CN100464189C - Magnetic separation direct chemiluminescence reagent and test method using the same - Google Patents

Abstract

The invention relates to a magnetic separation direct chemiluminescence reagent and a testing method thereof, mainly using isoluminol derivatives as luminescent markers, immune nano-magnetic microbeads coated with goat anti-FITC polyclonal antibodies as separation reagents, and using NaOH and H ₂ O ₂ reagents and test methods for direct magnetochemical luminescence as luminescent substrates; the reagents described in the present invention are stable, non-polluting, non-decomposed and not affected by environmental factors, and the test method is fast and the result is precise.

Description

Magnetic separation direct chemiluminescence reagent and test method using the reagent

technical field

The invention relates to a magnetic separation direct chemiluminescence reagent and a test method using the reagent, in particular to a reagent using isoluminol derivatives as markers and nano magnetic microbeads as separation materials.

Background technique

The rapid development of modern clinical medical technology requires the precise quantitative determination of trace active substances in more and more people's blood or urine to provide accurate basis for clinicians to diagnose diseases, formulate effective treatment plans, and evaluate treatment effects. , the methods to achieve accurate quantitative analysis of trace active substances in human serum or urine mainly include radioimmunoassay, enzyme immunoassay, enzymatic chemiluminescence or acridinium ester-labeled direct chemiluminescence method and triple pyridine ruthenium-labeled electrochemical Luminous method.

Radioimmunoassay uses the radioactive isotope I ¹²⁵ as a tracer, which is marked on the antigen or the corresponding antibody. On the gamma ray detector, the content of the substance to be tested is determined by measuring the intensity of the I ¹²⁵ radiation. This method Although the purpose of quantitative analysis of trace active substances has been well solved, its defects are obvious: as a tracer technology, radioisotope I ¹²⁵ will pollute the environment during the manufacture, storage and application of reagents. , causing serious harm to the human body; at the same time, due to the limitation of the half-life of I ¹²⁵ , the validity period of the radioimmunoassay reagent is only one month; the validity period of the radioimmunoassay reagent is also only one month; the non-specificity of the separation process leads to poor accuracy of the results , can not do emergency specimens.

The enzyme immunoassay method uses HRP horseradish peroxidase as a luminescent marker, plastic microwell plate as a carrier and separation technology, OPD _{o- phenylenediamine} , etc. Stop the solution, and analyze the concentration of the substance to be tested by measuring the absorbance value of the enzyme reaction product wavelength at 492nm. This method is mainly used for qualitative screening of a large number of human serum samples. The defect is that the color intensity of the chromogenic product varies with Time changes and cannot be fixed. The chromogenic substrate OPD is a strong carcinogen. At the same time, the sensitivity to a considerable part of trace hormones is not enough, the reaction time is too long, and it is difficult to standardize during the operation.

The enzyme-catalyzed chemiluminescent immunoassay method uses alkaline phosphatase AKP or horseradish enzyme HRP as a marker, and _uses AMPPD (adamantane) or luminol, _H2O2 as a luminescent substrate; its advantage is that the analyte is converted The optical signal can be measured, which significantly improves the sensitivity of the analysis. The disadvantage is that many factors that affect the enzyme activity, such as ambient temperature, incubation temperature and time, storage conditions, etc., will directly affect the measurement results, and the stability of the reagent is not good. At the same time, the luminescence reaction of this method is slow to start, and it needs to be incubated at 37° C. for a certain period of time to reach the luminescence platform, which will strongly affect the result of the luminescence measurement.

In addition, the direct chemiluminescence method labeled with acridinium ester and the electrochemiluminescence method labeled with ruthenium terpyridine bring defects such as unstable reagents, low accuracy of analysis results and insufficient test speed during the analysis process, which hinders the accurate analysis of trace amounts. The amount of active substance presents a hindrance.

Contents of the invention

The purpose of the present invention is to provide a reagent for magnetic separation direct chemiluminescence, especially a magnetic chemical direct chemiluminescence reagent using isoluminol derivatives as markers and nano-magnetic beads as separation materials. Luminescence reagent.

Another object of the present invention is a test method for direct chemiluminescent reagents using the above-mentioned magnetic separation.

To achieve the above object, the present invention is achieved by adopting the following technical solutions:

The magnetic separation direct chemiluminescent reagent includes a luminescent marker, a separation reagent, a FITC-antibody conjugate and a standard antigen or antibody, wherein the luminescent marker is an isoluminol derivative, and the isoluminol derivative The structural formula is:

或

or

Where: R1 is C ₂ H ₅ , C ₃ H ₇ or C ₄ H ₉ , R ₂ is NH ₂ -(CH ₂ ) ₄ , NH ₂ -(CH ₂ ) ₆ , NH ₂ -(CH ₂ ) ₈ or NH ₂ -(CH ₂ ) ₁₀ , preferably ABEI and AHEI, in which R1 is C ₂ H ₅ , R2 is NH ₂ -(CH ₂ ) ₄ or NH ₂ -(CH ₂ ) ₆ ;

The separation reagent is sheep anti-FITC coated nano-magnetic micro-beads. The nano-magnetic micro-beads are coated with Fe ₃ O ₄ or Fe ₂ O ₃ inside and contain -OH, -COOH, -NH ₂ Microbeads of active groups, preferably -COOH and -NH ₂ , the content of the active groups is 0.05-0.5eqm/g; the nano-magnetic microbeads coated with goat anti-FITC are on the monoclonal antibody or antigen mark.

The following steps that the present invention adopts are analyzed and tested:

A. Mix and incubate the monoclonal antibody labeled with isoluminol derivatives, FITC and the serum to be tested respectively;

B. After the immune reaction is completed, add goat anti-immune nano-magnetic beads coated with FITC polyclonal antibody;

C. Under the action of an external magnetic field, the antigen-antibody complex is specifically separated, and the bottom test tube is placed in the measurement chamber after being washed with a lotion;

D. Pump the excitation substrates NaOH and H ₂ O ₂ with an automatic syringe pump;

E. Use a photomultiplier tube to count the number of photons emitted by the mixed product in the test tube, and use an analyzer to analyze and obtain the result of the analyte.

In the present invention, the monoclonal antibody of the luminescent marker of the isoluminol derivative is obtained by linking the luminescent marker of the isoluminol derivative with carbon dichloride or N-hydroxysuccinic acid, and then with the monoclonal antibody In step C, the bottom of the test tube uses nano-magnetic microbeads as a carrier.

The described magnetic separation direct chemiluminescence immunoassay testing method can be divided into sandwich method and competition method, and its design formula is shown in Fig. 1 and Fig. 2 respectively, and Ab ₁ * and Ag* in the figure represent respectively labeling on Ab ₁ and Ag Isoluminol derivatives ABEI or AHEI, Ab ₂ ^FITC and Ab ^FITC represent small molecules labeled FITC on Ab ₂ and Ab respectively, FITC is fluorescent sulfur isothiocyanate,

表示在纳米磁珠表面联接抗FITC抗体。

Indicates that the anti-FITC antibody is attached to the surface of the nano magnetic beads.

According to the magnetic separation direct chemiluminescence analysis method of the present invention, its technical effect has the following aspects:

1. The use of isoluminol derivatives as luminescent markers completely overcomes the defect of easy hydrolysis of traditional acridinium esters. As a direct chemiluminescence mode, not only the analysis speed is fast, but it is close to the automatic direct chemiluminescence of 180 tests/ Hours, completely avoid the defect that the enzyme activity of enzymatic luminescence is easy to decrease;

2. Nano-immunomagnetic microbeads are used as the carrier of the antibody and the separation reagent of the antigen-antibody immune complex, which greatly shortens the time of the immune reaction. The time for the separation of the immune reaction is only 5 minutes, and the time for the entire immune reaction is only 20 minutes, and High accuracy and precision of test results;

3. The bridging antibody immune design technology of FITC and goat anti-FITC antibody coated nano-magnetic beads greatly simplifies the immunological design of the entire reagent system. With the support of the strong protein adsorption capacity on the surface of nano-magnetic beads, the nano-immunomagnetic Beads become a common reagent for all test items, simplifying production procedures.

Description of drawings

Figure 1 is a design diagram of the magnetic separation direct chemiluminescence immune sandwich method;

Figure 2 is the design diagram of the magnetic separation direct chemiluminescence immunocompetition method

Detailed ways

The present invention uses artificially synthesized isoluminol derivatives as luminescent markers, which are directly marked on antigens or antibodies, and NH ₂ on the side chain of the benzene ring of isoluminol is activated by CSCl ₂ to form isothiocyanate derivatives , through which the -N=C=S bond is directly linked to the amine group on the antigen or antibody, separated and purified, and the unlinked isoluminol-N=C=S is removed. The chemiluminescent reaction of isoluminol is carried out on the metal In the presence of Mn ²⁺ , Fe ²⁺ , ClO ^- , etc., the alkaline environment caused by the excitation reagent NaOH, through the oxidation reaction of isoluminol derivatives and the excitation reagent H ₂ O ₂ , produces visible light with a wavelength of 400-600nm, Constitute a typical direct chemiluminescent reaction.

In the present invention, the mensuration of the surface group of nano-magnetic microbeads is to disperse the magnetic beads in a 10 ^-2 M Nacl solution, and use potentiometric titration to measure the -COOH or _-NH content on the surface of the magnetic beads. ×10 ^{- 3} M NaOH solution, the titration end point is obtained by the double parallel line method, and the calculated surface carboxyl group, amine group or hydroxyl group content per gram of magnetic beads is 0.05-0.5eqm/g.

The used analyzer of analytical test method among the present invention comprises power supply circuit, automatic syringe pump 1 and 2, measurement room, light-emitting room, photomultiplier tube counter and output system, also is equipped with the Windows control software of computer and Chinese interface simultaneously, can Perform functions such as data entry, result summary, quality control, result storage and result inquiry, etc., can complete the programming of various analysis modes, quantitative or qualitative report results, automatic generation, storage, and update functions, and two-point automatic correction of the standard curve.

Example 1

1. Preparation of monoclonal antibody labeled with isoluminol derivatives:

Take three test tubes of 3.2mmol ABEI, dissolve them in 0.2ml secondary water, 3.5mmol CSCl ₂ in 0.3ml DMF, mix the two evenly, react at room temperature for 2h, take 10mg anti-CEA-α, anti-AFP-α and 10mg anti -PSA-α monoclonal antibody, adjust the volume to 1ml with pH9.5 carbonate buffer, add the above-mentioned activated ABEI solution, mix well, react at room temperature for 20h, and purify through G-25 gel column;

2. FITC-labeled CEA, AFP, PSA monoclonal antibody

Take 10mg of anti-CEA-β, AFP-β and PSA-β monoclonal antibodies, slowly adjust the volume to 1ml with pH9.5 carbon, add FITC100ug, react at room temperature for 20 hours, and purify through G-25 gel column;

3. Preparation of goat anti-FITC polyclonal antibody coated on the surface of nano-magnetic microbeads:

The preparation of nano-magnetic micro-beads is prepared according to the Chinese patent, the patent number is CN92105584.6, the surface of the magnetic beads is a group containing -NH ₂ , and its content is 0.17eq/g; take 10ml of magnetic beads, the concentration is 30mg/ml , wash twice with PBS0.01M of P7.4, and finally suspend in 10ml of PBS of 0.01MPH7.4, add 10-100μl of 50% glutaraldehyde solution to make the final concentration of glutaraldehyde 0.01-0.5%, add purification Goat anti-FITC IgG antibody 100μg-1000μg, react at 37°C for 2 hours, remove the supernatant on the magnet, wash three times with 0.01MPH7.4 PBS containing 0.01-1% BSA, and finally suspend in the solution, add 0.01- 0.5% _NaN3

4. Preparation of magnetic separation direct chemiluminescence reagent and result analysis

Take CEA, AFP and PSA standard products, 20 μl each of serum samples, add 40 μl of luminescent marker monoclonal antibody ABEI and FITC monoclonal antibody each, mix well and incubate at 37°C for 15 minutes in a water bath; wait for the immune reaction After the occurrence, add 40 μl of immune nano-magnetic beads separation reagent coated with goat anti-FITC polyclonal antibody on the surface, mix well, and bathe in water for 5 minutes. Wash 400 μl, mix well, separate for 4 minutes, pour off the supernatant; repeat the above steps for one separation, put the test tube directly into the measurement chamber of the analyzer, and pump the excitation substrate NaOH and H ₂ O ₂ into the excitation substrate to generate direct luminescence Reaction, while using photomultiplier tubes to measure the number of photons, the analyzer analyzes and prints out the results.

Select 20 clinical specimens and analyze and test according to the above experimental process. In this example, there are 2 cases of primary early liver cancer, 2 cases of liver cancer resection patients, 3 cases of rectal cancer, 1 case of prostate, 1 case of prostatic hypertrophy, and normal subjects. 11 cases, the results are listed in Table 1.

Table 1

Patients 4 ^# and 6 ^# are primary early liver cancer patients, and the AFP value continues to increase for a long time. 11 ^# and 14 ^# are patients who have undergone liver cancer resection and are undergoing radiotherapy and chemotherapy. The AFP concentration in serum has decreased, but there is still no The AFP value of patients 2 ^# , 8 ^# and 6 ^# also exceeded the normal value, but the clinical diagnosis was rectal cancer, and the CEA value increased significantly, especially In 16 ^# , CEA was as high as 1270mIU/ml, which may indicate that there is a certain connection or crossover between different tumor markers. 1 ^# and 8 ^# patients were prostatic and simple prostatic hypertrophy, and their PSA measurement values were slightly increased.

From the data analysis in Table 1, it can be seen that the results obtained by the magnetoluminescence immunoassay test method described in the present invention have good clinical consistency.

Example 2

The preparation of monoclonal antibody of isoluminol derivatives and the preparation method of goat anti-FITC polyclonal antibody coated on the surface of nano-magnetic microbeads are the same as in Example 1. The luminescent marker used in this example is AHEI, and the surface of the magnetic beads is The group containing -COOH, its content is 0.3eq/g;

Take TSH, T3, T4, FT3 and FT4 standard products, 20 μl each of serum samples, add 40 μl each of AHEI and FITC monoclonal antibodies of the luminescent marker monoclonal antibody, mix well and incubate at 37°C, in a water bath 15 minutes; after the immune reaction occurs, add 40 μl of immune nano-magnetic beads separation reagent coated with goat anti-FITC polyclonal antibody on the surface, mix well, and bathe in water for 5 minutes. Under the action of an external magnetic field, separate on the upper separator for 4 minutes, pour Supernatant; add 400 μl of washing solution, mix well, separate for 4 minutes, pour off the supernatant; repeat the above steps for one separation, put the test tube directly into the measurement chamber of the analyzer, and pump the excitation substrates NaOH and H ₂ with the automatic pump O ₂ undergoes a direct luminescent reaction, while using a photomultiplier tube to measure the number of photons, and the analyzer analyzes and prints out the results.

Selected 261 clinical specimens, including 37 cases of clinically confirmed hypothyroidism, 50 cases of newly diagnosed hyperthyroidism, 26 cases of follow-up hyperthyroidism, 6 cases of cured hyperthyroidism, 13 cases of thyroid tumor, 5 cases of goiter, 120 cases of normal controls, aged 18-68 Aged, male 45 cases, all subjects were collected blood from veins, separated serum, stored at -20°C, and completed the test within one week. The test results are listed in Table 2:

Table 2

The measurement results of the five hormones listed in Table 2 showed that in 37 patients with hypothyroidism, TDH values were all significantly increased, and T3, T4, T3, T4, FT3, FT4 were all significantly decreased, which was in line with clinical diagnosis. For 50 newly diagnosed patients with hyperthyroidism, TSH decreased significantly, and T3, T4, FT3, FT4 increased significantly. Statistical analysis of the five hormone measurements of 26 patients with hyperthyroidism in treatment showed that all T4 values tended to recover or approached normal values, T3 The rate of decline is obviously slower than the T4 value, and the results obtained by the magnetoluminescence immunoassay test method described in the present invention have good clinical consistency.

The test items of the reagents described in the examples of the present invention are the determination of thyroid hormones and tumor markers as examples, but the present invention is not limited to testing these two items.

Claims (9)

1. Magnetic separation of direct chemiluminescent reagents, including luminescent markers, separation reagents, FITC-antibody conjugates, standard antigens or antibodies and excitation substrates, characterized in that: the excitation substrates are NaOH solution and H ₂ O ₂ solution, wherein the NaOH solution contains Mn ²⁺ , Fe ²⁺ and ClO ^- ions, the luminescent label is an isoluminol derivative, and the structural formula of the isoluminol derivative is: or

Where: R1 is C ₂ H ₅ , C ₃ H ₇ or C ₄ H ₉ , R2 is NH ₂ -(CH ₂ ) ₄ , NH ₂ -(CH ₂ ) ₆ , NH ₂ -(CH ₂ ) ₈ or NH ₂ -(CH ₂ ) ₁₀ . 2. magnetic separation direct chemiluminescent reagent according to claim 1, is characterized in that: described separation reagent is the nano-magnetic microbead of goat anti-FITC coating, and described nano-magnetic microbead is that the inside is coated with Fe ₃ O ₄ or Fe ₂ O ₃ , microbeads containing -OH, -COOH or -NH ₂ active groups on the outside. 3. The magnetic separation direct chemiluminescent reagent according to claim 2, characterized in that: the goat anti-FITC-coated nano-magnetic microbeads are labeled on monoclonal antibodies or antigens. 4. The magnetic separation direct chemiluminescent reagent according to claim 1, characterized in that: the isoluminol luminescent markers are ABEI and AHEI, in which R1 is C ₂ H ₅ , R2 is NH ₂ - (CH ₂ ) ₄ or NH ₂ -(CH ₂ ) ₆ . 5 . The magnetic separation direct chemiluminescence reagent according to claim 2 , characterized in that: the groups contained on the surface of the nano-immunomagnetic beads are -COOH or -NH ₂ . 6 . The magnetic separation direct chemiluminescent reagent according to claim 5 , characterized in that: the content of groups on the surface of the nano-immunomagnetic beads is 0.05-0.5 eqm/g. 7. A test method using the above-mentioned magnetic separation direct chemiluminescence reagent, which comprises the following steps: A. Mix and incubate the monoclonal antibody labeled with isoluminol derivatives, FITC and the serum to be tested respectively; B. After the immune reaction is completed, add immune nano-magnetic beads coated with sheep anti-FITC polyclonal antibody; C. Under the action of an external magnetic field, the antigen-antibody immune complex is specifically separated, and after being washed with a lotion, the test tube is placed in the measuring chamber of the analyzer; D. Pumping the excitation substrate with an automatic syringe pump, the excitation substrate is NaOH solution and H ₂ O ₂ solution, wherein the NaOH solution contains Mn ²⁺ , Fe ²⁺ and ClO ^- ions; E. Use a photomultiplier tube to count the number of photons emitted by the mixed product in the test tube, and the analyzer analyzes to obtain the concentration of the analyte. 8. The magnetic separation direct chemiluminescent assay method according to claim 7, characterized in that: the monoclonal antibody of the isoluminol luminescent marker is obtained by combining the isoluminol luminescent marker with carbon dichloride or N - After linking with hydroxysuccinic acid, it is obtained by linking with monoclonal antibody. 9. The magnetic separation direct chemiluminescence analysis test method according to claim 7, characterized in that: in the step C, the bottom of the test tube is based on nano magnetic beads as a carrier.

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