TW200408808A - Method and kit for homocysteine assay - Google Patents

Abstract

A method is provided for assaying homocsyteine in a biological sample containing homocysteine and cysteine. A competing compound with an amino group (-NH2) is mixed with the biological sample. An aldehyde is added into the biological sample to form homocysteine complex with fluorescence response. The concentration of homocysteine in the biological sample is determined according to the fluorescence intensity.

Description

200408808 V. Description of the invention (l) The technical field to which the invention belongs The present invention relates to a determination method, and in particular to a method and a kit (ht) for detecting cysteine (Höffyl Cysteine) in a biological sample mouth. Homocysteine is a kind of amino acid produced by metabolism in the human body. Current research shows that cysteine can cause platelets to aggregate, cause atherosclerosis and increase blood clotting. Cysteine concentrations in the blood of older and postmenopausal women usually increase, which increases their risk of heart disease and stroke. In a 1969 study, McCully et al. First proposed cysteine in plasma? The relationship between agronomy and cardiovascular disease. The US Food and Drug Administration has imposed a requirement of 140 mg of folic acid per 100 grams of all nutritionally fortified cereal products since January 1998 to reduce the level of cysteine in the blood. Class A cystine is considered to be the leading risk factor for arteriosclerosis, and its mechanism of action may be due to oxidative damage. Human plasma usually contains both cysteine and cysteine. However, deamidine-like and cysteine (Cysteine) are both amino acids containing hydrogen sulfide (sulfhydryl), and each molecule contains an amine group (_〇2), a carboxyl group (-C00H) and a thiol group (-SH ) Three functional groups. They differ by only one carbon atom. As follows:

0648-9124TWF (Nl); 13910016; peggy.ptd page 6 200408808 V. Description of the invention (2)

Cysteine

HS ^ / COOH NH2

Homocysteine also has the same functional group, so it is easy for the two to interfere with each other during the measurement, and it is very difficult to separate them. Methods for determining cysteine include GC-MS, high performance liquid chromatography (HPLC), high performance wool, fine chromatography (HPCE), fluorescent polarization immunoassay (FPIA), or enzymes Immunoelectrophoresis (E IA) and so on. However, the disadvantages of chromatography are that it is time-consuming and labor-intensive, and it is also difficult to analyze low concentrations of cysteine in plasma. In addition, cysteine is easily oxidized, so it is difficult to accurately quantify the concentration of cysteine in humans. Non-amino acids of low-concentration blood pupa In U.S. Patent No. 62 652 20, a non-immunoassay method is disclosed. Cysteine and quasi-like compounds are derived from cis-1,4-dioxo-2 -butene group (CIS-1 '4-di 0X0-2-butene moiety) or its hydrolysis. React with different substances. According to the patent, the maximum emission wavelength of Cysteine adduct is 566 nm, and the maximum emission wavelength of Homocysteine adduct is 577 nm. Since the fluorescein reactions of cysteine adducts and cysteine-like adducts are quite similar, the method disclosed in this patent will cause difficulties in actually identifying these two adducts. In addition, when using HEPES buffer, cysteine and cysteine have similar maximum emission wavelengths of 52nm, so

200408808 V. Description of the invention (3) This is to prevent the interference of cysteine compounds and o-pht ha 1 aldehyde by adding antibodies labeled with an inhibitor. However, since the emitted fluorescence is broadband, the fluorescence signal between cysteine and cysteine is still not easy to distinguish. U.S. Patent No. 5 4 7 8 7 2 9 discloses a cystine-like immunological detection method. Tris (2-car box ye thy 1) phosphine is mainly used to reduce disu 1 fides in plasma, and then para-bromoacetamidobenzoic acid is added. (p-bromoacetyl benzoic acid) reacts with cysteine and cysteine. This method chemically modifies the analyte and its analogs to increase their immunogenicity and improve the recognition rate of their antibodies. In its reagent, hydrogen sulfide (sul fhydry 1 group) does not form a cyclic adduct with cysteine, but it forms a six-membered cyclic adduct with cysteine. The cystic acid content was then quantified by high performance liquid chromatography (HLPC). Another way is for the antibodies to form specific bonds with the modified cysteine after the presence of the ring. The modified cysteine adduct does not form an immunocomplex with it, so cysteine does not interfere with the quantification of cysteine. However, the method disclosed in this patent, the sample processing is quite complicated, and it is difficult to use the general clinical rapid detection. Most current methods for detecting cysteine are time-consuming or expensive. For example, Frantzen et al. Proposed the conversion of cysteine to S-adenosyl homocysteine in 1998, and the use of monoclonal antibodies to analyze S-adenosaurus by enzyme immunoassay (EIA) Cysteine content (Frantzen et al. Clin. Chem. (1 998) 44: 3Π-316 ·). Although this method

0648-9124TWF (Nl); 13910016; peggy.ptd page 8 200408808 V. Description of the invention (4) It can be operated automatically, but each test sample requires an average of 2.5 hours of test time. Dias et al. Proposed to aggregate the reduced blood with a derivatizing agent, for example: 4-aminosulfanyl group-qi-2,1,3-benzodiazepine (4-aminosulfonyl-7- fluoro-2, 1,3-benzoxydiazole) reacts with cysteine and cysteine to form a fluorescent adduct (Dias et al. Clin · Chem · (1 99 8) 44: 2 1 99-22 0 1 ·), Then the adducts of the two were separated by HPLC. However, this method takes more than 1 hour for each sample. U.S. Patent No. 60460 17 proposes a fast and sensitive test method for cysteine. This method uses a series of enzyme reactions including methionine synthase and SHMT to detect and quantify cystatin in a biological sample. It takes about 30 minutes. The disadvantage of this method is that it requires the use of radioisotopes. U.S. Patent No. 59 85540 discloses a cystine-like method for biological samples with high specificity. Among them, a cysteine-decomposing enzyme using 7-face amine cysteine synthetase (r-glutamylcysteine synthetase) can avoid the interference of cysteine or methionine in biological samples. . However, this method also requires considerable time to complete the entire measurement. US Patent No. 58 85767 discloses a method for quantifying the content of L-cysteine (1 ^ -110111007 5 16 1116) and / or 1-methionine in a solution. When L-cysteine and / or L-cystine and methionine 7-methionine gamma-lyase

0648-9124TWF (Nl); 13910016; peggy.ptd page 9 200408808 V. Description of the invention (5) During the reaction, 2-ketobutyrate will be produced. The content of L-cysteine and / or L-sulfanilate is calculated based on the content of 2-ketobutyric acid. However, in general biological samples, thallium thiamine is often present, so it cannot be excluded to get the exact L-cysteine content. U.S. Patent No. 563 1 27 discloses a combination of a cystine-like enzyme assay and an analysis kit. S-adenosyl- homocysteine hydrolase (SH-hydrolase) is mainly used to catalyze the reaction between cysteine and adenosine to form S-adenosyl cysteine ( S-adenosyl-homocysteine). However, the disadvantage of this method is that its equilibrium constant is only 106 M-1, which will cause significant errors for low concentrations of cysteine. In U.S. Patent No. 6,002,206, a method for determining cysteine in a sample is disclosed, which includes step (a) converting cysteine in the sample to cysteine thiolactone ( homocysteine thiolactone); step (b) reacting a compound containing a free thio group (thio) in the sample with a thio group scavenger; step (c) converting cysteine thiolactone to cysteine; (d ) Detection of cysteine content in the sample. However, the disadvantage of this method is that some steps are more complicated and require 30 minutes reaction at 8 (pc.) For the analysis of cysteine, the current method is still 'avoid' Cysteine interference, cysteine-like concentration. Therefore, for a clinical need for a fast and affordable test to accurately detect the biological sample, the invention aims to provide a fast (Homocysteme) Methods and kits for cystic acid during skin makeup

200408808 V. Description of the invention (6) Detection of biological samples with cysteine (Cysteine) Another object of the present invention is to provide an affordable cysteine-like detection method and kit, which is suitable for clinical detection of cysteines in biological samples. Cysteine Concentration 0 Another object of the present invention is to provide an accurate detection method and kit for cysteine, which can detect and quantify low concentrations of cysteine in biological samples. In order to achieve the above-mentioned objective, the present invention provides a method for detecting cystine-like acid, which comprises the following steps: (a) providing a competitor with the biological sample, wherein the competitor has an amine group; Compounds are added to (a) a solution to form a fluorescent complex with cysteine in the biological sample; and (c) detecting (b) the fluorescence intensity of the solution to estimate the cystamine in the biological sample Acid content. The present invention further provides a method for detecting cysteine, which is suitable for detecting a biological sample containing both cysteine and cysteine, comprising the following steps: (a) providing a competitor with the biological sample, Wherein, the competitor contains trismethylaminomethane (TRIS); (b) o-phthalaldehyde is added to (a) the solution forms fluorescein with cysteine in the biological sample; And (c) detecting the light intensity of the solution (b) to estimate the cysteine content in the biological sample. The present invention further provides a cysteine-like detection kit, which is suitable for detecting a biological sample containing one of cysteine and cysteine, including a competitor, and a compound containing at least one amino group (-NH2) ; And a reaction reagent 'can form glory adducts with cysteine, and after competing with this competitor

0648-9124TWF (Nl); 13910016; peggy.ptd Page 11 200408808 V. Description of the invention (7) No fluorescent radiation will be generated in a few minutes, but it can react with cysteine to generate fluorescent addition Thing. In order to make the above-mentioned objects, features, and advantages of the present invention more comprehensible, the following is described in detail with the accompanying drawings: Embodiments A general biological sample, such as plasma or urine, usually contains cysteine (Cysteine) and Homocysteine (Homocysteine), so if you want to detect the content of Homocysteine in plasma or urine, you must avoid the interference of cystine. Therefore, according to a method for determining cysteine according to the present invention, a competitor is added to the detection reagent. The detailed description is exemplified below. The medicines in the following examples were purchased from Merck,

Darmstadt), Aldrich Chemical Company, Milwaukee, Wis · and Sigma, St. Louis, Mo ·. Except for o-phthalaldehyde, which was formulated with 20% methanol, all other reaction solutions and samples were prepared with pure water as the matrix. Example 1 Three samples were prepared, each containing 1 ml of a solution containing Homocysteine, cysteine, and a blank test. ImM ethylenediaminetetraacetic acid (EDTA) and 18 mM greenhouse sodium were added. (Sodium borohydride, NaBH4) in 1 ml of 100 mM Tris- (hydroxymethyl) aminomethane (hereinafter referred to as TRIS) buffer solution, and stirred at room temperature for 2 minutes. Add 20% methanol or ethyl

0648-9124TWF (Nl); 13910016; peggy.ptd Page 12 200408808 V. Description of the invention (8) Alcohol-made 18 mM phthalic acid (0-phthalaldehyde, hereinafter referred to as OPA) to form a fluorescent complex ( f luorescence complex) ° Detect the absorption between 3 and 500 mD1 of the sample at a wavelength of 5 25nm. Its spectral absorption distribution is shown in Figure 1, and its maximum absorption wavelength is 436 nm. The fluorescence reaction of the sample was excited by excitation light with a wavelength of 436 nm, and its fluorescence spectrum was detected between 450 and 600 nm, and its maximum wavelength was 5 24 nm. The cysteine adduct fluorescence reaction is strongest after 4 minutes. In contrast, the fluorescence reaction of the cysteine adduct disappeared after 4 minutes of reaction. Therefore, the cysteine and cysteine adducts in the sample can be distinguished by their light absorption characteristics, as shown in Figure 2A. Figure 2A shows the fluorescence emission spectrum of three samples measured at a wavelength of 450 to 600 nm in the fourth minute. Curve I represents a blank test solution without added cysteine or cysteine, that is, a solution containing only 0PA. Curve 11 is the cysteine-added solution, that is, the solution contains cysteine and OPA. Curve 11 I is the sample with cysteine added, that is, the sample contains cysteine and OPA. Because trimethanol aminomethane (TR IS) has an amine group (-NH2) that can react with OPA, the amine group (-NH2) of TRIS will react with cysteine (Homocysteine) and cysteine (Cysteine) The amine groups compete together for the OPA reaction. The reaction between 0PA and TRIS is as follows:

0648-9124TWF (Nl); 13910016; peggy.ptd page 13 200408808 V. Description of the invention (9) (OPA) -C-0 + H2N0 (CH20H ^ (TRIS)

-CH = NC (ChfeOH) " CHsNHCCC ^ O% and the reaction between cysteine and cysteine and OPA is as follows OPA + Homocysteine ΟΌ-Η +

HS- \ .COOH

nh2

OPA + cysteine

+

CH

〇 After the reaction between OP A and TRIS in the blank test sample, the product did not produce fluorescence absorption, as shown in curve I in Figure 2A. Still referring to Figure 2A, the curve IBfil 0648-9124TWF (Nl); 13910016; peggy.ptd Page 14 200408808 5. In the description of the invention (ίο) II, TR IS competes with Cysteine (Cysteine) and reacts with OPA Under the catalysis of sodium borohydride and methanol, TR IS is superior to cysteine and forms a stable compound with excess OPA in the sample, so curve II in Figure 2A does not show fluorescence absorption. Still referring to Figure 2A, Homocysteine preferentially forms stable compounds with TRIS than OPA, and shows a clear fluorescence absorption peak, as shown in curve I 11. If the TRIS buffer in the example is filled with a phosphate buffer or N-2 -hydroxyethyl p-diazahexyl-N-2-ethanesulfonic acid (N-2-Hydroxyethylpiperazine-2-N ' -ethanesulfonic acid (hereinafter referred to as HEPES), the fluorescence reactions are shown in Figures 2B and 2C, respectively. In Figure 2B, it can be seen that the blank test curve I containing only OPA has no fluorescence absorption. However, curve II containing cysteine and curve 11 I containing cysteine both generate fluorescence absorption, showing that the response of cysteine and cysteine to OPA is higher than that of linate, so the phosphate buffer solution It cannot replace cysteine and react preferentially with OPA, so it is not suitable as a competitor. Similarly, according to Fig. 2C, a blank test containing only OPA has curve I showing some degree of absorption. And the curve containing cysteine and cysteine! j and Π I have more obvious fluorescence absorption, showing that the HEPES buffer solution cannot replace cysteine and preferentially react with OPA, which is even less suitable as a competitor. Therefore, comparing Figures 2A to 2C, it can be seen that the TRIS buffer solution containing amine groups is the best competitor. When the TRIS concentration is high enough, it can replace cysteine with OPA, but its reaction stability is Lower than Cysteine and therefore does not affect Cysteine response. So ‘TRIS’ amines compete

0648-9124TWF (Nl); 13910016; peggy.ptd page 15 200408808 V. Description of the invention (11) The agent can effectively exclude the effect of cysteine on the determination of cysteine. According to the research of the present invention, because the five-membered cycloadduct formed by cysteine and OPA is less stable, it will be replaced by TR IS, while cysteine can quickly form stable and OPA with high fluorescence emission. Six-membered ring, so by adding appropriate competitors, such as TR IS, you can more clearly distinguish cystine and cysteine, making the quantification of cysteine more accurate. In another embodiment, Acetamide (CH3C0NH2) containing an amine group is selected as a competitor. The reaction result is shown in FIG. 5. The competitor solution is 100 mM acetamide, which contains 1 mM EDTA and 100 mM sodium borohydride. The curve π is, after adding i8mM cysteine and cysteine, let stand for two minutes, then 100 mM OPA was added. After the reaction, the fluorescence emission spectrum of cysteine is shown as curve II, and the fluorescence emission spectrum of cysteine is shown as curve I I I. From the graph in Figure 5, it can be seen that when acetamide is used as a competitor, the fluorescence emission of cysteine at a wavelength of about 450 nm is extremely low, so the fluorescence emission value at a wavelength of 450 nm can be measured to estimate Cysteine concentration. Embodiment 2 The detection time required by the method according to the present invention will be described below with practical examples. Cysteine-containing samples were added in equal amounts to 100 mM TRIS buffer containing 1 mM EDTA and 18 mM sodium hydrogenated side (NaBH4) and reacted for two minutes. Next, the above solution was mixed with 18 mM OPA prepared in a 20% methanol solution to form a fluorescent adduct. After different reaction times (1, 5, 10, 20, 30, and 50 minutes), it is excited by excitation light with a wavelength of 436 nm

0648-9124TWF (Nl); 13910016; peggy.ptd page 16 200408808 V. Description of the invention (12) and 4 test π 0 between 4 5 0 ~ 6 0 0 n m. * Eight μ fluorescence reacts first. As shown in Figure 3A, the fluorescence reflection f gradually increases from the 1st clock to the 5th minute with the strongest fluorescent response: As the reaction time lengthens, the fluorescent response gradually weakens to the 50th knife ^ ^ It is approaching gentle. Figure 3B shows a sample containing cysteine.

In an equal manner, add 100 mM TRIS buffer solution containing lmM Sin and quilted sodium borohydride (MB. After two minutes of homogeneous reaction by stirring, the above solution and 1001 ^ formulated in a 20% methanol solution. 〇Mixed to form a fluorescent adduct, and detected the fluorescence signal at ~ 5 minutes after mixing. As can be seen from Figure 3B, as the concentration of 〇pA increased to 100 mM, fluorescence emission The intensity reaches its maximum at the first minute, and then the fluorescence emission intensity decreases slowly from the fifth to the fifth minute. Therefore, as shown in Figure 3 and Figure 38, the method for determining cysteine of the present invention, in order to ensure that After the reaction is completed, the fluorescence is detected at about 4 minutes, that is, the second minute after the addition of 0pa, so the measurement of the entire sample can be completed after 4 minutes, which is quite fast and convenient. Taking one embodiment as an example, Add 100 mM TRIS buffer containing ImM EDTA and 18 mM sodium borohydride (NaBH4) to the cysteine-containing solution, stir for 2 minutes, then add a 20% methanol solution containing i8mM 0PA, and measure after 2 minutes. Its fluorescent signal. Three following detailed description. In a preferred embodiment, the cysteine class detection kit comprising a detection kit according to its competitive detection method of the present invention an embodiment of one class cystamine acid

IHI 0648-9124TWF (Nl); 13910016; peggy.ptd Page 17 200408808

Agent is a competition reagent between cysteine and cysteine. The competition test contains amine (-NM compounds, preferably: amines = ethylamine, ethylamine, ethyl Acetamide or TR IS buffer, etc. In a preferred embodiment, the competitor is 100 mM TRIS. The detection kit also contains a fluorescent agent capable of generating fluorescence with cysteine The reaction reagent of the adduct is, for example, an aldehyde-based reagent, preferably 100-o-phthalaldehyde (OPA), which is formulated with 20% methanol or ethanol, and is used to communicate with cystoids. Glycine, cysteine, and amine groups in the competitor do not generate fluorescent radiation after reacting with the competitor, but only generate fluorescent radiation with cysteine. Among them, the competitor and the fluorescent The reaction stability of the reagent is higher than that of the cysteine and the fluorescent reactant, and lower than that of the cysteine and the fluorescent reactant. According to the above cysteine detection kit, cystamine can be performed Acid calibration line and cysteine detection in samples, detailed description below. Calibration line-standard addition Standard Addition) Add 50, 100, and 150 / z M of cysteine to the sample to be tested, such as human serum, and 1 ml of iodol per 1 ml (mL) of serum sample. mM TRIS buffer containing ImM EDTA and 18 mM sodium borohydride. After 2 minutes of room temperature reaction, the above solution was added with 1 ml of 100 mM 0PA solution prepared with 20% ethanol. After 2 minutes of reaction, The fluorescence reaction was excited at a wavelength of 436 nm, and the maximum fluorescence emission value was measured at a wavelength of 525 nm. The results are shown in Table 1 below.

200408808 V. Description of the invention (14) Table 1 Cysteine concentration (μΜ) Cysteine labeled concentration I Serum Addition FI Intensity Intensity Δ Intensity Intensity) Δ Intensity 0 0.534 0.000 1.779 0.024 50 0.545 0.011 1.891 0.136 100 0.552 0.018 2.028 0.273 150 0.560 0.026 2.063 0.308 Blank serum strength: 1.221 Blank reagent strength (including TRIS buffer and 〇PA): 0.534 Table 1, the obtained calibration curve is shown in Figure 4. The curve I shown in Figure 4 is the calibration curve for the standard concentration of cysteine, y = 0.0002x + 0.001, and the curve II is the calibration curve for serum standard after adding cysteine, y = 0.00 2 + 0. 0 369. Sample detection Take 1 ml of a serum or urine sample and add 1 ml of TRIS buffer, which contains lmM EDTA and 18 mM sodium borohydride. After the above samples had reacted for 2 minutes at room temperature, 1 ml of a 100 mM solution of 0PA in 20% ethanol was added. After 2 minutes of reaction, the fluorescence reaction was excited at a wavelength of 43 6 nm, and the maximum fluorescence emission value was measured at a wavelength of 5 2 5 nm. The radiation value was compared with the above-mentioned calibration line to calculate the cysteine content in the sample. The fluorescence reaction is derived from three components: (1) the product of the reaction between cysteine and OPA, (2) the background interference of the reagent (buffer or opa solution), and (3) the background interference of the test sample (such as serum or urine). liquid). Therefore, in the actual detection of cysteine concentration in biological samples, it is necessary to exclude the background values of the latter two.

0648-9124TWF (Nl); 13910016; peggy.ptd Page 19 200408808

The standard concentration curve of cysteine in Table I is based on pure water. When the cysteine concentration is 0, the fluorescence reaction should come from the test 1 agent $ interference ', and its reading can be regarded as the background value of the reagent. In the method of detecting κ in serum standard addition, human serum containing unknown concentrations of cysteine is used as the base for cysteine standard addition, and the standard concentration of cysteine is added back to the company / month. Cysteine concentration. In this method, the color of Jinqing may form background interference. Therefore, the serum blank test group (helmet TR buffer solution and OPA) was used to remove the background interference of serum. ..., although the present invention is disclosed as above with a preferred embodiment, it is not intended to define the present invention. Anyone skilled in the art can make some changes and retouch without departing from the scope of the present invention and the target range. Therefore, the present invention The ^ ^ Wai Dang shall be determined as defined in the scope of the attached patent application. "crossbar for yoking horses

0648-9124TWF (Nl); 13910016; peggy.ptd Page 20 200408808 Brief Description of the Drawings Figure 1 shows an absorption spectrum of a cysteine-0PA complex at an emission wavelength of 525 nm in one example. Figures 2A to 2C do not show the fluorescence emission spectra of cysteine and cysteine when different competition buffers are used according to an embodiment of the present invention. Figure 3A shows the fluorescence spectrum of cysteine in TRIS buffer at different reaction times according to an embodiment of the present invention. Figure 3B shows the fluorescence spectrum in 1 to 5 minutes. Figure 4 shows a calibration curve of standard cysteine and serum standard additions according to an embodiment of the present invention. Fig. 5 shows the fluorescence emission spectra of cysteine and cysteine when acetamide is used as a competitor according to an embodiment of the present invention.

0648-9124TWF (Nl); 13910016; peggy.ptd Page 21

Claims (1)

200408808 6. Scope of patent application 1. A method for detecting cysteine (Homocysteine), suitable for detecting a biological sample, comprising the following steps: (a) providing a competitor with the biological sample, wherein the competitor has a Amine group; (b) adding an aldehyde compound to (a) a solution to form a fluorescein complex with cysteine in the biological sample; and (c) detecting (b) the fluorescence intensity of the solution to estimate Cystoid content in the biological sample. 2. The detection method of cysteine (Homocysteine) as described in item 1 of the scope of the patent application, wherein the competitor is tris (tris) aminoacetic acid (TRIS), acetamide, ethylamine ( ethylamine) or one of the amines. 3. The method for detecting cysteine (Homocysteine) according to item 2 of the scope of the patent application, wherein the amine-based compound is a long-chain or branched or cyclic compound containing at least one amine group. 4. The method for detecting cysteine (Homocystei ne) as described in item 1 of the scope of the patent application, wherein the biological sample is one of serum or urine. 5. The detection method of cysteine (Homocystei ne) according to item 4 of the scope of the patent application, wherein the biological sample contains cysteine and cysteine. 6. The method for detecting cysteine (Homocystei ne) as described in item 5 of the scope of the patent application, wherein the fluorescence generating agent forms a fluorescent complex with cysteine and cysteine (Cysteine) and Not with this competitor 0648-9124TWF (Nl); 13910016; peggy.ptd page 22 200408808 Formation of fluorescent complexes. 7. The detection method of cysteine (Homocysteine) as described in item 1 of the scope of the patent application, wherein the fluorescence generating agent is o-phthalaldehyde with a concentration of 1 to 100. Ben-8. According to the method for detecting cysteine (Homocysteine) as described in item 1 of the scope of the patent application, wherein the body of steps (a) to (c) is 4 to 15 minutes. Big 9 · According to the method for detecting cysteine (Homocysteine) described in item 1 of the scope of the patent application, wherein the steps (a) to (b) are separated by 2 minutes. θ ^ 1 〇 According to the method for detecting homocysteine (Homocysteine) as described in item 5 of the scope of patent application, wherein steps (b) to (c) are generally separated by 2 minutes. ~ 11. · A method for detecting cysteine (Homocysteine), suitable for the detection of biological samples containing both cysteine (Cysteine) and cysteine (Cyste i ne), including the following steps: (a) provide A competitor is mixed with the biological sample, wherein the competitor contains trismethylaminomethane (TRIS); (b) o-phthalaldehyde is added to (a) the solution and cystoids in the biological sample Glycine forms a fluorescent complex; and (c) detects (b) the fluorescence intensity of the solution to estimate the cysteine-like content in the biological sample. 1 2 · The detection method for cysteine (Homocystei ne) according to item 11 in the scope of the patent application, wherein the competitor further comprises ethylenediamine 0648-9124TWF (Nl); 13910016; peggy · ptd page 23 200408808 6. Scope of patent application ~ Tetraacetic acid (EDTA) and sodium borohydride. 1 3. According to a method for detecting a deamino acid (Homocysteine) according to item 12 in the scope of the patent application, wherein the concentration of the trimethylolaminomethane (TRIS) buffer solution is approximately 100 mM, and the ethylenediamine The concentration of tetraacetic acid (to eight) is approximately 1 mM, and the concentration of sodium borohydride is approximately 8 mM. 14. The detection method for cysteine (Homocyste ne) as described in item 12 of the scope of the patent application, wherein the o-phthalaldehyde is prepared in a 20% ethanol solution at a concentration of 1 100 mM 〇1 5. The detection method of cysteine (Homocysteine) according to item 14 in the scope of the patent application, wherein the body of steps (a) to (c) is 4 to 15 minutes. A 16. The method for detecting cysteine (Homocysteine) according to item 15 of the scope of the patent application, wherein steps (a) to (b) are separated by 2 minutes. Roughly 17 · According to the method for detecting cysteine (Homocysteine) described in item 16 of the scope of patent application, wherein the interval between steps (b) to ((:) is 2 minutes. Roughly 1 8 · According to the scope of patent application The method for detecting cysteine (Homocysteine) according to item 11, wherein the biological sample is one of the main fluids. Α Take urine 19. A cysteine (Homocysteine) detection kit is suitable for detecting One of Cysteine and Cysteine is colder than contains ... 200408808 VI. Patent application scope A competitor, including a compound containing at least one amine group (-Njj2); and a reaction reagent 'can react with cysteine to generate fluorescent radiation' will not react with the competitor Fluorescent radiation is generated, and fluorescent radiation is generated with cysteine. 20 · According to Homocysteine detection kit according to item 19 in the scope of the patent application, wherein the reaction stability of the competitor and the reaction reagent is higher than that of the cysteine and the reaction reagent, but the reaction stability is low. The reaction of the deamidated acid with the reaction reagent. 21 · According to Homocysteine test kits described in item 19 of the scope of the patent application, wherein the competitor is Tris-Methanolamine (TRIS), acetamide, ethylamine ) Or one of the amines. 22 · The cysteine (Homocysteine) detection kit according to item 21 of the patent application scope, wherein the competitor further comprises ethylenediamine tetraacetic acid (EDTA) and sodium borohydride. 23. The cysteine (Homocysteine) detection kit according to item 22 of the scope of the patent application, wherein the concentration of the trisamine aminomethane (TRIS) buffer is approximately 100 mM, and the ethylenediaminetetraacetic acid (EDTA) The concentration was generally ImM, and the sodium borohydride concentration was approximately 18 mM. 24. The Homocysteine detection kit according to item 21 of the patent application scope, wherein the reaction reagent is an aldehyde compound. 25 · According to the cystine type as described in item 24 of the scope of patent application 0648-9124TWF (Nl); 13910016; peggy.ptd Page 25 200408808 Six. Patent application scope (Homocysteine) detection kit, the aldehyde compound is o-phthalaldehyde 〇2 6. According to the scope of patent application Homocysteine detection kit according to item 24, wherein the o-phthalaldehyde is prepared in an ethanol solution at a concentration of 1 to 100 mM. 0648-9124TWF (Nl); 13910016; peggy.ptd p.26