CN117783513A - CEACAM6 latex turbidimetry detection kit adopting integrated method of transmission and scattering - Google Patents
CEACAM6 latex turbidimetry detection kit adopting integrated method of transmission and scattering Download PDFInfo
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Abstract
The invention discloses a CEACAM6 latex turbidimetry detection kit by a transmission and scattering integrated method, and belongs to the technical field of biological detection. The invention discloses a CEACAM6 latex turbidimetry detection kit by a transmission and scattering integrated method, which comprises a first reagent R1 and a second reagent R2; r1 comprises Hepes-NaOH buffer solution, naCl, polyethylene glycol 12000, tween-20 and a mouse monoclonal antibody blocker; r2 comprises buffer solution, tween-20, 300nm carboxyl latex, 450nm carboxyl latex, protective agent and CEACAM6 monoclonal antibody. The detection speed of the CEACAM6 latex turbidimetry detection kit adopting the integrated method of transmission and scattering is equal to that of a traditional biochemical analyzer, and compared with a chemiluminescence method, the detection speed is high and the cost is low; compared with fluorescence and colloidal gold chromatography and microfluidic methods, the detection repeatability is better.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a CEACAM6 latex turbidimetry detection kit by a transmission and scattering integrated method.
Background
A large number of researches show that carcinoembryonic antigen related cell adhesion molecule 6 (Carcinoembryonic antigen-related cell adhesion molecule, CEACAM6) plays a plurality of roles in the occurrence and development of tumors, including the aspects of promoting proliferation, migration and invasion of tumor cells, inhibiting apoptosis of tumor cells, promoting angiogenesis, inducing drug resistance and the like. CEACAM6 is the most characteristic biological marker of many invasive tumors throughout the carcinoembryonic antigen gene family (fig. 1, 2).
The university of double denier affiliated Huashan hospital in 2020 first found that five LUAD-LM patients had CSF-CTC with high expression of CEACAM6 based on single cell RNA sequencing study; later, the latest research of the central laboratory of Huashan hospital affiliated to the university of Kanji medical science affiliated to the first hospital of the university of Kanji medical science in combination with the complex denier university shows that the level of CEACAM6 in the cerebrospinal fluid of LUAD-LM (lung adenocarcinoma pia mater metastasis) is higher than that of normal people. In serum, the levels of CAECAM6 were higher in LUAD patients than in healthy control groups, with the levels of CAECAM6 being highest in LM patients. Serum CEACAM6 is higher than CEA (0.95vs 0.64,p<0.001) in distinguishing LUAD patients from LM and non-LM. The area under the curve (AUC) of serum CEACAM6 diagnostic of LUAD-LM was higher than CEA, CYFRA 21-1 and NSE, showing that CEACAM6 can be a potential biomarker for LUAD-LM.
The integrated detection method of the transparent scattering is a new methodology developed in 2020 and updated on the basis of the original latex immunoturbidimetry, and the integrated instrument of the transparent scattering (such as Hitachi3500 and the like) developed in 2013 by Hitachi high-new company in Japan is marketed in 2015 and the registration certificate of NMPA medical equipment is obtained in 2019 and 2 months. The transmission and scattering integrated machine is added with a scattered light detection module based on transmission detection of a traditional biochemical analyzer, as shown in fig. 3, namely the same detection is carried out, the detection of transmitted light and scattered light is completed within 0.7s, signal values are calculated respectively, and then the results of the two are fused through software. Common knowledge in the IVD field is that the scattering detection sensitivity is high, but the detection range is very narrow, and high values cannot be distinguished; the transmission detection sensitivity is low, the low-value detection repeatability is poor, but the detection range is wide, and high values can be effectively distinguished; the integrated method of transmission and scattering is used for obtaining the detection with high sensitivity and high linearity range by fusing results, using scattering results in low concentration area and transmission results in high concentration area, inheriting the advantages of transmission and scattering immunity turbidimetry, and avoiding the disadvantages. The integrated method of the transmission and scattering can improve the sensitivity by one order of magnitude only through the hardware capability and the software algorithm of the integrated machine of the transmission and scattering.
Therefore, the provision of a CEACAM6 latex turbidimetry detection kit by a transmission and scattering integrated method is a problem to be solved urgently by those skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a CEACAM6 latex turbidimetry detection kit by a transparent scattering integrated method, which is used for detecting CEACAM6 in body fluids such as serum, cerebrospinal fluid and the like.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a CEACAM6 latex turbidimetry detection kit adopting a transmission and scattering integrated method comprises a first reagent R1 and a second reagent R2;
the first reagent R1 comprises the following components in concentration: 25-200mM Hepes-NaOH buffer pH 6.8-7.5, naCl 200-1000mM, polyethylene glycol 120002g/L, tween-20.05% -2%, murine monoclonal antibody blocker 20-40ml/L;
the second reagent R2 comprises the following components in concentration: 50-200mM buffer solution pH 7.0-7.6, tween-20-16 ml/L,300nm carboxyl latex 1.6-2.4g/L,450nm carboxyl latex 1.0-1.8g/L, protective agent 10-90g/L, CEACAM6 monoclonal antibody 20-40mg/L.
Further, the first reagent R1 and the second reagent R2 also include a preservative, which is 1g/L sodium azide or 0.5ml/L PC300.
Further, in the second reagent R2, the buffer solution is Hepes-NaOH buffer solution, phosphate buffer solution or glycine-Tris buffer solution.
Further, in the second reagent R2, the protective agent is at least one of 10-30g/L sucrose, 10-30g/L trehalose and 10-30g/L fructose.
Further, the CEACAM6 latex turbidimetry detection kit adopting the integrated method of transmission and scattering also comprises a calibrator and a quality control product;
the calibrator comprises the following components in concentration: 50mM Hepes-NaOH buffer pH7.4, CEACAM6 antigen 100-2000 pg/ml (100 pg/ml, 300pg/ml, 800pg/ml, 1500pg/ml, 2000 pg/ml), sodium chloride 900mM, tween-20 10ml/L, preservative;
the quality control product comprises the following components in concentration: 50mM Hepes-NaOH buffer pH7.4, CEACAM6 antigen 200-500 pg/ml (200 pg/ml, 500 pg/ml), sodium chloride 900mM, tween-20 10ml/L, preservative;
the preservative is 1g/L sodium azide or 0.5ml/L PC300.
Furthermore, the CEACAM6 latex turbidimetry detection kit adopting the integrated method of transparent scattering is applied to detection of CEACAM 6.
Compared with the prior art, the invention discloses and provides a CEACAM6 latex turbidimetry detection kit by a transparent scattering integrated method, which has the following beneficial effects:
(1) Current diagnosis of LM depends on clinical manifestations, cytological examination of cerebrospinal fluid, and imaging examination. Diagnostic gold standard is positive for cerebrospinal cytology. However, if the detection is carried out by an artificial microscope, the efficiency is lower, the precision is not high, and the detection has a great relationship with the personnel capacity level; moreover, cancer cells in cerebrospinal fluid are scarce, which is a major limitation of diagnosis. One retrospective study included 334 patients with advanced lung cancer, of which 22% were diagnosed as LM by cerebrospinal fluid cytological examination only, 35% were diagnosed by Magnetic Resonance Imaging (MRI) only, and 42% were used by a combination of both. Thus, there is an urgent need for novel biomarkers and detection methods for LM diagnosis.
After the reagent is generated according to the formula, the reagent is tested on a Hitachi3500 full-automatic biochemical analyzer by using a transparent scattering integrated method, and the detection range can reach 50-2000pg/ml; the coefficient of variation CV in the batch was 8.0% (n=10).
(2) As shown in fig. 4, the detection level of the tumor marker is usually in the nanogram or picogram level, and CEACAM6 is used as the analyte with the concentration of picogram level, so the high low-end sensitivity requirement is almost unsatisfied on the common biochemical detection except the chemiluminescence method, but the chemiluminescence method is time-consuming, and the consumable is expensive and the cost is extremely high.
The invention adopts a transmission and scattering integrated method, and can ensure the detection with high sensitivity at the low end, wide linear range and wide safety area range simultaneously by fusing the results, using the scattering result in the low concentration area and using the transmission result in the high concentration area. Compared with a chemiluminescence method, the hardware detection speed of the integrated transmission and scattering method is high and the cost is low, wherein the hardware detection speed of the integrated transmission and scattering method is equal to that of a traditional biochemical analyzer; compared with fluorescence and colloidal gold chromatography and a microfluidic method, the detection repeatability is better; in addition, the machine can be matched with a test assembly line for use, and is beneficial to laboratory automation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing the structural and affinity interactions of CEACAMs;
FIG. 2 is a drawing of the biological markers that CEACAM6 is the most characteristic of many invasive tumors;
FIG. 3 is a diagram of a perspective scattering integrated machine;
FIG. 4 is a graph showing the detection levels of tumor markers under different detection methods;
FIG. 5 is a graph showing a 20 degree scattering calibration curve for example 1 of the present invention;
FIG. 6 is a graph showing a transmission channel calibration curve according to example 1 of the present invention;
FIG. 7 is a graph showing a linear fit of example 1 of the present invention;
FIG. 8 is a graph of a linear fit of comparative example 1 of the present invention;
FIG. 9 is a graph of a linear fit of comparative example 2 of the present invention;
FIG. 10 is a graph showing a linear fit of example 2 of the present invention;
fig. 11 is a graph showing a linear fit of example 3 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A CEACAM6 latex turbidimetry detection kit adopting a transmission and scattering integrated method comprises a first reagent R1 and a second reagent R2;
the first reagent R1 is formulated as follows:
Hepes-NaOH buffer 50mM, pH7.0
Sodium chloride 500mM
Polyethylene glycol-120002 g/L
Tween-20 ml/L
40ml/L of murine monoclonal antibody blocker (Phpeng organism E-010)
Preservative 0.5ml/L PC300
The preparation method of the first reagent R1 comprises the following steps:
taking 800ml of purified water, weighing the materials in the formula according to the volume of 1L, sequentially adding the materials into pure water, fully dissolving the materials, fixing the volume to 1L, and filtering the materials with a 450nm filter membrane for later use.
The formula of the second reagent R2 is as follows:
Hepes-NaOH buffer 50mM, pH7.4
Tween-20 16ml/L
300nm carboxyl latex 2.4g/L (Suzhou Chemicals, inc.)
450nm carboxyl latex 1.8g/L (Suzhou Du Biotechnology Co., ltd.)
Protective agent (sucrose 30g/L, trehalose 10g/L, fructose 10 g/L)
CEACAM6 mouse monoclonal antibody 40mg/L (Yiqiaoshenzhou biotechnology Co., ltd.)
Preservative 0.5ml/L PC300
The preparation method of the second reagent R2 comprises the following steps:
(1) And (3) cleaning latex: the latex microspheres were centrifuged 2 times with 100mM MES-NaOH buffer, pH 5.5; taking 10ml of 10% mass concentration latex microspheres of 300nm and 450nm respectively, namely 1g of each of the two latex microspheres, centrifuging at 22000rpm for 15min, removing supernatant, redissolving by using 10ml of MES-NaOH buffer solution, centrifuging again, removing supernatant redissolving, and completing cleaning for later use;
(2) Latex activation: taking 10ml of the washed latex as an example, weighing 50mg of NHS (N-hydroxysuccinimide), 30mg of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), dissolving with 5ml of the MES-NaOH buffer for washing, adding 1ml of each to 10ml of the washed two microspheres, and magnetically stirring and uniformly mixing at room temperature for 10min;
(3) Cleaning after activation: centrifuging the two latices after the activation in the step (2) at 22000rpm for 15min, removing supernatant, and adding 20ml of 50mM Hepes-NaOH buffer solution with pH of 7.4 for re-dissolution for later use;
(4) Antibody coupling: two CEACAM6 murine monoclonal antibody portions were added to the above activated two latex solutions reconstituted with Hepes-NaOH buffer, with 1.2ml of antibody (2.4 g latex VS 40mg antibody) added to 300nm group; 1.2ml of antibody (1.8 g of latex VS 40mg of antibody) was added to the 450nm group and magnetically stirred at room temperature for 3 hours to complete the antibody coupling.
(5) Sealing and cleaning: after centrifugation at 22000rpm for 15min, the whole supernatant was removed, and the 300nm and 450nm groups were reconstituted with blocking washes (50 mM pH7.4 Hepes-NaOH buffer, tween-2016 ml/L, sucrose 30g/L, trehalose 10g/L, fructose 10g/L, 0.5ml/L PC 300) 42ml (latex final concentration about 4.8 g/L), 54ml (latex final concentration about 3.6 g/L), respectively, the two latices were reconstituted at volume 1:1 (the final concentration is 300nm carboxyl latex 2.4g/L,450nm carboxyl latex 1.8 g/L) is prepared as the finished product R2.
Comparative example 1
A CEACAM6 latex turbidimetry detection kit adopting a transmission and scattering integrated method comprises a first reagent R1 and a second reagent R2;
the first reagent R1 is formulated as follows:
Hepes-NaOH buffer 50mM, pH7.0
Sodium chloride 500mM
Polyethylene glycol-120002 g/L
Tween-20 ml/L
40ml/L of murine monoclonal antibody blocker (Phpeng organism E-010)
Preservative 0.5ml/L PC300
The preparation method of the first reagent R1 comprises the following steps:
taking 800ml of purified water, weighing the materials in the formula according to the volume of 1L, sequentially adding the materials into pure water, fully dissolving the materials, fixing the volume to 1L, and filtering the materials with a 450nm filter membrane for later use.
The formula of the second reagent R2 is as follows:
Hepes-NaOH buffer 50mM, pH7.4
Tween-20 16ml/L
300nm carboxyl latex 2.4g/L (Suzhou Chemicals, inc.)
450nm carboxyl latex 1.8g/L (Suzhou Du Biotechnology Co., ltd.)
Protective agent (sucrose 30g/L, trehalose 10g/L, fructose 10 g/L)
CEACAM6 mouse monoclonal antibody 15mg/L (Yiqiaoshenzhou biotechnology Co., ltd.)
Preservative 0.5ml/L PC300
The preparation method of the second reagent R2 comprises the following steps:
(1) And (3) cleaning latex: the latex microspheres were centrifuged 2 times with 100mM MES-NaOH buffer, pH 5.5; centrifuging 10ml of 10% mass concentration latex microspheres at 300nm and 450nm at 22000rpm for 15min, removing supernatant, redissolving by using 10ml of MES-NaOH buffer solution, centrifuging, removing supernatant redissolving, and cleaning for later use;
(2) Latex activation: taking 10ml of the washed latex as an example, weighing 50mg of NHS (N-hydroxysuccinimide), 30mg of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), dissolving with 5ml of the MES-NaOH buffer for washing, adding 1ml of each to 10ml of the washed two microspheres, and magnetically stirring and uniformly mixing at room temperature for 10min;
(3) Cleaning after activation: centrifuging the two latices after the activation in the step (2) at 22000rpm for 15min, removing supernatant, and adding 20ml of 50mM Hepes-NaOH buffer solution with pH of 7.4 for re-dissolution for later use;
(4) Antibody coupling: two CEACAM6 murine monoclonal antibody portions were added to the above activated two latex solutions reconstituted with Hepes-NaOH buffer, with the 300nm group added with 0.45ml antibody (2.4 g latex VS15mg antibody); 0.45ml of antibody (1.8 g of latex VS15mg of antibody) was added to the 450nm group and magnetically stirred at room temperature for 3 hours to complete the antibody coupling.
(5) Sealing and cleaning: after centrifugation at 22000rpm for 15min, the whole supernatant was removed, and the 300nm and 450nm groups were reconstituted with blocking washes (50 mM pH7.4 Hepes-NaOH buffer, tween-2016 ml/L, sucrose 30g/L, trehalose 10g/L, fructose 10g/L, 0.5ml/L PC 300) 42ml (latex final concentration about 4.8 g/L), 54ml (latex final concentration about 3.6 g/L), respectively, the two latices were reconstituted at volume 1:1 (the final concentration is 300nm carboxyl latex 2.4g/L,450nm carboxyl latex 1.8 g/L) is prepared as the finished product R2.
Comparative example 2
A CEACAM6 latex turbidimetry detection kit adopting a transmission and scattering integrated method comprises a first reagent R1 and a second reagent R2;
the first reagent R1 is formulated as follows:
Hepes-NaOH buffer 50mM, pH7.0
Sodium chloride 500mM
Polyethylene glycol-120002 g/L
Tween-20 ml/L
10ml/L of murine monoclonal antibody blocker (Phpeng organism E-010)
Preservative 0.5ml/L PC300
The preparation method of the first reagent R1 comprises the following steps:
taking 800ml of purified water, weighing the materials in the formula according to the volume of 1L, sequentially adding the materials into pure water, fully dissolving the materials, fixing the volume to 1L, and filtering the materials with a 450nm filter membrane for later use.
The formula of the second reagent R2 is as follows:
Hepes-NaOH buffer 50mM, pH7.4
Tween-20 16ml/L
300nm carboxyl latex 2.4g/L (Suzhou Chemicals, inc.)
450nm carboxyl latex 1.8g/L (Suzhou Du Biotechnology Co., ltd.)
Protective agent (sucrose 30g/L, trehalose 10g/L, fructose 10 g/L)
CEACAM6 mouse monoclonal antibody 50mg/L (Yiqiaoshenzhou biotechnology Co., ltd.)
Preservative 0.5ml/L PC300
The preparation method of the second reagent R2 comprises the following steps:
(1) And (3) cleaning latex: the latex microspheres were centrifuged 2 times with 100mM MES-NaOH buffer, pH 5.5; centrifuging 10ml of 10% mass concentration latex microspheres at 300nm and 450nm at 22000rpm for 15min, removing supernatant, redissolving by using 10ml of MES-NaOH buffer solution, centrifuging, removing supernatant redissolving, and cleaning for later use;
(2) Latex activation: taking 10ml of the washed latex as an example, weighing 50mg of NHS (N-hydroxysuccinimide), 30mg of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), dissolving with 5ml of the MES-NaOH buffer for washing, adding 1ml of each to 10ml of the washed two microspheres, and magnetically stirring and uniformly mixing at room temperature for 10min;
(3) Cleaning after activation: centrifuging the two latices after the activation in the step (2) at 22000rpm for 15min, removing supernatant, and adding 20ml of 50mM Hepes-NaOH buffer solution with pH of 7.4 for re-dissolution for later use;
(4) Antibody coupling: two CEACAM6 antibodies were added to the above activated two latex solutions reconstituted with Hepes-Naoh buffer, with 1.5ml of antibody (2.4 g latex VS 50mg antibody) added to 300nm group; 1.5ml of antibody (1.8 g of latex VS 50mg of antibody) was added to the 450nm group and magnetically stirred at room temperature for 3 hours to complete the antibody coupling.
(5) Sealing and cleaning: after the two latices are centrifuged at 22000rpm for 15min, the whole supernatant is removed, and a 300nm group and a 450nm group are respectively added with a sealing cleaning solution (50 mM pH7.4 Hepes-NaOH buffer solution, tween-2016 ml/L, sucrose 30g/L, trehalose 10g/L, fructose 10g/L and 0.5ml/L PC 300) 42ml (the final concentration of the latices is about 4.8 g/L) and 54ml (the final concentration of the latices is about 3.6 g/L), and the two latices are mixed according to the volume of 1:1 and then are uniformly mixed at room temperature for 2 hours for standby (the final concentration of the 300nm carboxyl latex is 2.4g/L and the 450nm carboxyl latex is 1.8 g/L), thus obtaining the finished product R2.
Example 2
The first reagent R1 is formulated as follows:
Hepes-NaOH buffer 100mM, pH7.5
Sodium chloride 700mM
Polyethylene glycol-120002 g/L
Tween-20 ml/L
Mouse monoclonal antibody blocker 30ml/L (Phpeng organism E-010)
Preservative 0.5ml/L PC300
The preparation method of the first reagent R1 comprises the following steps:
taking 800ml of purified water, weighing the materials in the formula according to the volume of 1L, sequentially adding the materials into pure water, fully dissolving the materials, fixing the volume to 1L, and filtering the materials with a 450nm filter membrane for later use.
The formula of the second reagent R2 is as follows:
Hepes-NaOH buffer 100mM, pH7.0
Tween-20 ml/L
300nm carboxyl latex 2.0g/L (Suzhou degree Biotechnology Co., ltd.)
450nm carboxyl latex 1.5g/L (Suzhou Du Biotechnology Co., ltd.)
Protective agent (sucrose 20g/L, trehalose 20g/L, fructose 20 g/L)
CEACAM6 mouse monoclonal antibody 20mg/L (Yiqiaoshenzhou biotechnology Co., ltd.)
Preservative 0.5ml/L PC300
The preparation method of the second reagent R2 comprises the following steps:
(1) And (3) cleaning latex: the latex microspheres were centrifuged 2 times with 100mM MES-NaOH buffer, pH 5.5; taking 10ml of 10% mass concentration latex microspheres of 300nm and 450nm respectively, namely 1g of each of the two latex microspheres, centrifuging at 22000rpm for 15min, removing supernatant, redissolving by using 10ml of MES-NaOH buffer solution, centrifuging again, removing supernatant redissolving, and completing cleaning for later use;
(2) Latex activation: taking 10ml of the washed latex as an example, weighing 50mg of NHS (N-hydroxysuccinimide), 30mg of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), dissolving with 5ml of the MES-NaOH buffer for washing, adding 1ml of each to 10ml of the washed two microspheres, and magnetically stirring and uniformly mixing at room temperature for 10min;
(3) Cleaning after activation: centrifuging the two latices after the activation in the step (2) at 22000rpm for 15min, removing supernatant, and adding 20ml of 100mM Hepes-NaOH buffer solution with pH of 7.0 for re-dissolution for later use;
(4) Antibody coupling: two CEACAM6 murine monoclonal antibody portions were added to the above activated two latex solutions reconstituted with Hepes-NaOH buffer, with the 300nm group added with 0.6ml antibody (2.0 g latex VS20mg antibody); 0.6ml of antibody (1.5 g of latex VS20mg of antibody) was added to the 450nm group and magnetically stirred at room temperature for 3 hours to complete the antibody coupling.
(5) Sealing and cleaning: after centrifugation at 22000rpm for 15min, the whole supernatant was removed, and the 300nm group and 450nm group were reconstituted with a blocking wash (100mM pH7.0 Hepes-NaOH buffer, tween-2010 ml/L, sucrose 20g/L, trehalose 20g/L, fructose 20g/L, 0.5ml/L PC 300) 35ml (latex final concentration about 4.0 g/L), 45ml (latex final concentration about 3.0 g/L), respectively, the two latices being reconstituted at a volume of 1:1 (the final concentration is 300nm carboxyl latex 2.0g/L,450nm carboxyl latex 1.5 g/L) is prepared as the finished product R2.
Example 3
The first reagent R1 is formulated as follows:
Hepes-NaOH buffer 200mM, pH6.8
Sodium chloride 1000mM
Polyethylene glycol-120002 g/L
Tween-20 ml/L
Murine monoclonal antibody blocker 20ml/L (Phpeng organism E-010)
Preservative 1g/L sodium azide
The preparation method of the first reagent R1 comprises the following steps:
taking 800ml of purified water, weighing the materials in the formula according to the volume of 1L, sequentially adding the materials into pure water, fully dissolving the materials, fixing the volume to 1L, and filtering the materials with a 450nm filter membrane for later use.
The formula of the second reagent R2 is as follows:
Hepes-NaOH buffer 200mM, pH7.6
Tween-20 5ml/L
300nm carboxyl latex 1.6g/L (Suzhou Chemicals, inc.)
450nm carboxyl latex 1.0g/L (Suzhou Du Biotechnology Co., ltd.)
Protective agent (sucrose 10g/L, trehalose 30g/L, fructose 30 g/L)
CEACAM6 mouse monoclonal antibody 30mg/L (Yiqiaoshenzhou biotechnology Co., ltd.)
Preservative 1g/L sodium azide
The preparation method of the second reagent R2 comprises the following steps:
(1) And (3) cleaning latex: the latex microspheres were centrifuged 2 times with 100mM MES-NaOH buffer, pH 5.5; taking 10ml of 10% mass concentration latex microspheres of 300nm and 450nm respectively, namely 1g of each of the two latex microspheres, centrifuging at 22000rpm for 15min, removing supernatant, redissolving by using 10ml of MES-NaOH buffer solution, centrifuging again, removing supernatant redissolving, and completing cleaning for later use;
(2) Latex activation: taking 10ml of the washed latex as an example, weighing 50mg of NHS (N-hydroxysuccinimide), 30mg of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), dissolving with 5ml of the MES-NaOH buffer for washing, adding 1ml of each to 10ml of the washed two microspheres, and magnetically stirring and uniformly mixing at room temperature for 10min;
(3) Cleaning after activation: centrifuging the two latices after the activation in the step (2) at 22000rpm for 15min, removing supernatant, and adding 20ml 200mM Hepes-NaOH buffer solution with pH of 7.6 for re-dissolution for later use;
(4) Antibody coupling: two CEACAM6 murine monoclonal antibody portions were added to the above activated two latex solutions reconstituted with Hepes-NaOH buffer, with the 300nm group added with 0.9ml antibody (1.6 g latex VS 30mg antibody); 0.9ml of antibody (1.0 g of latex VS 30mg of antibody) was added to the 450nm group and magnetically stirred at room temperature for 3 hours to complete the antibody coupling.
(5) Sealing and cleaning: after centrifugation at 22000rpm for 15min, the whole supernatant was removed, and the 300nm group and 450nm group were each added with a blocking wash (200mM pH7.6 Hepes-NaOH buffer, tween-205 ml/L, sucrose 10g/L, trehalose 30g/L, fructose 30g/L, sodium azide 1 g/L) 28ml (final latex concentration about 3.2 g/L), 30ml (final latex concentration about 2.0 g/L) and reconstituted, the two latices being 1 by volume: 1 (1) and uniformly mixing for 2 hours at room temperature for standby (the final concentration of the carboxyl latex is 300nm is 1.6g/L, and the final concentration of the carboxyl latex is 450nm is 1.0 g/L), thus obtaining the finished product R2.
Example 4 calibration Curve acquisition
Dilution preparation (1L): taking 800ml of purified water, respectively weighing 11.9155g of hepes,52.598g of NaCl and 10ml of Tween-20,0.5ml proclin300, putting into the purified water, fully dissolving, regulating the pH to 7.4+/-0.1 by using NAOH, fixing the volume to 1L, using a filter membrane with the thickness of 0.22 mu m, and filtering to obtain the calibrator and quality control diluent.
CEACAM6 antigen (purchased from the company of biotechnology, san. Sedge) was formulated into calibrators and quality controls at the following concentrations:
calibration material: 0.0pg/ml, 100pg/ml, 300pg/ml, 800pg/ml, 1500pg/ml, 2000pg/ml;
quality control product: 200pg/ml, 500pg/ml.
The calibration curves were obtained by calibrating the reagents of example 1, comparative example 2, example 2 and example 3 (hereinafter referred to as reagent A, reagent B, reagent C, reagent D and reagent E) and the calibration quality control upper machine, respectively.
Wherein example 1 scatter 20 degree calibration curve is shown in FIG. 5; example 1 a transmission channel calibration curve is shown in figure 6.
The instrument is a Hitachi integrated machine for transmission and scattering, hitachi3500 is taken as an example, and the method is a transmission and scattering integrated method;
and (5) loading parameters: sample size 20. Mu.l, R1 addition 150. Mu.l; the addition amount of R2 is 50 μl;
a transmission wavelength of 600nm; a scattering angle of 20 degrees; a two-point end point method; 18-34 read points;
scattering calibration points 0.0, 100, 300, 800;
transmission calibration point: 0.0, 100, 300, 800, 1500, 2000;
the transmission scattering fusion zone is 100-300pg/ml;
results less than 300pg/ml report scattering results, results greater than or equal to 300pg/ml report transmission results, and results within the fusion zone report scattering results preferentially.
The calibration data are shown in tables 1-5.
TABLE 1
TABLE 2
TABLE 3 Table 3
TABLE 4 Table 4
TABLE 5
Example 5 Linear Range detection
High value samples approaching the upper limit of the linear range (2000 pg/ml) were diluted in proportion approaching the lower limit of the linear range (50 pg/ml). Repeating the detection with the reagents A, B, C, D and E for each concentration sample for 2 times, calculating average value, performing straight line fitting on the average value and dilution ratio, and calculating linear coefficient R 2 . The results are shown in tables 6-10 and FIGS. 7-11.
TABLE 6
Note that: results less than 300pg/ml report scattering results, results greater than or equal to 300pg/ml report transmission results, and results within the fusion zone report scattering results preferentially.
TABLE 7
Note that: results less than 300pg/ml report scattering results, results greater than or equal to 300pg/ml report transmission results, and results within the fusion zone report scattering results preferentially.
TABLE 8
Note that: results less than 300pg/ml report scattering results, results greater than or equal to 300pg/ml report transmission results, and results within the fusion zone report scattering results preferentially.
TABLE 9
Note that: results less than 300pg/ml report scattering results, results greater than or equal to 300pg/ml report transmission results, and results within the fusion zone report scattering results preferentially.
Table 10
Note that: results less than 300pg/ml report scattering results, results greater than or equal to 300pg/ml report transmission results, and results within the fusion zone report scattering results preferentially.
From the above linear dilution results, examples 1, 2 and 3 satisfy the conditions that the deviation of the measured linear dilution value from the estimated value is less than + -5% and the linear correlation coefficient R is within the range of 50pg/mL to 2000pg/mL 2 The linear relationship is good, and the linear relationship is more than 0.9995. Comparative example 1 had a low high value test, comparative example 2 had a low value dilution disproportionate phenomenon, and the deviation was large.
Example 6 repeatability investigation
The first reagent R1 and the second reagent R2 prepared in examples 1, 2 and 3 and comparative examples 1 and 2, respectively, were repeatedly measured 10 times for 100pg/ml of the sample, and the average value (M) and Standard Deviation (SD) of the 10 measured results were calculated to obtain the coefficient of variation according to the following formula:
CV＝SD/M×100％。
wherein: CV is the coefficient of variation; SD is the standard deviation of 10 measurements; m is the average of 10 measurements.
The results are shown in tables 11 and 12.
TABLE 11
Table 12
As can be seen from the results of tables 11, 12, the repeatability results at 100pg/ml for example 1, example 2 and example 3 are all better than for comparative example 1 and comparative example 2; the repeatability results of example 1 at 100pg/ml are better than those of examples 2 and 3; and as mentioned above in example 1, the CV of the transmission channel is close to 10% due to insufficient sensitivity, but the CV of the scattering channel is within 2%, further explaining the advantage and improvement of the integrated method of transmission and scattering compared with the conventional transmission detection. In combination with the linear dilution results, the subsequent stability experiments were performed on the basis of the reagents of example 1.
EXAMPLE 7 stability observations
1) Acceleration stability: the first reagent R1 and the second reagent R2 prepared in example 1 were placed in a 37℃incubator and a 4℃refrigerator, respectively (R1 and R2 were each placed in a 37℃incubator and R1 and R2 were each placed in a 4℃refrigerator and a group), and the 37℃reagent was taken out and tested simultaneously with the refrigerated reagent on days 3, 5 and 8, respectively. And (3) respectively testing high/low value quality control products (the concentration is 500pg/ml and 200pg/ml respectively), repeating the test for 3 times, taking an average value, comparing the average value of the test result of the accelerating reagent with the test result of the refrigerating reagent, and calculating the relative deviation (%). The results are shown in Table 13.
Note that: after the heat accelerating reagent is taken out from the incubator, the temperature and the refrigerated reagent are required to be balanced to the same temperature for testing (the experiment result caused by different temperatures is avoided).
TABLE 13
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2) Stability in opening bottles: after the first reagent R1 and the second reagent R2 prepared in example 1 were opened, they were placed in a full-automatic biochemical analyzer (2-8deg.C), high/low quality controls (500 pg/ml and 200pg/ml, respectively) were tested, and the opened reagent and the refrigerated reagent were taken out and tested simultaneously on days 3, 5, 7 and 15. The test was repeated 3 times, the average value was taken, the average value of the test results of the open-bottle reagent was compared with the test results of the chilled reagent, and the relative deviation (%) was calculated. The results are shown in Table 14.
TABLE 14
As can be seen from the results in tables 13 and 14, the reagent of the present invention was stable for 7 days in a 37℃high temperature environment and for 14 days in a 4℃unsealing condition, indicating excellent stability.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. The CEACAM6 latex turbidimetry detection kit adopting a transmission and scattering integrated method is characterized by comprising a first reagent R1 and a second reagent R2;
the first reagent R1 comprises the following components in concentration: 25-200mM Hepes-NaOH buffer pH 6.8-7.5, naCl 200-1000mM, polyethylene glycol 120002g/L, tween-20.05% -2%, murine monoclonal antibody blocker 20-40ml/L;
the second reagent R2 comprises the following components in concentration: 50-200mM buffer solution pH 7.0-7.6, tween-20-16 ml/L,300nm carboxyl latex 1.6-2.4g/L,450nm carboxyl latex 1.0-1.8g/L, protective agent 10-90g/L, CEACAM6 monoclonal antibody 20-40mg/L.
2. The integrated transmission and scattering CEACAM6 latex turbidimetry detection kit according to claim 1, wherein the first reagent R1 and the second reagent R2 further comprise a preservative, and the preservative is 1g/L sodium azide or 0.5ml/L PC300.
3. The integrated transmission and scattering CEACAM6 latex turbidimetry detection kit according to claim 1 or 2, wherein the buffer solution in the second reagent R2 is Hepes-NaOH buffer solution, phosphate buffer solution or glycine-Tris buffer solution.
4. The kit for detecting the specific turbidity of CEACAM6 latex by a transparent scattering integrated method according to claim 3, wherein the protective agent in the second reagent R2 is at least one of 10-30g/L sucrose, 10-30g/L trehalose and 10-30g/L fructose.
5. The integrated transmission and scattering CEACAM6 latex turbidimetry detection kit of claim 4, further comprising a calibrator and a quality control material;
the calibrator comprises the following components in concentration: 50mM Hepes-NaOH buffer pH7.4, CEACAM6 antigen 100-2000 pg/ml, sodium chloride 900mM, tween-20 10ml/L, preservative;
the quality control product comprises the following components in concentration: 50mM Hepes-NaOH buffer pH7.4, CEACAM6 antigen 200-500 pg/ml, sodium chloride 900mM, tween-20 10ml/L, preservative;
the preservative is 1g/L sodium azide or 0.5ml/L PC300.
6. The application of the CEACAM6 latex turbidimetry detection kit in detection of CEACAM6 by a transparent scattering integrated method as claimed in claim 1.
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