CN114563564A - Detection reagent and detection method for sialidation carbohydrate chain antigen KL-6 - Google Patents
Detection reagent and detection method for sialidation carbohydrate chain antigen KL-6 Download PDFInfo
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- CN114563564A CN114563564A CN202111527429.2A CN202111527429A CN114563564A CN 114563564 A CN114563564 A CN 114563564A CN 202111527429 A CN202111527429 A CN 202111527429A CN 114563564 A CN114563564 A CN 114563564A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4725—Mucins, e.g. human intestinal mucin
Abstract
The invention discloses a detection reagent and a detection method for a sialidalized sugar chain antigen KL-6, wherein the detection reagent comprises an R1 buffer solution, an R2 reagent and KL-6 antigen standard products with different concentrations, and a preparation method for the R2 reagent comprises the following steps: (1) cleaning latex particles; (2) activating styrene latex particles; (3) antibody label coupling; (4) sealing; (5) and (5) storing. Drawing a standard curve according to the detection result delta A of the KL-6 standard substance with different concentrations, detecting the sample to obtain the delta A of the sample, and comparing the delta A of the sample with the standard curve to obtain the concentration of the sialylated glycoantigen KL-6 in the sample. The reagent and the detection method for determining KL-6 by using the latex immunoturbidimetry are simple in process, simple and convenient to operate, high in detection speed and comparable in precision and repeatability to those of the imported kit.
Description
Technical Field
The invention belongs to the technical field of biochemical detection, and particularly relates to a detection reagent and a detection method for a sialylated glycoantigen KL-6.
Background
KL-6 (sialidated sugar chain antigen) is a transmembrane high molecular weight mucin discovered by Kohno of Japanese in 1985, and contains a sialylated sugar chain with a molecular weight of about 200KD, which is mainly a tandem repeat sequence consisting of 20 amino acid residues and has a spatial structure epitope recognized by a specific antibody. In normal lung tissue, it is mainly expressed in the cytoplasm and cell membrane of alveolar cell and bronchial epithelial cell, and partially expressed in the cytoplasm of bronchiolar basal cell and Clara cell and bronchial gland, and also partially expressed in epithelial cells of organ tissues such as pancreas, stomach and mammary gland. Various studies on KL-6 conducted in Japan and China show that it has positive clinical significance as a serological index of Interstitial Lung Disease (ILD), can be used for diagnosis and determination of treatment strategies for interstitial pneumonia including drug-induced interstitial pneumonia, collagen imbalance-induced interstitial pneumonia and the like, and is used for diagnosing patients with lung cancer, pancreatic cancer and the like.
In scientific research, the common detection methods of KL-6 mainly comprise ELISA, fluorescence immunochromatography, chemiluminescence and latex immunoenhancement turbidimetry, ELSA is a reliable method, but the operation and the timeliness of the ELSA cannot meet the detection of a large number of clinical samples, and the two current clinical KL-6 kits in China are both magnetic micro-particle chemiluminescence methods, so that the cost is high, the operation is complex, a special chemiluminescence detector needs to be reconfigured, and in contrast, the latex immunoenhancement turbidimetry is the most common biochemical analysis method, the popularity of biochemical analyzers in hospitals and inspection institutions is high, the operation is relatively simple and convenient, the sensitivity and the linear range are both in the required range, and the method is the first choice for KL-6 batch detection. But the KL-6 detection product registration of the methodology is not available in China.
The two methods for preparing the reagent by the latex immunoturbidimetry which are widely used at present mainly comprise two methods, one method is to mix two strains of antibodies according to a certain proportion and then carry out microsphere marking together, because the marking conditions required by different antibodies are different, the precision and precision of the method are not high, and the utilization rate of the antibodies is low, while the other method is to couple two strains of monoclonal antibodies on latex microspheres with different particle sizes respectively and mix the monoclonal antibodies uniformly according to a certain proportion, so that the method involves repeated centrifugal ultrasonic and heavy suspension operations, the process is too complex, and the method is not beneficial to amplification production.
Disclosure of Invention
The invention aims to provide a detection reagent and a detection method for a salivation sugar chain antigen KL-6, and solves the problems that the existing detection process and steps are too complex and are not beneficial to amplification production.
In order to solve the technical problems, the invention is realized by the following technical scheme:
a detection reagent for salivary liquefaction sugar chain antigen KL-6 comprises an R1 buffer solution, an R2 reagent and KL-6 antigen standard products with different concentrations, wherein the concentration of the KL-6 antigen standard products is 0, 250, 500, 1000, 2500 and 5000U/mL;
the formula of the R1 buffer solution is as follows:
adjusting pH to 7.0 + -0.05, diluting to constant volume to 1L volumetric flask, filtering with 0.22 μm filter membrane, and storing at 2-8 deg.C;
the preparation method of the R2 reagent comprises the following steps:
(1) cleaning latex particles; measuring 0.5ml of styrene latex particles with solid content of 10 percent, adding the styrene latex particles into 3.0ml of cleaning buffer solution, centrifuging, rotating at 20000 rpm for 30 minutes, removing supernatant, and carrying out ultrasonic resuspension on latex microparticle precipitates by using 3.0ml of activation buffer solution;
(2) activating styrene latex particles: adding an activating agent into the washed latex particles, uniformly mixing, activating the latex particles, and activating at 37 ℃ for 0.5 hour;
(3) antibody labeling and coupling: adding the anti-human KL-6 antibody into the activated styrene latex particle solution, and uniformly mixing for 2-4h at 37 ℃ by a shaking table;
(4) and (3) sealing: after the marking is finished, centrifuging, 20000 rpm for 30 minutes, removing supernatant, taking 5.0ml of blocking buffer solution to carry out resuspension ultrasonic on the latex particle sediment, and then blocking for 1 hour;
(5) and after the sealing is finished, centrifuging for 30 minutes at 20000 rpm, removing the supernatant, adding 150ml of latex preservation solution, and carrying out ultrasonic resuspension to obtain a milky white solution, namely the reagent R2.
Further, the formulation of the latex preservative solution in the step (5) is as follows,
further, the configuration of the kl-6 antigen standard: various concentrations of anti-human KL-6 standards were prepared in phosphate buffer containing 1% BSA, 0.8ml/L Proclin 300, pH 6.5-7.5, sterile filtered.
Further, the anti-human kl-6 antibody includes, but is not limited to, monoclonal antibodies or polyclonal non-affinity IgG, affinity IgG or IgY antibodies of rabbit, sheep, mouse, chicken and duck origin.
Further, the styrene latex particles are 50-150 nm.
Further, in the step (1), the washing buffer and the activation buffer are both phosphate buffer.
Further, the activator in step (2) was 2% EDAC 0.1ml, 4% NHS 0.2 ml. EDAC and NHS are difunctional crosslinkers. NHS for N-hydroxy succinimide.
Further, the blocking buffer in step (4) was 1% BSA.
A method for detecting saliva liquefaction sugar chain antigen KL-6 adopts a detection reagent of saliva liquefaction sugar chain antigen KL-6, comprises the following steps,
drawing a standard curve according to the detection result delta A of the KL-6 standard substance with different concentrations, detecting the sample to obtain the delta A of the sample, and comparing the delta A of the sample with the standard curve to obtain the concentration of the sialylated oligosaccharide antigen KL-6 in the sample.
The invention has the following beneficial effects:
the reagent and the detection method for determining KL-6 by using the latex immunoturbidimetry are simple in process, simple and convenient to operate, high in detection speed and comparable in precision and repeatability to those of the imported kit.
The screened KL-6 antibody is marked on the latex microsphere with a specific particle size, KL-6 in samples such as serum and the like and the latex microsphere marked with the antibody are subjected to antigen-antibody reaction, the absorbance of the latex microsphere is detected under a specific wavelength, and the change degree of the absorbance and the KL-6 content in the samples form a certain linear relation.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used for describing the embodiments will be briefly introduced below.
FIG. 1: the standard curves of different kits of the invention are compared.
FIG. 2: the invention correlation check graph.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
Instrument and reagent
The main detecting instrument: the content of Hitachi 7180 is as follows,
the main reagents are as follows: human KL-6 antigen, anti-human KL-6 antibody, 50-150nm styrene latex particles, and a comparison KL-6 kit. The KL-6 antibody, the antigen and the control kit are purchased from Japan hydrographic medical science and technology Limited. Comprises R1 and R2, wherein R1 is buffer solution, and different concentrations of kl-6 standard substance (0, 250, 500, 1000, 2500, 5000U/mL) 2.1R1 are prepared
Preparing buffer solution according to the following table proportion, adjusting the pH to 7.0 +/-0.05, fixing the volume to 1L volumetric flask, filtering with a 0.22um filter membrane, labeling, and storing at 2-8 ℃.
2.2 preparation of R2
2.2.1 latex particle washing: 0.5ml of latex microparticles containing 10% solids were weighed into 3.0ml of washing buffer, centrifuged, 20000 rpm for 30 minutes, the supernatant removed, and the latex microparticle pellet resuspended in 3.0ml of activation buffer using ultrasound.
2.2.2 latex particle activation: 0.1ml of 2% EDAC and 0.2ml of 4% NHS (N-hydroxysuccinimide) were weighed out and added to the washed latex microparticles, and the latex microparticles were uniformly mixed and activated. 37 degrees activation for 0.5 hour. The activating buffer used in this step is phosphate buffer.
2.2.3 antibody labeling conjugation: adding the anti-human KL-6 antibody into the activated latex particle solution, and uniformly mixing for 2-4h at 37 ℃ by a shaking table.
2.2.4 blocking: after the labeling was completed, centrifugation was started at 20000 rpm for 30 minutes, the supernatant was removed, and 5.0ml of blocking buffer was taken to resuspend the latex microparticle pellet with ultrasound and then blocked for 1 hour. The blocking buffer used was 1% BSA.
2.2.5 after the sealing is finished, centrifuging, 20000 rpm for 30 minutes, removing supernatant, adding 150ml of latex preservation solution, and carrying out ultrasonic resuspension to obtain milky white solution which is the reagent R2, wherein the formula of the latex preservation solution is as follows
2.3 preparation of KL-6 Standard substance
Different concentrations of KL-6 standards were prepared in 0.8ml/L Proclin 300pH 6.5-7.5 phosphate buffer containing 1% BSA and sterile filtered.
2.4 filling R1 and R2 into proper actual bottles respectively according to the proportion of 4:1, and matching with standard products with different concentrations for standby.
Detection method
A detection instrument: hitachi 7180 full-automatic biochemical analyzer
The instrument parameters are as follows:
the method comprises the following steps: temperature of end-point method: 37 deg.C
Dominant wavelength: wavelength number 570 sub-wavelength: 800
Sample size/R1/R2: 6ul/180ul/60ul
The reaction direction is as follows: forward reaction time: for 10min
The calculation method comprises the following steps: multi-point calibration
The detection steps are as follows:
secondly, evaluating the performance of the kit
1, linearity:
linear correlation coefficient r2: the high value specimens close to the upper limit of the linear region were diluted with the low value specimens close to the lower limit of the linear region in the range of [ 100-. The linear regression equation was calculated using the dilution concentration (Xi) as an independent variable and the measurement result mean (yi) as a dependent variable. And (3) calculating a correlation coefficient r of the linear regression according to the formula (1), wherein the obtained result meets the requirement that r is more than 0.99.
The present kit and the control kit were used to draw standard curves (3 measurements per concentration, average Δ a) respectively, and the results are shown in table 1 and fig. 1.
TABLE 1 comparison of fitted curves for different kits
2 precision
Repeatability: adopting high and low quality control products, respectively and continuously measuring for 10 times by using two reagents, respectively calculating the average value and standard deviation of the measured values, and calculating the variation Coefficient (CV) in batch according to the formula (2), wherein the CV of the obtained result is less than or equal to 8.0 percent. (the results are shown in Table 2)
In the formula: SD is standard deviation, and the calculation formula is
dn is the deviation of the measured values at the same level, and the calculation formula is
xn is measured at the same level for each time;
Repeatability test results (Table 2)
According to the detection results, the variation coefficients of the detection results of the low-value sample and the high-value sample of the kit and the control kit are less than 8%, and the clinical requirements are met.
3 degree of accuracy
Taking a high-level quality control product and a low-level quality control product, repeatedly measuring for 3 times by using the same batch of reagent, recording the test result as (xi), calculating the relative deviation (Bi) according to the formula (3), wherein the 3 times of results all accord with the corresponding deviation between the measured value and the target value of the quality control product which is less than or equal to 15.0%; if 2 times of results in 3 times of results meet the requirements and 1 time of results do not meet the requirements, continuously testing for 20 times again, and respectively calculating the relative deviation (Bi) according to the formula (3), and if the results are more than or equal to 19 times of results meet the requirements, the accuracy is verified to meet the requirement that the relative deviation between the measured value and the target value of the quality control product is less than or equal to 15.0 percent.
In the formula: xi is the result of the determination;
t is the target value.
The theoretical concentration of the low-value sample is 400U/ml, and the theoretical concentration of the high-value sample is 2500U/ml
The results are as follows: (Table 3)
The coefficient of variation is less than 15 percent
4 correlation
Randomly extracting 30 serum samples, respectively detecting the myoglobin content in the samples by using an R2-3 kit contrast kit method, calculating the correlation coefficient by using a CORREL function, wherein the correlation coefficient is more than or equal to 0.9, and then showing that the kit provided by the invention is suitable for detecting KL-6
The results are shown in fig. 2, the correlation coefficient of the test results of the kit and the control kit is greater than 0.99, and the kit and the control kit can be used for detecting the clinical KL-6 sample.
The embodiments were chosen and described in order to best explain the principles of the invention and its practical applications, to thereby enable others skilled in the art to best understand the invention and utilize the invention.
Claims (9)
1. A detection reagent for a sialylated sugar chain antigen KL-6 is characterized in that:
comprises R1 buffer solution, R2 reagent and kl-6 antigen standard products with different concentrations, wherein the concentration of the kl-6 antigen standard products is 0, 250, 500, 1000, 2500 and 5000U/mL;
the formulation of the R1 buffer solution is as follows:
adjusting pH to 7.0 + -0.05, diluting to constant volume to 1L volumetric flask, filtering with 0.22 μm filter membrane, and storing at 2-8 deg.C;
the preparation method of the R2 reagent comprises the following steps:
(1) cleaning latex particles; measuring 0.5ml of styrene latex particles with solid content of 10 percent, adding the styrene latex particles into 3.0ml of cleaning buffer solution, centrifuging, rotating at 20000 rpm for 30 minutes, removing supernatant, and carrying out ultrasonic resuspension on latex microparticle precipitates by using 3.0ml of activation buffer solution;
(2) activating styrene latex particles: adding an activating agent into the washed latex particles, uniformly mixing, activating the latex particles, and activating at 37 ℃ for 0.5 hour;
(3) antibody labeling and coupling: adding the anti-human KL-6 antibody into the activated styrene latex particle solution, and uniformly mixing for 2-4h at 37 ℃ by a shaking table;
(4) and (3) sealing: after the marking is finished, centrifuging, 20000 rpm for 30 minutes, removing supernatant, taking 5.0ml of blocking buffer solution to carry out resuspension ultrasonic on the latex particle sediment, and then blocking for 1 hour;
(5) and after the sealing is finished, centrifuging for 30 minutes at 20000 rpm, removing the supernatant, adding 150ml of latex preservation solution, and carrying out ultrasonic resuspension to obtain a milky white solution, namely the reagent R2.
2. The reagent for detecting sialylated glycoantigen KL-6 according to claim 1, wherein the reagent comprises: the formula of the latex preservative fluid in the step (5) is as follows,
3. the reagent for detecting sialylated glycoantigen KL-6 according to claim 1, wherein the reagent comprises: preparation of kl-6 antigen standard: various concentrations of anti-human KL-6 standards were prepared in phosphate buffer containing 1% BSA, 0.8ml/L Proclin 300, pH 6.5-7.5, sterile filtered.
4. The reagent for detecting sialylated glycoantigen KL-6 according to claim 1, wherein the reagent comprises: the anti-human kl-6 antibody includes but is not limited to monoclonal antibody or polyclonal non-affinity IgG antibody, affinity IgG antibody or one or two of IgY antibody from rabbit, sheep, mouse, chicken and duck.
5. The reagent for detecting sialylated glycoantigen KL-6 according to any of claims 1 to 4, wherein the reagent comprises: the styrene latex particles are 50-150 nm.
6. The reagent for detecting sialylated glycoantigen KL-6 according to claim 5, wherein the reagent comprises: in the step (1), the washing buffer solution and the activation buffer solution are both phosphate buffer solutions.
7. The reagent for detecting sialylated glycoantigen KL-6 according to claim 6, wherein the reagent comprises: the activating agent in step (2) was 2% EDAC 0.1ml, 4% NHS 0.2 ml.
8. The reagent for detecting sialylated glycoantigen KL-6 according to claim 7, wherein the reagent comprises: the blocking buffer in step (4) was 1% BSA.
9. A method for detecting a sialylated sugar chain antigen KL-6 adopts a detection reagent of the sialylated sugar chain antigen KL-6 of any one of claims 1 to 8, and is characterized in that: comprises the following steps of (a) carrying out,
drawing a standard curve according to the detection result delta A of the KL-6 standard substance with different concentrations, detecting the sample to obtain the delta A of the sample, and comparing the delta A of the sample with the standard curve to obtain the concentration of the sialylated glycoantigen KL-6 in the sample.
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