CN103293323B - A kind of interstitialcellstimulating hormone (ICSH) nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box and preparation method thereof - Google Patents

Abstract

The invention discloses a kind of interstitialcellstimulating hormone (ICSH) nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box, described kit comprises: interstitialcellstimulating hormone (ICSH) calibration object; Coupling has the nano magnetic microparticle suspending liquid of Streptavidin; Biotin labeled interstitialcellstimulating hormone (ICSH) antibody; Interstitialcellstimulating hormone (ICSH) abzyme bond, enzyme used is horseradish peroxidase, horseradish peroxidase purity RZ >=3.0, activity >=250U/mL; Interstitialcellstimulating hormone (ICSH) quality-control product; Chemical luminescence for liquid A liquid and B liquid; 20 times of concentrated washing lotions; Reaction tube.The invention also discloses the preparation method of kit of the present invention in addition.Kit of the present invention susceptibility compared with available reagent box is high, can measure that concentration range is wide, the reagent term of validity is long, simple to operate, detect automaticity advantages of higher.

Description

Luteinizing hormone nano magnetic particle chemiluminescent immunoquantitative detection kit and preparation method thereof

technical field

The invention relates to the field of immunoanalysis medicine. Specifically, the invention provides a luteinizing hormone (LH) nano magnetic particle chemiluminescent immunoquantitative detection kit and a preparation method thereof.

Background technique

Luteotropic hormone (LH) is secreted by basophils of the pituitary gland. In men, it can stimulate the Leydig cells of the testis to secrete male hormones, and in women, it can stimulate the ovaries to secrete female estrogen. Luteinizing hormone (LH) cooperates with follicle stimulating hormone (FSH) in women to maintain the menstrual cycle of the ovaries, leading to ovulation and corpus luteum formation. The production of LH is controlled by the hypothalamic gonadotropin-releasing hormone, and is also regulated by positive and negative feedback from the ovary. The combined detection of LH and FSH is mainly used to identify primary (ovarian) or secondary (pituitary) amenorrhea in women; it is used to identify primary or secondary hypofunction of testicles in men; it can also identify true amenorrhea in prepubertal children Or pseudoprecocious puberty.

The release peak of LH in the menstrual cycle is closely related to ovarian ovulation. Once the LH peak appears, it indicates ovarian ovulation in 24-36 hours. Therefore, the peak serum LH can be monitored during the menstrual cycle to determine the best time for conception. The test results are expressed in milliinternational units/milliliter (MIU/ML).

The current method for detecting luteinizing hormone (LH) is time-resolved immunofluorescence analysis (patent application number: 200810043552.5), using long-term fluorescent markers such as rare earth metals (Eu, Tb, Sm, Dy) to mark, and through bright-light resolution Non-specific fluorescence, but this method requires the use of rare earth metals, which is costly, difficult to label, and not easy to obtain.

In addition, the most commonly used methods for detecting LH are chemiluminescence immunoassay and magnetic particle chemiluminescence immunoassay. Compared with the previous radioimmunoassay and enzyme-linked immunoassay, they have advantages in detection sensitivity, detection range, detection time and automatic operation. The improvement is greatly improved, and there is no pollution, which is favored by clinical workers.

The existing technology generally adopts the fluorescein system (patent application number: 200910089583.9, 201210571305.9), the validity period of the kit of this system is short, unstable, and the measured value is still not accurate enough;

Another technology (patent application number: 201110257444.X) is to directly link antibodies on magnetic particles, which is difficult to operate and not easy to obtain.

Contents of the invention

The problem to be solved by the present invention is to provide a chemiluminescent immunoassay quantitative detection kit for luteinizing hormone and its preparation method, which avoids the shortcomings of radioimmunoassay reagents such as short validity period, radioactive pollution, and cumbersome operation, and solves the problem of low sensitivity, The measured value is not accurate enough, the detection range is narrow, the cost is high, and the shelf life is short.

In order to solve the above-mentioned technical problems, the technical scheme adopted in the present invention is: luteinizing hormone nano-magnetic particle chemiluminescent immunoquantitative detection kit, comprising: luteinizing hormone calibrator; Microparticle suspension; biotin-labeled luteinizing hormone antibody; luteinizing hormone antibody enzyme conjugate, the enzyme used is horseradish peroxidase, horseradish peroxidase purity RZ≥3.0, activity≥250U/mL ; Luteinizing hormone quality control product, the quality control product includes a low-value quality control product with a concentration of 5mIU/mL and a high-value quality control product with a concentration of 120mIU/mL; chemiluminescent liquid A liquid and B liquid, A liquid is 5mmol/L, pH8 .6 Tris-HCl damping fluid, and contain final concentration 0.7g/L luminol and final concentration 0.08g/L p-iodophenol in this damping fluid; B liquid is 0.675g/L carbamide peroxide; 20 times concentrated washing liquid; reaction tube.

Furthermore, the nano-magnetic particle is iron ferric oxide coated with amino or carboxyl active groups on the surface, and the particle diameter is 10-50nm.

Further, the material of the reaction tube is transparent polystyrene, polyethylene, polypropylene or glass.

The preparation method of kit comprises the following steps:

(1) Preparation of luteinizing hormone calibrator:

Prepare the luteinizing hormone antigen with goat serum to prepare the concentrated stock solution of the calibrator, calibrate with the national calibrator, and dilute the concentrated stock solution with goat serum to the working concentration, respectively 0, 2, 10, 25, 100, 250mIU /mL is the concentration of the calibrator;

(2) Preparation of luteinizing hormone quality control products:

Dilute the above concentrated stock solution to 5mIU/mL and 120mIU/mL with goat serum respectively, and calibrate with the national calibrator; 5mIU/mL is used as a low-value quality control product, and 120mIU/mL is used as a high-value quality control product;

(3) Preparation of magnetic nanoparticle-streptavidin suspension:

A. Preparation of ferroferric oxide nano-magnetic particles

Precipitation method was used to prepare ferroferric oxide nano-magnetic particles. The specific preparation method was as follows: 1) FeCl ₃ 6H ₂ O and FeCl ₂ 4H ₂ O were added to distilled water at a molar ratio of 2:1, and stirred vigorously to dissolve; 2) Add 0.5M ammonia water to the above iron salt solution under nitrogen atmosphere, adjust the pH to 9-10, the reaction temperature is 65°C, and the reaction time is 45min; 3) After the reaction, wash with distilled water until neutral, discard the supernatant, and dry at 60°C dry to obtain 10-50nm iron ferric oxide nano-magnetic particles;

B. Coupling of carboxyl groups on the surface of nanomagnetic beads

Coupling is carried out by the dispersion polymerization method, and the specific preparation method is as follows: Ultrasonic dispersion of the nano-magnetic particles prepared above in a 10% polyethylene glycol (PEG8000) solution to obtain a magnetic fluid solution is added to the magnetic fluid solution at a volume ratio of 1: 10 Add absolute ethanol, stir for 30 minutes, then transfer to a three-neck flask equipped with a stirrer, condenser, and nitrogen inlet, add the cross-linking agent N,N'-methylenebisacrylamide; under the protection of nitrogen, heat up to 60±1°C, stir at constant temperature for 30 minutes, then add benzoyl peroxide in turn, the dosage is 3% of the magnetic fluid, the stirring speed is about 500rpm, the volume of styrene is the same as that of the magnetic fluid solution, and the volume of acrylic acid is 1/ of that of the magnetic fluid solution 4. Keep the nitrogen flow, and keep the other conditions unchanged. React for 8-10 hours. The obtained product is left to stand, washed repeatedly with distilled water, then adjusted to pH = 1 with hydrochloric acid, soaked for 24 hours, and left standing; then washed repeatedly with distilled water to remove uncoated Coated Fe ₃ O ₄ magnetic powder, put the precipitated product in a vacuum drying oven at 50°C and dry for 24 hours to obtain nano-magnetic particles with carboxyl groups on the surface;

C, the preparation of magnetic nanoparticle-streptavidin suspension, prepare 1L, the method is as follows:

Take 100mL of 0.1M 2-morpholineethanesulfonic acid (MES) buffer solution, add 10mg of nano-magnetic particles with carboxyl groups on the surface, stir at room temperature for 40min, then add 3.5mg of streptavidin, and then add 8mg/mL carbodiimide (EDC) solution, react at 2-8°C for 1 hour, wash with 0.01M PBS buffer for 3 times, and finally dissolve to 1L with 0.01M PBS;

(4) Preparation of biotin-labeled luteinizing hormone antibody

Take 0.5 mg of luteinizing hormone antibody and dialyze with borate buffer at 2-8°C for 1-3 hours; add 25 μg of biotin to the dialyzed antibody and add dimethyl sulfoxide at the same time to make dimethyl sulfoxide The final mass concentration is 5-10%, shake slowly, and react in the dark for 3 hours; add 250uL of 1M ammonium chloride solution to the above solution, and react in the dark for 30-60min at room temperature; dialyze with 0.01M PBS solution at 2-8°C for 2 days , change the liquid 3-5 times during the period;

(5) Preparation of luteinizing hormone antibody-enzyme conjugate

After coupling the luteinizing hormone antibody with horseradish peroxidase by the modified sodium periodate oxidation method, dilute it to a working concentration of 1:3000-5000 with enzyme diluent, and add 15% enzyme stabilizer, Store at 2-8°C;

The steps of the improved sodium periodate oxidation method include:

A: HRP activation

1) Prepare 10mg/mL HRP solution;

2) Prepare 12.8mg/mL sodium periodate NaIO ₄ solution;

3) Mix the above 1) and 2) prepared solutions in a volume ratio of 1:1, and react at 4°C in the dark for 30 minutes;

4) Prepare an aqueous ethylene glycol solution with a concentration of 20uL/mL, mix it with the above solution 3) in the same volume, react at room temperature in the dark for 20 minutes, the activation is complete, and store at -20°C (the storage time does not exceed 3 months).

B. Luteinizing hormone monoclonal antibody labeling

1) Put the raw material to be marked into a dialysis bag, and dialyze it for 30 minutes with 0.05M carbonate buffer solution with pH9.6;

2) Mix the labeled material with the activated HRP at a mass ratio of 1:2, and then dialyze with 0.05M carbonate buffer at 4°C for 24 hours (change the medium 2-3 times during the period);

3) Prepare a NaBH ₄ aqueous solution with a concentration of 2mg/mL, mix according to the ratio of 1mgHRP plus 80uL of the prepared NaBH ₄ aqueous solution, and react at 4°C for 2 hours in the dark;

4) Dialyze the labeling solution completed in step 3) with 0.01M PBS at 4°C for 24 hours, add an equal volume of glycerol, and store at -20°C.

The enzyme diluent includes 10mL/L 2M NaOH, 15g/L NaCl, 10g/LBSA, 5g/L Dextran T-2000 (purchased from Sigma), 1.05g/L Triton X-100 (purchased from Sigma), 2.5mL /L Gentamicin Sulfate, 1mL/L Carmine (Carmine is a solid powder, prepared to a concentration of 40mg/mL before use), 2g/L Tween-20 (purchased from Sigma), 1mL/L ProClin300 (purchased from Sigma Corporation);

(6) Preparation of 20 times concentrated lotion

20 times concentrated lotion includes 58g/L disodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/LTween-20 and 2% Proclin300;

(7) Preparation of chemiluminescence solution A and B

A solution is 0.7g/L luminol, 0.08g/L p-iodophenol, buffer solution is 5mmol/L Tris-HCl of pH 8.6, and is stored away from light; B solution is 0.675g/L carbamide peroxide, and Prepare with water; liquid A and liquid B are mixed 5 minutes before use;

(8) Assembly: Assemble the above reagents into a box and store at 2-8°C;

(9) Carry out physical inspection on the kit prepared by this method, and measure the accuracy, linearity, precision, specificity, sensitivity, measured value and stability of the quality control product of the dose-response curve.

The principle of the present invention is to measure LH in serum or plasma by double-antibody sandwich method, add biotin-LH antibody conjugate to the avidin-nano magnetic particle suspension, and through the affinity reaction of avidin and biotin , Form magnetic particle-avidin-biotin-luteinizing hormone antibody complex, add sample and enzyme, through antigen-antibody reaction, form magnetic particle-avidin-biotin-luteinizing hormone antibody-luteinizing hormone Luminol-luteinizing hormone antibody-HRP complex, the complex is adsorbed on the bottom of the test tube with a magnetic field, the free components are washed away, and the substrate working solution is added. Under the action of the oxidant, HRP catalyzes luminol to generate an amino group in an excited state When the phthalate ion returns to the ground state, it releases a photon of 425nm, and the luminescence value RLU of each sample well is measured in the 5th minute. The RLU of the sample was positively correlated with the concentration of luteinizing hormone in the sample. The concentration of luteinizing hormone in the sample is quantified according to the Log(X)-Log(Y) mathematical model established by the concentration of luteinizing hormone in the calibrator and the corresponding RLU, so as to detect the content of luteinizing hormone in human serum and plasma .

The Luteinizing Hormone Nano Magnetic Particle Chemiluminescent Immunoquantitative Assay Kit invented by this patent has the following advantages:

(1) High sensitivity, the analytical sensitivity of this kit is not higher than 0.2mIU/mL.

(2) The specificity is good. This product has no cross-reaction to human chorionic gonadotropin (200000mIU/mL), thyroid-stimulating hormone (500μIU/mL), and follicle-stimulating hormone (500mIU/mL).

(3) The precision is good, the imprecision within a batch is not higher than 5%, and the imprecision between batches is not higher than 10%.

(4) Low cost. Compared with similar products on the market, this kit has good performance, low cost, and has clinical application value.

(5) Good stability. This product can be stored at 37°C for more than 7 days, and at 2-8°C for 1 year.

Description of drawings

Fig. 1 is a comparison chart of the assay results of Luteinizing Hormone measured by the kit of the present invention and Roche's assay of Luteinizing Hormone, where the ordinate is the Luteinizing Hormone value measured by the kit, and the abscissa is the Roche kit's determination of Luteinizing Hormone. Luteinizing hormone value, the correlation coefficient of the two methods (r) = 0.9601, the linear equation y = 1.1034x-0.669.

Detailed ways

Example 1: Preparation of Luteinizing Hormone Nano-Magnetic Particle Chemiluminescent Immunoquantitative Assay Kit

(1) Preparation of luteinizing hormone calibrator:

The luteinizing hormone antigen (manufactured by Fitzgerald Company) was prepared with goat serum (purchased from Zhengzhou Yikang Bioengineering Co., Ltd.) to make a calibrator concentrated stock solution, and the national calibrator (batch number: 150531-0211, specification: 530mIU/bottle) For calibration, dilute the concentrated stock solution with goat serum to the working concentration, respectively 0, 2, 10, 25, 100, 250mIU/mL, which is the concentration of the calibrator;

(2) Preparation of luteinizing hormone quality control products:

Dilute the above concentrated stock solution to 5mIU/mL and 120mIU/mL with goat serum respectively, and calibrate with the national calibrator; 5mIU/mL is used as a low-value quality control product, and 120mIU/mL is used as a high-value quality control product;

(3) Preparation of magnetic nanoparticle-streptavidin suspension:

A. Preparation of ferroferric oxide nano-magnetic particles

Precipitation method was used to prepare ferroferric oxide nano-magnetic particles. The specific preparation method was as follows: 1) FeCl ₃ 6H ₂ O and FeCl ₂ 4H ₂ O were added to distilled water at a molar ratio of 2:1, and stirred vigorously to dissolve; 2) Add 0.5M ammonia water to the above iron salt solution under nitrogen atmosphere, adjust the pH to 9-10, the reaction temperature is 65°C, and the reaction time is 45min; 3) After the reaction, wash with distilled water until neutral, discard the supernatant, and dry at 60°C dry to obtain 10-50nm iron ferric oxide nano-magnetic particles;

B. Coupling of carboxyl groups on the surface of nanomagnetic beads

The dispersion polymerization method is used for coupling, and the specific preparation method is as follows: take the nano-magnetic particles prepared above and ultrasonically disperse them in a 10% PEG8000 solution to obtain a magnetic fluid solution, and add absolute ethanol to the magnetic fluid solution at a volume ratio of 1:10, After stirring for 30 minutes, move it into a three-necked flask equipped with a stirrer, condenser, and nitrogen inlet, and add the crosslinking agent N,N'-methylenebisacrylamide; under the protection of nitrogen, raise the temperature to 60±1°C, Stir at constant temperature for 30 minutes, then add benzoyl peroxide sequentially, the dosage is 3% of the magnetic fluid dosage, the stirring speed is about 500rpm, the volume of styrene is the same as that of the magnetic fluid solution, the volume of acrylic acid is 1/4 of the magnetic fluid solution, and the nitrogen gas flow is maintained. The rest of the conditions remain unchanged, react for 8-10h, the product obtained stands still, wash repeatedly with distilled water, then adjust the pH=1 with hydrochloric acid, soak for 24h, let stand; then wash repeatedly with distilled water to remove uncoated Fe ₃ O ₄ Magnetic powder, put the precipitated product into a vacuum drying oven and dry at 50°C for 24 hours to obtain nano-magnetic particles with carboxyl groups on the surface;

C, the preparation of magnetic nanoparticle-streptavidin suspension, prepare 1L, the method is as follows:

Take 100mL of 0.1M MES buffer solution, add 10mg of nano-magnetic particles with carboxyl groups on the surface, stir at room temperature for 40min, then add 3.5mg of streptavidin, then add 8mg/mLEDC solution, react at 2-8°C for 1h, then use 0.01 Wash 3 times with M PBS buffer, and finally dissolve to 1L with 0.01M PBS;

(4) Preparation of biotin-labeled luteinizing hormone antibody

Take 0.5 mg of luteinizing hormone antibody and dialyze with borate buffer at 2-8°C for 1-3 hours; add 25 μg of biotin to the dialyzed antibody and add dimethyl sulfoxide at the same time to make dimethyl sulfoxide The final mass concentration is 5-10%, shake slowly, and react in the dark for 3 hours; add 250uL of 1M ammonium chloride solution to the above solution, and react in the dark for 30-60min at room temperature; dialyze with 0.01M PBS solution at 2-8°C for 2 days , change the liquid 3-5 times during the period;

(5) Preparation of luteinizing hormone antibody-enzyme conjugate

After coupling the luteinizing hormone antibody with horseradish peroxidase by the modified sodium periodate oxidation method, use the enzyme diluent (the composition includes 5g/LMES, 10mL/L2M NaOH, 15g/L NaCl, 10g/ LBSA, 5g/L Dextran T-2000 (Dextran T-2000) (purchased from Sigma), 1.05g/L polyethylene glycol octylphenyl ether (Triton X-100) (purchased from Sigma), 2.5mL/L Gentamicin Sulfate, 1mL/L Carmine (Carmine is a solid powder, prepared to a concentration of 40mg/mL before use), 2g/L Tween-20 (purchased from Sigma), 1mL/L ProClin300 ( (purchased from Sigma Company)) was diluted to a working concentration of 1:3000-5000, and 15% enzyme stabilizer was added, and stored at 2-8°C;

(6) Preparation of 20 times concentrated lotion

20 times concentrated lotion includes 58g/L disodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/L Tween-20 and 2% Proclin300;

(7) Preparation of chemiluminescence solution A and B

A solution is 0.7g/L luminol, 0.08g/L p-iodophenol, buffer solution is 5mmol/L Tris-HCl of pH 8.6, and is stored away from light; B solution is 0.675g/L carbamide peroxide, and Prepare with water; liquid A and liquid B are mixed 5 minutes before use;

(8) Assembly: Assemble the above reagents into a box and store at 2-8°C;

(9) Carry out physical inspection on the kit prepared by this method, and measure the accuracy, linearity, precision, specificity, sensitivity, measured value and stability of the quality control product of the dose-response curve.

Embodiment 2: the inspection of kit of the present invention

(1) Physical inspection: liquid components should be clear without sediment or floc; other components should have no package damage.

(2) Accuracy: The kit calibration product and the national standard product series are analyzed and determined at the same time, and the double-logarithmic mathematical model is used for fitting, requiring that the two dose-response curves do not deviate significantly from parallel (t test, |t|<2.447) ; With the national standard substance of luteinizing hormone as the reference substance, the average value of the ratio between the measured value and the labeled value of the kit calibrator should be within the range of 0.90 to 1.10 by using a double-logarithmic mathematical model.

(3) Linearity of the dose-response curve: Fitting with a double-reading mathematical model, the absolute value of the correlation coefficient r of the dose-response curve within the concentration range of 0.2-250mIU/mL is not less than 0.9900.

(4) Analytical sensitivity: The analytical sensitivity of the kit is not higher than 0.2mIU/mL.

(5) Precision: 10 wells were measured in parallel for high-value and low-value quality control products, and the average concentration of the measurement results was calculated ( ) and standard deviation (SD), within-assay imprecision Use 3 batches of products to carry out 3 tests, calculate the average concentration of assay result ( ) and standard deviation (SD), inter-assay imprecision The results should meet the within-assay imprecision (CV%) should not be higher than 5%; inter-assay imprecision (CV%) should not be higher than 10%.

(6) Measured value of quality control products: parallel determination of high and low value quality control products in 10 wells, fitting with Log(X)-Log(Y) mathematical model, the measured value of quality control products should be within the allowable range, The measured value of the low-value quality control product is 4-6mIU/mL, and the measured value of the high-value quality control product is 96-144mIU/mL.

(7) Specificity:

Cross-reactivity meets the requirements of the table below:

(8.) Stability: Store at 37°C for 7 days, and the measured value should meet the above requirements.

Embodiment 3: the using method of kit of the present invention

(1) Equilibrate the test kit at room temperature (18-25°C) for 30 minutes.

(2) Preparation of lotion: dilute the concentrated lotion with distilled water at 1:20 (1mL of lotion plus 19mL of distilled water). If the concentrated lotion has crystals, the concentrated lotion can be placed at room temperature or 37°C, and then diluted after the crystals dissolve.

(3) Preparation of luminous liquid: Take an appropriate amount of luminous liquid A and luminous liquid B and mix them in equal volumes 5 minutes before use.

(4) Number the reaction tubes, add 25-50uL calibrator or serum samples, 50uL magnetic particle-streptavidin suspension, 50uL biotin-luteinizing hormone antibody conjugate, 50-100uL Luteinizing hormone antibody enzyme conjugate, shake reaction at 37°C for 30 minutes, place the test tube rack on a magnetic separator for separation for 5 minutes, then pour out the supernatant, add 500uL washing solution, mix well, and separate on a magnetic separator , pour out the washing solution, repeat 3 times, add 100uL of chemiluminescent substrate solution to each tube, mix well, place in the dark for 5min, measure the luminescence value (RLU) of each tube on a tube chemiluminescence instrument, and use the calibrator The Log value of the concentration is the abscissa, and the Log of the luminescence value is the ordinate, and a standard curve is drawn, and the concentration of luteinizing hormone can be calculated according to the luminescence value of the serum sample.

Embodiment 4: the methodological evaluation result of this kit

Detection range: the range is 0.2 ~ 250mIU/mL, for samples with a concentration greater than 250mIU/mL, it should be diluted before measurement.

Sensitivity: 0.2mIU/mL.

Precision: less than 5%.

Accuracy: The average recovery rate is in the range of 0.90 to 1.10.

Specificity: The cross-reaction coefficient with thyroid-stimulating hormone (TSH), human chorionic gonadotropin (hCG), and follicle-stimulating hormone (FSH) is less than 1%.

Measured values of quality control products: the measured values of low-value quality control products (QcL) and high-value quality control products (QcH) are within the allowable range, the measured values of low-value quality control products are 4-6mIU/mL, and the measured values of high-value quality control products are within the allowable range. The measured value is 96-144mIU/mL.

Stability: The reagent components in the kit were placed at 37°C for 7 days, and the stability was good.

Embodiment 5: the clinical comparative experiment of this kit

The kit of this patent invention has been clinically evaluated. The total number of samples in this clinical trial is 120 cases. First, after testing with the Luteinizing Hormone Roche Detection Kit, the kit (chemiluminescence) of this patent invention is used for measurement. The results show that the linear equation is y=1.1034x-0.669 and the correlation coefficient R=0.9601. It can be seen that the kit prepared by this method has a good consistency with the measured value of the hospital. Use SPSS13.0 statistical analysis software to carry out t-test on the correlation coefficient (test level α=0.05), P<0.001, the degree of correlation between the luteinizing hormone values measured by the two methods is significant, it can be seen that the two methods measured The value of luteinizing hormone is closely related, indicating that the diagnostic ability of the kit is strong, and it can be popularized for clinical application.

In order to determine the clinical reference value of this kit, 1033 normal human serum and plasma samples were tested with this kit, and the results showed that the reference value (reference range) of this kit was male: 1-11mIU/mL; female: Follicular phase: 1-17mIU/mL, ovulation phase: 24-98mIU/mL, luteal phase: 0.5-19mIU/mL, menopause: 14-54mIU/mL.

Claims (1)

1. an interstitialcellstimulating hormone (ICSH) nano magnetic particulate chemistry electrochemiluminescent immunoassay immue quantitative detection reagent box, described kit comprises:

1) interstitialcellstimulating hormone (ICSH) calibration object, concentration is 0,2,10,25,100,250mIU/mL;

2) coupling has the nano magnetic microparticle suspending liquid of Streptavidin, and nano magnetic particulate is the tri-iron tetroxide that surface wraps up with amino or carboxyl-reactive group, particle diameter 10-50nm;

3) biotin labeled interstitialcellstimulating hormone (ICSH) antibody;

4) interstitialcellstimulating hormone (ICSH) abzyme bond, enzyme used is horseradish peroxidase, horseradish peroxidase purity RZ >=3.0, activity >=250U/mL;

5) interstitialcellstimulating hormone (ICSH) quality-control product; Quality-control product comprises the low value quality-control product of concentration 5mIU/mL and the high level quality-control product of 120mIU/mL;

6) chemical luminescence for liquid A liquid and B liquid; A liquid is the Tris-HCl damping fluid of 5mmol/L, pH8.6, and containing final concentration 0.7g/L luminol and final concentration 0.08g/L p-iodophenol in this damping fluid; B liquid is 0.675g/L urea peroxide;

7) 20 times of concentrated washing lotions;

8) reaction tube, the material of described reaction tube is transparent polystyrene, transparent polyethylene, transparent polypropylene or clear glass; It is characterized in that,

(1) preparation of interstitialcellstimulating hormone (ICSH) calibration object:

Interstitialcellstimulating hormone (ICSH) antigen lowlenthal serum is mixed with the dense liquid storage of calibration object, calibrates with national calibration object, dense liquid storage lowlenthal serum is diluted to working concentration, is respectively 0,2,10,25,100,250mIU/mL;

(2) preparation of interstitialcellstimulating hormone (ICSH) quality-control product:

With lowlenthal serum, above-mentioned dense liquid storage is diluted to 5mIU/mL and 120mIU/mL respectively, and calibrates with national calibration object; Using 5mIU/mL as low value quality-control product, 120mIU/mL is as high level quality-control product;

(3) preparation of nano magnetic particulate-Streptavidin suspending liquid:

Prepared by A, ferriferrous oxide nano magnetic particle

Adopt the precipitation method to prepare ferriferrous oxide nano magnetic particle, concrete preparation method is as follows: 1) by FeCl ₃6H ₂o and FeCl ₂4H ₂o joins in distilled water with mol ratio 2:1, dissolved with vigorous agitation; 2) add 0.5M ammoniacal liquor in a nitrogen environment in above-mentioned iron salt solutions, adjust pH9-10, temperature of reaction 65 DEG C, reaction time 45min; 3), after reaction terminates, be washed with distilled water to neutrality, abandon supernatant, in 60 DEG C of oven dry, obtain the ferriferrous oxide nano magnetic particle of 10-50nm;

The coupling of B, nanometer magnetic bead surface carboxyl groups

Dispersion copolymerization method is adopted to carry out coupling, concrete preparation method is as follows: the nano magnetic microparticulate ultrasound getting above-mentioned preparation is dispersed in 10%PEG8000 solution, obtain magnetic fluid solution, in magnetic fluid solution, 1:10 adds absolute ethyl alcohol by volume, after stirring 30min, moves into stirrer, condenser pipe, in the three-necked bottle of nitrogen inlet, add crosslinking chemical N, N '-methylene-bisacrylamide; Under the protection of nitrogen, be warming up to 60 ± 1 DEG C, constant temperature stirs 30min, add benzoyl peroxide successively afterwards, consumption is magnetic fluid consumption 3%, and stirring rate is about 500rpm, volume of styrene is with magnetic fluid solution, and acrylic acid volume is 1/4 of magnetic fluid solution, keeps stream of nitrogen gas, all the other conditions remain unchanged, reaction 8-10h, and products therefrom leaves standstill, use distilled water cyclic washing, regulate pH=1 with hydrochloric acid again, soak 24h, leave standstill; Use distilled water cyclic washing again, remove not coated Fe ₃o ₄magnetic, puts into dry 24h at vacuum drying chamber 50 DEG C the product precipitated, obtains the nano magnetic particulate that surface is associated with carboxyl;

The preparation of C, nano magnetic particulate-Streptavidin suspending liquid, preparation 1L, method is as follows:

Get 100mL0.1M MES damping fluid, add the nano magnetic particulate that 10mg surface is associated with carboxyl, stirring at room temperature 40min, add 3.5mg Streptavidin afterwards, then 8mg/mLEDC solution is added, after 2-8 DEG C of reaction 1h, with 0.01M PBS buffer solution 3 times, be finally settled to 1L with 0.01M PBS;

(4) preparation of biotin labeled interstitialcellstimulating hormone (ICSH) antibody

Get 0.5mg interstitialcellstimulating hormone (ICSH) antibody, to dialyse at 2 ~ 8 DEG C 1 ~ 3h with borate buffer solution; Antibody after dialysis is added 25 μ g biotins, adds dimethyl sulfoxide (DMSO) simultaneously, make dimethyl sulfoxide (DMSO) final mass concentration be 5-10%, slowly vibrate, lucifuge reaction 3h; 250 μ L1M ammonium chloride solutions are added, reacting at normal temperature without light 30-60min in above-mentioned solution; Dialyse 2 days at 2 ~ 8 DEG C with 0.01MPBS solution, period changes liquid 3-5 time;

(5) preparation of interstitialcellstimulating hormone (ICSH) abzyme bond

After adopting improvement sodium periodate oxidation that interstitialcellstimulating hormone (ICSH) antibody and horseradish peroxidase are carried out coupling, be diluted to working concentration 1:3000-5000 with enzyme dilution, and added 15% enzyme stabilizers, be stored in 2 ~ 8 DEG C; Enzyme dilution comprises 10mL/L2M NaOH, 15g/L NaCl, 10g/LBSA, 5g/L Dextran T-2000,1.05g/L Triton X-100,2.5mL/L gentamicin sulphate, 1mL/L is carmine, and famille rose is powder solid, is mixed with concentration 40mg/mL and uses later, 2g/L Tween-20,1mL/L ProClin300; 5g/LMES;

The preparation of (6) 20 times of concentrated washing lotions

20 times of concentrated washing lotions comprise 58g/L sodium hydrogen phosphate, 5.92g/L sodium dihydrogen phosphate, 180g/L NaCl, 10mL/L Tween-20 and 2%Proclin300;

(7) preparation of chemical luminescence for liquid A liquid and B liquid

A liquid is 0.7g/L luminol, 0.08g/L p-iodophenol, and damping fluid is the 5mmol/L Tris-HCl of pH8.6, keeps in Dark Place; B liquid is 0.675g/L urea peroxide, prepares with process water; The 5min mixing before use of A liquid and B liquid;

(8) assemble: mentioned reagent is assembled into box, is stored in 2 ~ 8 DEG C;

(9) to adopting the kit prepared of the method to carry out physical examination, the measured value of linear, precision, the specificity of accuracy, dose-response curve, sensitivity, quality-control product and stability are measured.