正 、 、, invention description (11) After the reaction of the coloring agent, the interpretation result is called indirect display carrier. The bottom plate used in the present invention is a waterproof plastic sheet or waterproof paper. The sample absorbent pad and the absorbent pad used in the present invention are not limited in material, but the higher the water absorption, the better. The display carrier fiber block used in the present invention is a water insoluble fiber. The invention will be elucidated below by way of non-limiting examples. Example 1 Cultivation of anti-finalized glycoprotein antibody

A purified antigen (for example, a final glycated protein antigen) is directly obtained by immunizing a rabbit or a goat (for example, a plurality of antibodies), or immunized with a mouse to prepare a fusion tumor cell, thereby obtaining the same (for example, Strain antibody). Example 2 Immunoagglutination assay of final glycated protein antigen

The carrier particles are shown to be diluted to a concentration of 3%, and the carrier is selected to be 8 micron polystyrene particles or other colored particles. The anti-finalized protein antibody obtained in Example 1 was diluted with a phosphate buffer to a concentration of 2 mg/ml. 1 ml of each was placed in a glass tube and allowed to stand for 8 hours. One more gram of bovine albumin was added to the glass tube and mixed, and allowed to stand for 8 hours. After centrifugation at 12 rpm for 30 minutes, the supernatant was removed and repeated. Add 2% bovine albumin solution to a total of 20 ml. Further, a homogeneous suspension was prepared by sonication treatment to prepare a display carrier suspension. Prepare three negative standard serums with a final glycated protein antigen content of

Page 15 1283750 Case No. 93108448 曰 Amendment 5, invention description (12) Weak positive standard serum, the final glycated protein antigen content is 5 units / ml and a strong positive standard serum, the final deuterated protein antigen content is 1 6 units / ml and an unknown sample to be tested. Each 100 μl of serum sample was placed on an individual test piece. Next, on each sample test original, add 50 μl of the display carrier suspension and spread it out. After 3-5 minutes, the results were visually interpreted. Results: 颞 display vector agglutination suspension θ negative standard blood 淸 0 unit / ml P negative standard blood 淸 1 unit / ml # — ^ negative standard blood 淸 2.5 units / ml θ — weakly positive blood sputum sample 5 / position / 奎Ascending θ Strong positive standard blood stasis 16 units / 奎升p 十ρ Unknown sample to be tested π 10 ^ There is agglutination reaction, which is positive for the final glycated protein antigen test. Those who did not have agglutination responded negatively for the final glycated protein antigen test. When the minimum positive reaction value is set at 5 units/ml, it is unknown that the sample to be tested is positive, indicating that the final glycated protein antigen contained in the sample to be tested has a concentration of 5 units/ml. Example 3 Immunoassay of final glycated protein antigen

930313002.ptc Page 16 1283750 Case No. 9310844« V. INSTRUCTIONS (13) Take the non-carrier particle Q solution. The display carrier can be used 〇 other final glycosylated protein antibodies at different wavelengths can be used, and 4 g of bovine albumin mixed for 30 minutes, and the supernatant liquid is removed to a total of 1 liter. The carrier suspension is again shown. Prepare six standard gold clearing/ml, 1 unit/millimeter, 8 unit/ml, and 1 6 each within 250 microliters of tube, each within 20 microliters, and then take 20 microliters of unknown The colorimetric analyzer firstly adds 20 μl of the standard serum colorimetric test tube, and then uses the colorimetric analysis for 240 seconds to measure the OD value once again, which means that the positive reaction value is determined for each reaction. OD value, draw the standard curve drawn and the concentration of the sample to be tested is unknown.

• 2 grams, placed in 1 liter of distilled water to make a suspension • 3 micron white polystyrene particles (also measured as particles). Add 3 〇 香 技 混合 and mix and let stand for 18 hours. Add again and let stand for 18 hours. Repeat three times in succession using 120 rpm. Adding 丨% albumin solution to a uniform suspension by sonication treatment, making the final glycated protein liter, 2 units/millimeter/ml and a display carrier suspension of the aforementioned standard serum plus test sample, added to 3 4 〇奈The meter wavelength and unknown sample to be tested determine the OD value (the secondary OD value (absorbance value) should be 0 D difference. 5 units / ml, into a standard curve. Calculate the unknown to be measured as 9 units / ml antigen containing liter, 4 unknowns should not be added to the other than the other, and the absorbance should be the same. The first amount is 〇 single unit / ml sample to be tested. The same color test color test tube color inside the military officer. Zero. When respectively Immediately after the individual), and subtracted the second standard of the standard ik clear income can be based on the quantitative concentration of the standard blood sample. ' Positive.

1283750 Case No. 93108448 曰五、发明说明(14) 0秒 OD値240秒 OD 値^ Reaction OD値 “Quasi-blood 淸 0.86^ 0.85^ 0.0柃0^3 Meng Zhun Blood 淸 0.92# 0.87^ 0.05^ 1^ Standard Blood stasis 0.98p 0.82# 0.16^ 2^ k quasi-blood 淸1 ·02ρ 0.83# 0.1如如定血血淸Εβ 0.94^ 0.69^ 0.25^ k准血淸F# 0.99必0.69^ 0.3^ Associate unknown sample to be tested 1.07^ 0.81^ 0.26^ 9p According to the present embodiment, it is also possible to perform only the negative standard blood rise and the positive standard blood * 5 units/ml to perform the qualitative rigid determination. If the test sample 之D value is greater than or equal to the positive standard The OD value of the blood & ml is positive, otherwise it is negative. / Example 4 is 3% blue by the immunoassay strip. The vector can be selected. 〇 · Glycosylated protein antibody Place 1 g of bovine albumin at 1 2000 rpm, centrifuge for 30% into 2% bovine albumin, and shake it to a uniform suspension. 4 cm wide and dry at room temperature with 10% sucrose. After the 'stained in the final saccharification of the carrier containing the desiccant micro-micron acid buffer glass tube mixed glass tube Inner clock, and go to the % sucrose solution float, made, 15 5 cm showed the carrier to hang, and then placed in the color of the sealed envelope. Place the gram / mA set 8 small static set 8 continuous heavy ml suspension suspension liquid fiber into the dry Medium. Dry under °C.

930313002.ptc Page 18 Protein antigen pellets or other polystyrene particles diluted to 2 mils, and mixed statically, and removed from the liquid, the liquid to the total amount of 2 〇 显 显 显 显 显 显 显 显 显 显 显, take out the cold dryer and store in 4 particles 'the rabbit resistance: the final concentration. Time. Add another hour. Use multiple times. Plus. Then, it is dried in a complete immersion box for 2 hours, and the case number is 93108448 1283750. 曰5, invention description (15) directly placing a predetermined amount of the display carrier suspension on the sample absorbent pad or can also be placed Porous fiber 臈 is the first $, but it is not conducive to mass production. It is '4.5 cm thick acid fiber membrane at the first 8: a spray-fixed mouse anti-final glycosylated protein antibody (M_eAnt i A ) The bath line is also called the interpretation section. The anti-rabbit w (a ntl-rabblt IgG) was spray-fixed at the 34th cent. This spray line is in contrast to the line. A few pieces of the nitrocellulose membrane were then infiltrated into the white egg-containing buffer solution. Infiltrate at least 2 pull-type - ~, p, six. v, too, *,, Z %. The receiver takes out the nitrocellulose membrane and clears it in the dry box. It is placed in a dry box and dried at room temperature. Then it sticks to the bottom of the plastic. The bottom of the bottom plate is ticked at the 2nd centimeter, so that it is completely aligned and covered. The dryer was dried for 2 hours and placed in a sealed bag containing desiccant and stored at 4 t:. The sample absorbent pad is 15 cm long and 2 cm wide. The material of this sample squeegee or squeegee is not limited, but the higher the absorbability, the better, and the size of the sample squeegee is variable. The carrier fiber block is placed on the front end of the porous fiber membrane and on the bottom plate, and the carrier fiber block and the nitrocellulose membrane are overlapped and connected, and the sample absorption pad is adhered to the display carrier fiber block and the bottom plate, and The display ί 3 5 t blocks are connected to each other. Finally, the absorbent pad is adhered to the end of the bottom end of the porous fiber and the quasi-film element. At least a portion of the surface of the absorbent pad is superposed on each other. Then cut into a loose 〇 · 5 cm test strip finished product. Should be on the sample squeegee, contact each other. Sample: area

930313002.ptc Put the finished product of the test strip into a waterproof device box, this 1283750 month 曰 _

_______ 93108448 V. Description of invention (16) should be smaller than the sample squeegee. The interpretation section and the control line of the test strip shall be located in the hole of the reaction zone of the device box, visible to the naked eye. " , the other side of the judgment section is the sample absorbent pad. The carrier fiber block is displayed and the nitrocellulose membrane and the sample absorbent pad are in contact with each other. After reading the section ^ on the makeup line. The control line is followed by an absorbent pad. When the sample to be tested is added, the result can be directly judged by using the naked eye in the pore of the reaction zone. As soon as a sample of 15 〇 microliters of negative liquid serum is added to the sample well, the blue member carrier will move to the reaction line (interpretation section and control line) due to the capillary principle and enter the absorption pad at the last end. Since this negative sample does not contain the final glycated protein antigen, the blue display vector immobilized anti-finalized glycoprotein cannot bind to the anti-finalized protein antibody immobilized by the interpretation segment, and two = two blue lines visible to the naked eye. The blue display vector will bind to the control of each of the anti-rabbit IgG antibodies to form a macroscopic line γ which is a negative reaction. The absorbent pad will absorb all of the flowing liquid: it continues to form a capillary reaction. The addition of 1 to the interpretation of the serum sample arrival will be repeated with the color line in the direct response of the segment reaction, the most segmented segment of the product before the final saccharification. On the remaining line, it is the end of the test strip; in addition to the use of Shengyang, the upper saccharification, the interpretation of the protein anti-blue is the Sanming finished product' can complete the S-type test on the sandwich

930313002.ptc page 20 in the sample carrier in the absence of antibodies, will be visible with the display carrier transfer protein antibody eye blue 'and will be bound, of course, as the previous serum sample fixed anti-most protein antigen junction area The original binding is immobilized on the segment, and the carrier is positively treated by the anti-therapeutic method, and the sample is pre-formed as described above to complete the glycation of the protein. When the blue anti-final sugar forms a piece of meat, the segment is read and the test result is to be tested. 1283750 Case No. 93108448 Year Month_Day Article V. Invention Description (17). The use of competition law is also feasible. According to the fourth embodiment, the experiment of the competition method can be performed by replacing the fixed substance sprayed on the interpretation section, for example, the final glycated protein antigen. To determine the anti-final glycosylated antibody, simply change the substance immobilized on the blue display vector to the final glycated protein antigen, and change the immobilized substance to the final glycosylated protein antibody to compete against the final glycated protein antibody. Determination of the law. If the blue display carrier is the final glycated protein antigen, the interpretation of the segmental immobilization substance is replaced with an anti-anti-final glycosylated antibody or an anti-human I g G antibody (referred to as a second antibody), and the final glycated protein can be carried out. Sandwich assay for antibodies. '' Specific reagents and methods to test whether it can exist, thus allowing medical personnel to carry out complications or block complications

In summary, it has been known by the present invention that the final glycated protein of diabetes is prevented from continuing in the earliest period.

1283750 案号 93108448 Λ_Ά 曰Revision diagram simple description FIG. 1 is a three-dimensional structure diagram of the immunochromatographic test strip of the present invention; FIG. 2 is a three-dimensional outline view of a waterproof device box; The immunochromatographic test strip shown in the figure; and Fig. 3 is a schematic cross-sectional view showing the overall test strip of the present invention in the waterproof device case shown in Fig. 2. <Explanation of Symbols> 10 Test Strips 11 Squeegee 12 Display Carrier Fiber Block 13 Porous Fiber Membrane 14 Interpretation Section 15 Control Section 16 Absorption Pad 17 Base Plate 18 Test Strip 1 0 Front End 19 Test Strip 1 0 Rear End 20 Waterproof box 21 sample hole 22 reaction area hole

930313002.ptc Page 22

Claims (1)

1283750 Emperor No. 9310? Um Year of the game ^ month / f day repair (Car Month - H VI. Patent scope 1) A measuring solution used to determine the final glycated protein, which includes the solid-growth phenomenon of glycated egg 2 According to the application body is a 3. According to the body of the application 4. According to the application of the substance as the final sugar 5. According to the application of the substance as the final sugar 6. The type of measurement and measurement of the absorption, the existence of the species is indicated by the waiting, Judging white antibody, please patent the species, please patent, small system, clearing the patent, final glycation protein reading, patent anti-finalization protein, final species display, determine whether this luminosity is determined or not. The suspension of the carrier shows the carrier sample. The presence or absence of the sample to be tested is in the range of the first color of the particle. The first item in the range of 〇·01 μm is the first antibody to the antigen. Scope 1 The glycosylation protein anti-antigen. Suspension display carrier sample test sample device for determining the final saccharified egg liquid; after contact with the affinity substance on the surface, the final glycated protein is resistant; a condensed or anti-final assay solution, wherein the display contains the assay solution, which is between ~60 microns. The assay solution, which can be used to determine the assay solution to be tested, the body, It can be used to determine the test solution to be used, and the display carrier that is in contact with the test liquid on the surface has a white antigen or is resistant to the end of the test. Affinity test sample 930313002.ptc page 23 7. The assay solution according to claim 6, wherein the display carrier is a microparticle or an enzyme. 8. The measuring liquid according to claim 7, wherein the microparticles are a kind of granules, lipid granules, dye granules, polyethylene glycol granules, NADH microparticles, NAD microparticles, polymeric microparticles or carbon microparticles. 9. The assay solution described in Section 6 of the patent application, wherein the affinity: two is the final glycated protein antigen can be used to determine the antibody to be tested for glycosylated protein. · ^ 10: ί1ί ί 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 1L a measuring solution for determining a final glycated protein, comprising: a competitor, a display carrier suspension; and an affinity substance disposed on the surface of the display carrier; and mixing a sample to be tested with the competitor After the presence or absence of the body, the presence or absence of the antibody against the glycosylated protein can be determined by a device for measuring absorbance. ',' 12· According to the scope of the patent application, the sex of the rabbit, the field of the animal, the person who is the person, the person who is the person, the person who is the person, the 亲: ! protein antigen, the competitor is the final glycated protein resistance 1 3 The ruin is used to measure the final glycated protein antibody in the sample to be tested. The test solution described in Item 1 of the patent scope of the patent application, the anti-finalized protein is anti-finalized protein anti- 疋 卞 取 取 取 糖 930313002.ptc Page 24 Amendment 1283750, —— nickname 93108448 _± VI. Scope of application of the patent antibody, which can be used to determine the final sputum protein antigen in the sample to be tested. A test solution for determining a final deuterated protein, comprising: a competitor, a suspension of a display carrier; and an affinity substance immobilized on the surface of the display carrier; After the sample is mixed with the competitor, and then, after contact with the carrier, whether or not agglutination occurs to determine the presence of the final glycated protein antigen or the anti-finalized glycoprotein antibody in the sample to be tested is $15·= Π二所;, assay solution, &quot; affinity protein prion, the competitor is the final glycated protein anti-suppressed. The assay solution described in 4, ... affinity white antibody,; to: prion antibody, the competitor is the final glycated protein antigen in the final glycated egg d 疋 test sample. 1283750 Case No. 93108448 曰 Amendment 6. Designated representative figure (1), representative figure of the case is: ____1____ figure (2), the representative figure of the representative figure of the case is a simple description: 10 test strip 11 absorbent pad 12 display carrier Block 13 Porous fiber membrane 14 Interpretation section 15 Control section 16 Absorption 塾 17 Base plate 18 Test strip 1 0 Front end 19 Test strip 10 rear end 930313002.ptc Page 3