CN117368495A - Method and kit for detecting content of human chorionic gonadotrophin beta subunit - Google Patents
Method and kit for detecting content of human chorionic gonadotrophin beta subunit Download PDFInfo
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Classifications
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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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
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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
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
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- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a method and a kit for detecting the content of beta subunit of human chorionic gonadotrophin, wherein the detection method comprises the steps of preparing the kit; reading after the first reaction is finished by using a quartz needle biosensor, and reading a first reading signal; after the quartz needle is put into the kit for cleaning, continuing to read after the quartz needle biosensor finishes the second reaction, and reading a second reading signal; repeatedly reading the calibration materials with different concentrations and recording a first reading signal and a second reading signal of the calibration materials with each concentration; sequentially obtaining a high-concentration section signal standard curve and a low-concentration section signal standard curve according to the first reading signal and the second reading signal; and detecting the signal value of the sample to be detected, and back-calculating the sample to be detected by utilizing the high-concentration section signal standard curve and/or the low-concentration section signal standard curve to obtain the concentration of the sample to be detected. The scheme realizes the simplicity and convenience of detecting the content of the beta subunit of the human chorionic gonadotrophin, and the stability and accuracy of the detection result.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a method and a kit for detecting the content of beta subunit of human chorionic gonadotrophin.
Background
Human chorionic gonadotrophin subunit (human chorionic gonadotropin, HCG) is a glycoprotein hormone secreted by placental syngeneic trophoblasts, which reflects the functional status of the trophoblast villus. The HCG structure includes two subunits, α and β, the α chain and β chain being linked by 11 or 12 disulfide bonds, the α chain being similar to Luteinizing Hormone (LH), follicle Stimulating Hormone (FSH) and Thyroid Stimulating Hormone (TSH). beta-HCG is a molecular beta subunit in HCG, consists of 145 amino acids, has a relative molecular mass of 23KD and can be detected in blood. In the normal pregnancy period, the concentration is lower in early pregnancy, the concentration is rapidly increased from the 5 th week of pregnancy to the 8-10 th week of pregnancy, the serum concentration reaches the peak of approximately 200000IU/L, the concentration is reduced in 11-16 weeks, the concentration is basically stabilized to delivery, and HCG in blood is basically disappeared in about 1 week after delivery, so that the serum concentration value span is extremely large according to the pregnancy period.
The existing detection methods include: immunochromatography and chemiluminescence, wherein an antibody or antigen is usually immobilized on a carrier (such as nitrocellulose membrane) while another antibody is labeled with a tracer or fluorescent substance (such as colloidal gold particles, lanthanoid and chelating agent labeled microspheres, color microspheres, etc.) or an antigen is immobilized on glass fiber, and when a substance to be detected is contained in a sample, the labeled antigen or antibody reacts with the antibody on the nitrocellulose membrane while gold particles, fluorescent substance or color microspheres remain on the nitrocellulose membrane of the carrier, and the intensity of fluorescence or color development substance on the T-band (T-line) is in a linear relationship with the concentration of the substance to be detected. The product detection flux is low, manual sample application operation is generally required, and the product detection flux is easily affected by other factors, such as temperature in detection environment, moderate degree, evenness of a membrane strip, dirt degree, sample consistency, viscosity and the like.
The chemiluminescent method adopts horseradish peroxidase (HRP) to mark antibody (or antigen), after immunoreaction with the sample to be tested and solid phase carrier in the reaction system, forms solid phase coated antibody-antigen to be tested-enzyme (HRP) marked antibody complex, at this time, luminol luminescent agent, H2O2 and chemiluminescent reinforcing agent are added to produce chemiluminescence, through HRP catalytic reaction, plane conjugated structure is produced in the presence of reinforcing agent, so that the luminous intensity is enhanced many times. Since the functional groups introduced by the respective derivatives of luminol are different, there is also a difference in enhancement of the light emission amplitude. In addition, the HRP catalytic reaction is limited by the activity of HRP enzyme, and the enzyme is gradually deactivated in a long-term liquid environment, resulting in a decrease in the detection luminescence value, and thus, the stability is slightly poor.
In the prior art, the immunological detection of human chorionic gonadotrophin beta subunit has common detection methods such as immunochromatography, chemiluminescence method and the like, the detection upper limit of a detection kit is mostly not more than 10000IU/L under the influence of the detection method and a solid phase carrier form, and if the concentration of a sample exceeds the detection upper limit of the kit, the sample is required to be diluted and then detected, for example, the application publication number is CN114002444A, the invention patent of the name of beta-human chorionic gonadotrophin detection kit discloses a beta-human chorionic gonadotrophin detection kit which comprises a reagent R1, a reagent R3 and a reagent R4; the reagent R1 is provided with streptavidin-coated magnetic particles and Tris buffer solution, the reagent R3 is provided with acridinium ester-marked murine anti-human HCG antibody and phosphate buffer solution, the reagent R4 is provided with biotin-marked murine anti-human HCG antibody and morpholine ethane sulfonic acid MES, the upper detection limit of the reagent kit is 8000mIU/mL, whether the concentration of HCG+beta in a sample exceeds 8000mIU/mL needs to be judged first, and when the concentration of HCG+beta in the sample exceeds 8000mIU/mL, the sample needs to be diluted by sample diluent (the recommended dilution factor is 100 times) and then detected.
Before the re-test of the diluted sample, whether the concentration of the sample exceeds the detection upper limit of the kit is judged, so that the authorized bulletin number is CN108152505B, which is named as an immunoassay method, a system for identifying immunoassay and a kit, and an immunoassay method is provided for judging whether the sample needs to be diluted and re-measured; and (3) reading twice by adopting a chemiluminescent immunoassay method, recording the amplification of a signal value obtained by the reading twice, and preparing a standard curve, wherein in the follow-up detection, if the amplification of the reading twice of a sample to be detected of the target antigen or antibody to be detected is larger than the maximum value of the standard curve, the sample needs to be diluted and then is measured, and the upper detection limit of the sample is 10000mIU/ml.
Therefore, in the prior art, it is necessary to determine whether the concentration of the sample exceeds the detection upper limit value of the kit, if the concentration of the sample exceeds the detection upper limit value of the kit, the sample needs to be diluted and then measured, when the concentration of the sample is too high, the final result can be obtained by overlapping the signal values measured each time after repeated tests, and whether the sample exceeds the concentration upper limit value, the signal values of the sample are read and the final signal values of the sample are calculated for multiple times.
Disclosure of Invention
Therefore, in order to solve the problems, the invention provides a method and a kit for detecting the content of human chorionic gonadotrophin beta subunit, which are used for realizing the simplicity and convenience of detecting the content of human chorionic gonadotrophin beta subunit and the stability and accuracy of detection results.
The invention is realized by the following technical scheme:
the method for detecting the content of human chorionic gonadotrophin beta subunit comprises the following steps:
s1: preparing a human chorionic gonadotrophin beta subunit content detection kit;
the kit comprises a quartz needle biosensor, a reagent hole 1 for containing a sample, a reagent hole 2 containing an anti-human chorionic gonadotrophin beta subunit antibody marked with biotin, a reagent hole 3 containing a streptavidin fluorescent substance marked with polysaccharide, and a cleaning hole 1, a cleaning hole 2, a cleaning hole 3, a cleaning hole 4, a cleaning hole 5, a cleaning hole 6, a cleaning hole 7, a cleaning hole 8 and a cleaning hole 9 which are reading holes;
s2: placing the calibrator in a reagent hole 1 of a kit, sequentially placing the calibrator in the reagent hole 1, a reagent hole 2 and a reagent hole 3 by using a quartz needle biosensor, reading after the first reaction is finished, and reading a first reading signal;
S3: after the quartz needle is put into the reagent box for cleaning, the quartz needle biosensor is continuously put into the reagent hole 2 and the reagent hole 3 in sequence, reading is carried out after the second reaction is completed, and a second reading signal is read;
s4: repeating the steps S2 and S3 for the calibration materials with different concentrations, and sequentially recording a first reading signal and a second reading signal of the calibration materials with each concentration;
s5: sequentially obtaining a high-concentration section signal standard curve and a low-concentration section signal standard curve according to the first reading signal and the second reading signal obtained in the step S4;
s6: detecting a signal value of a sample to be detected, and back-calculating the sample to be detected by utilizing a high-concentration section signal standard curve and/or a low-concentration section signal standard curve to obtain the concentration of the sample to be detected;
in the step S2 and the step S3, each reaction is carried out, before the quartz needle biosensor is placed in the next reagent hole, the quartz needle biosensor is placed in a cleaning hole for cleaning.
Preferably, the quartz needle is Dan Yingzhen modified by aminopropyl, and the bottom surface of the quartz needle biosensor is dried after being coated with the capture antibody.
Preferably, the reaction time of the first reaction is less than the reaction time of the second reaction.
Preferably, the rotational speed of the quartz needle biosensor in the first reaction is lower than the rotational speed of the quartz needle biosensor in the second reaction.
Preferably, in step S4, "repeating steps S2 and S3 for different concentrations of calibrator" means that steps S2 and S3 are performed on a plurality of known different concentrations of human chorionic gonadotrophin β subunit antigen calibrator, respectively, having a concentration within the upper detection limit of the kit.
Preferably, the step S5 further includes: and (3) according to the first reading signal and the second reading signal of the calibrator with each concentration recorded in the step S4, preparing a plurality of first reading signals into a high-concentration section signal standard curve, preparing a plurality of second reading signals into a low-concentration section signal standard curve, and setting decision points of the high-concentration section signal standard curve and the low-concentration section signal standard curve.
Preferably, the step S2 includes:
s2.1: placing a quartz needle biosensor into a sample in the reagent hole 1 to react for 10s-20s at a rotating speed of 1000rpm-1200rpm;
s2.2: washing the reacted biosensor in the washing hole 7 for 5s-10s at 1000rpm-1200rpm;
s2.3: placing the cleaned biosensor into a reagent hole 2 for reaction for 10s-20s, wherein the rotating speed is 1000rpm-1200rpm;
S2.4: washing the reacted biosensor in the washing hole 5 for 5s-10s at 1000rpm-1200rpm;
s2.5: placing the cleaned biosensor into a reagent hole 3 for reaction for 10s-20s, wherein the rotating speed is 1000rpm-1200rpm;
s2.6: the circularly reacted biosensor is put into the washing hole 9 for reading, and the first reading signal is read.
Preferably, the step S3 includes:
s3.1: placing the reacted biosensor into a cleaning hole 3, and cleaning the biosensor in the cleaning hole 4 for 5s-10s at a rotating speed of 1000rpm-1200rpm;
s3.2: placing the cleaned biosensor into a reagent hole 2 for reaction for 40s-80s, wherein the rotating speed is 1200rpm-1500rpm;
s3.3: washing the reacted biosensor in the washing hole 5 for 5s-10s at 1000rpm-1200rpm;
s3.4: placing the cleaned biosensor into a reagent hole 3 for reaction for 10s-20s, and rotating at 1000rpm-1200rpm;
s3.5: washing the reacted biosensor in the washing hole 1 and the washing hole 2 for 5s-10s at 1000rpm-1200rpm;
s3.6: the circularly reacted biosensor is put into the washing hole 9 for reading, and the second reading signal is read.
Preferably, the step S6 includes:
s6.1: placing a sample to be detected into a reagent hole 1 of a reagent kit, sequentially placing a quartz needle biosensor into the reagent hole 1, a reagent hole 2 and a reagent hole 3, reading after finishing the first reaction, and reading a first reading signal;
S6.2: after the quartz needle is put into the reagent box for cleaning, the quartz needle biosensor is continuously put into the reagent hole 2 and the reagent hole 3 in sequence, reading is carried out after the second reaction is completed, and a second reading signal is read;
s6.3: if the sample is lower than the concentration value of the resolution point, only reading the signal for the second time, and putting the sample on a low-concentration section signal standard curve for back calculation, and comparing to obtain a sample concentration value;
if the sample is higher than the concentration value of the resolution point, only the first reading signal can be read, the sample is put on a signal standard curve of a high concentration section for back calculation, and the concentration value of the sample is obtained after comparison;
if the sample is near the resolution point, the first reading signal and the second reading signal can be read at the same time, and the sample is put into the low-concentration section signal standard curve and the high-concentration section signal standard curve to be calculated back, two sample concentration values are obtained by comparison, and the average value of the two sample concentration values is calculated to be used as the sample concentration value.
The human chorionic gonadotrophin beta subunit content detection kit comprises a Dan Yingzhen biosensor, a reagent hole 1 for accommodating a sample, a reagent hole 2 for accommodating an anti-human chorionic gonadotrophin beta subunit antibody marked with biotin, a reagent hole 3 for accommodating a streptavidin fluorescent substance marked with polysaccharide, and a cleaning hole 1, a cleaning hole 2, a cleaning hole 3, a cleaning hole 4, a cleaning hole 5, a cleaning hole 6, a cleaning hole 7, a cleaning hole 8 and a cleaning hole 9 which are reading holes;
The bottom surface of the quartz needle biosensor is dried after being coated with a capture antibody, the capture antibody comprises an anti-FITC antibody and a fluorescein-labeled human chorionic gonadotrophin beta subunit antibody, wherein the concentration ratio of the anti-FITC antibody to the fluorescein-labeled human chorionic gonadotrophin beta subunit antibody is 2:3, a step of;
in the anti-human chorionic gonadotrophin beta subunit antibody labeled with biotin in the reagent well 2, the ratio of biotin to the anti-human chorionic gonadotrophin beta subunit antibody is (5-10): 1, and the biotin-labeled anti-human chorionic gonadotrophin beta subunit antibody is diluted to 1ug/mL with a preservation solution at room temperature;
the polysaccharide-labeled streptavidin fluorescent material in the reagent well 3 was diluted to 5ug/mL with a preservative solution at room temperature.
The technical scheme of the invention has the beneficial effects that:
1. in the kit adopted by the scheme, the reaction time and the rotating speed of the probe in each reagent are conveniently controlled by putting the quartz needle modified by the aminopropyl into each reagent hole in the kit for reaction, the reaction time is short in the high concentration section, the rotating speed is low, the reaction time is long in the low concentration section, the rotating speed is higher, the high concentration and low concentration samples are fully distinguished by combining with the saturation, the low-end detection sensitivity and the detection range are improved, the signal values of different concentration sections can be obtained in the same detection in the same kit by adjusting the reaction time and the rotating speed of the two reactions, and compared with the prior art, the detection upper limit is improved by 30 times, the concentration upper limit of beta-HCG can be included, the signal values can be avoided from being detected repeatedly by multiple dilutions and detection, and the detection time and the detection cost are saved.
2. The method has the advantages that the samples with different concentrations in the detection upper limit value are repeatedly detected in advance, standard curves of the corresponding concentrations and signal values of the high concentration section and the low concentration section are respectively obtained, when the sample to be detected is tested, the concentration of the sample to be detected can be obtained by comparing the standard curves after the reaction of the kit and the reading of the signals for two times, the detection process is simplified, the detection convenience is greatly improved, meanwhile, multiple dilution, reading and calculation are not needed, the gradual inactivation of HRP enzyme in a long-time liquid environment is avoided, and the accuracy and the stability of the detection result are improved.
3. The surface of a Dan Yingzhen biosensor in the kit is modified by APS in advance, APS is used as a hydrophobic medium, the effectiveness of antibody binding is ensured, and the adsorption of other impurity proteins or impurities to a quartz needle can be removed, so that the density of the antibody on the surface of a quartz probe is high, the detection accuracy effect is good, the capturing antibody is coated by Dan Yingzhen modified by APS, the capturing antibody comprises an anti-FITC antibody (fluorescein antibody) and a fluorescein-labeled human chorionic gonadotrophin beta subunit antibody, the concentration of the fluorescein-labeled human chorionic gonadotrophin beta subunit antibody can be measured by fluorescein, and the anti-FITC antibody can be subjected to covalent binding reaction with the fluorescein specificity of the fluorescein-labeled human chorionic gonadotrophin beta subunit antibody, so that the capturing antibody is firmly and uniformly fixed on the surface of the Dan Yingzhen biosensor through hydrophobic binding.
4. The human chorionic gonadotrophin beta subunit detection antibody marked with biotin in the kit is combined with the polysaccharide skeleton coupled biotin-SA mark, the detection background is unchanged through biotin avidin amplification, the detection signal is increased by tens of hundreds of times through circulation enhancement, and the detection signal level is greatly improved, so that the human chorionic gonadotrophin beta subunit with lower concentration can be detected, and the detection sensitivity is improved.
Drawings
Fig. 1: the linear range diagram of a preferred embodiment of the present invention provides a kit for detecting the beta subunit content of chorionic gonadotrophin;
fig. 2: the present inventors have performed a hook-effect diagram of a preferred embodiment of a chorionic gonadotrophin beta subunit content detection kit;
fig. 3: the present inventors have compared the high concentration signal standard curve with the low concentration signal standard curve according to a preferred embodiment of the method for detecting chorionic gonadotrophin beta subunit content.
Detailed Description
So that the manner in which the objects, advantages and features of the invention are attained and can be more clearly understood, a more particular description of the preferred embodiments will be rendered by the following non-limiting description thereof. The embodiment is only a typical example of the technical scheme of the invention, and all technical schemes formed by adopting equivalent substitution or equivalent transformation fall within the scope of the invention.
Furthermore, the terms "first," "second," and the like in this description are used for descriptive purposes only and are not to be construed as indicating or implying a ranking of importance, or as implicitly indicating the number of technical features shown. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the present invention, the meaning of "plurality" means two or more, unless specifically defined otherwise.
The invention discloses a method for detecting the content of beta subunit of human chorionic gonadotrophin, which comprises the following steps:
s1: preparing a human chorionic gonadotrophin beta subunit content detection kit;
the kit comprises a quartz needle biosensor, a reagent hole 1 for containing a sample, a reagent hole 2 containing an anti-human chorionic gonadotrophin beta subunit antibody marked with biotin, a reagent hole 3 containing a streptavidin fluorescent substance marked with polysaccharide, and a cleaning hole 1, a cleaning hole 2, a cleaning hole 3, a cleaning hole 4, a cleaning hole 5, a cleaning hole 6, a cleaning hole 7, a cleaning hole 8 and a cleaning hole 9 which are reading holes;
s2: placing the calibrator in a reagent hole 1 of a kit, sequentially placing the calibrator in the reagent hole 1, a reagent hole 2 and a reagent hole 3 by using a quartz needle biosensor, reading after the first reaction is finished, and reading a first reading signal;
S3: after the quartz needle is put into the reagent box for cleaning, the quartz needle biosensor is continuously put into the reagent hole 2 and the reagent hole 3 in sequence, reading is carried out after the second reaction is completed, and a second reading signal is read;
s4: repeating the steps S2 and S3 for the calibration materials with different concentrations, and sequentially recording a first reading signal and a second reading signal of the calibration materials with each concentration;
s5: sequentially obtaining a high-concentration section signal standard curve and a low-concentration section signal standard curve according to the first reading signal and the second reading signal obtained in the step S4;
s6: detecting a signal value of a sample to be detected, and back-calculating the sample to be detected by utilizing a high-concentration section signal standard curve and/or a low-concentration section signal standard curve to obtain the concentration of the sample to be detected;
in the step S2 and the step S3, each reaction is carried out, before the quartz needle biosensor is placed in the next reagent hole, the quartz needle biosensor is placed in a cleaning hole for cleaning.
In some embodiments, the quartz needle is an aminopropyl modified Dan Yingzhen and the bottom surface of the quartz needle biosensor is baked after coating with capture antibodies.
The reaction time of the first reaction is smaller than that of the second reaction, and the rotating speed of the quartz needle biosensor in the first reaction is lower than that of the quartz needle biosensor in the second reaction.
In some embodiments, in step S4, "repeating steps S2 and S3 for different concentrations of calibrator" means that steps S2 and S3 are performed on a plurality of known different concentrations of human chorionic gonadotrophin β subunit antigen calibrator, respectively, having a concentration within the upper detection limit of the kit.
In some embodiments, step S5 of the method for detecting human chorionic gonadotrophin β subunit content further comprises: according to the first reading signal and the second reading signal of the calibration material with each concentration recorded in the step S4, a plurality of first reading signals are made into a high-concentration section signal standard curve, a plurality of second reading signals are made into a low-concentration section signal standard curve, and a decision point of the high-concentration section signal standard curve and the low-concentration section signal standard curve is set, wherein the decision point can select one concentration in a section with good separation degree in the two standard curves.
When the concentration value of the sample to be measured is located in the section with good separation degree in the two standard curves, the signal values of the first reading signal and the second reading signal are relatively close, and when the concentration value of the sample to be measured is located outside the section with good separation degree in the two standard curves, the difference between the signal values of the first reading signal and the second reading signal is obvious, so that the concentration value of the sample to be measured can be obviously distinguished to be higher than or lower than the concentration value of the decision point, the decision of the section with good separation degree in the two standard curves and the selection of the decision point can be adjusted according to actual requirements, and the details are omitted.
In one embodiment, the step S2 includes:
s2.1: placing a quartz needle biosensor into a sample in the reagent hole 1 to react for 10s-20s at a rotating speed of 1000rpm-1200rpm;
s2.2: washing the reacted biosensor in the washing hole 7 for 5s-10s at 1000rpm-1200rpm;
s2.3: placing the cleaned biosensor into a reagent hole 2 for reaction for 10s-20s, wherein the rotating speed is 1000rpm-1200rpm;
s2.4: washing the reacted biosensor in the washing hole 5 for 5s-10s at 1000rpm-1200rpm;
s2.5: placing the cleaned biosensor into a reagent hole 3 for reaction for 10s-20s, wherein the rotating speed is 1000rpm-1200rpm;
s2.6: and placing the circularly reacted biosensor into a cleaning hole 9 for reading, reading a first reading signal, determining each point signal of the calibrator for subsequent preparation of a high-concentration section signal standard curve, and back calculating the concentration of the sample to be measured on the standard curve to obtain the concentration in the range.
In one embodiment, the step S3 includes:
s3.1: placing the reacted biosensor into a cleaning hole 3, and cleaning the biosensor in the cleaning hole 4 for 5s-10s at a rotating speed of 1000rpm-1200rpm;
s3.2: placing the cleaned biosensor into a reagent hole 2 for reaction for 40s-80s, wherein the rotating speed is 1200rpm-1500rpm;
S3.3: washing the reacted biosensor in the washing hole 5 for 5s-10s at 1000rpm-1200rpm;
s3.4: placing the cleaned biosensor into a reagent hole 3 for reaction for 10s-20s, and rotating at 1000rpm-1200rpm;
s3.5: washing the reacted biosensor in the washing hole 1 and the washing hole 2 for 5s-10s at 1000rpm-1200rpm;
s3.6: and (3) placing the circularly reacted biosensor into a cleaning hole 9 for reading, reading a second reading signal, and determining each point signal of the calibrator for subsequent preparation of a low-concentration section signal standard curve, wherein the concentration of the sample to be measured in the range is calculated on the standard curve.
In some embodiments, the step S6 includes:
s6.1: placing a sample to be detected into a reagent hole 1 of a reagent kit, sequentially placing a quartz needle biosensor into the reagent hole 1, a reagent hole 2 and a reagent hole 3, reading after finishing the first reaction, and reading a first reading signal;
s6.2: after the quartz needle is put into the reagent box for cleaning, the quartz needle biosensor is continuously put into the reagent hole 2 and the reagent hole 3 in sequence, reading is carried out after the second reaction is completed, and a second reading signal is read;
s6.3: if the sample is lower than the concentration value of the resolution point, only reading the signal for the second time, and putting the sample on a low-concentration section signal standard curve for back calculation, and comparing to obtain a sample concentration value;
If the sample is higher than the concentration value of the resolution point, only the first reading signal can be read, the sample is put on a signal standard curve of a high concentration section for back calculation, and the concentration value of the sample is obtained after comparison;
if the sample is near the resolution point, the first reading signal and the second reading signal can be read at the same time, at the moment, the sample is respectively put on a low-concentration section signal standard curve and a high-concentration section signal standard curve for back calculation, two sample concentration values are obtained by comparison, and the average value of the two sample concentration values is calculated as the sample concentration value; wherein, two concentration value endpoints of the section with good separation in the two standard curves when the decision point is selected can be selected as the concentration critical value of the section located near the decision point.
The invention also discloses a kit for detecting the content of human chorionic gonadotrophin beta subunit, which in some embodiments comprises a quartz needle biosensor, a reagent well 1 for containing a sample, a reagent well 2 containing a biotin-labeled anti-human chorionic gonadotrophin beta subunit antibody, a reagent well 3 containing a polysaccharide-labeled streptavidin fluorescent substance, and a washing well 1, a washing well 2, a washing well 3, a washing well 4, a washing well 5, a washing well 6, a washing well 7, a washing well 8 and a washing well 9, wherein the washing well 9 is a reading well;
The bottom surface of the quartz needle biosensor is dried after being coated with a capture antibody, the capture antibody comprises an anti-FITC antibody and a fluorescein-labeled human chorionic gonadotrophin beta subunit antibody, wherein the concentration ratio of the anti-FITC antibody to the fluorescein-labeled human chorionic gonadotrophin beta subunit antibody is 2:3, a step of;
in the anti-human chorionic gonadotrophin beta subunit antibody labeled with biotin in the reagent well 2, the ratio of biotin to the anti-human chorionic gonadotrophin beta subunit antibody is (5-10): 1, and the biotin-labeled anti-human chorionic gonadotrophin beta subunit antibody is diluted to 1ug/mL with a preservation solution at room temperature;
the polysaccharide-labeled streptavidin fluorescent material in the reagent well 3 was diluted to 5ug/mL with a preservative solution at room temperature.
In a preferred embodiment, the preparation of the quartz needle biosensor in the kit comprises the following steps:
preparing 30ug/mL of anti-FITC antibody and 20ug/mL of fluorescein labeled human chorionic gonadotrophin beta subunit antibody;
APS modified Dan Yingzhen is placed in 30ug/mL of anti-FITC antibody solution to react for 800s-1000s at a speed of 400rpm-600rpm, then placed in a blocking solution to block for 50s-80s at a speed of 400rpm-600rpm, then placed in 10mM PBST solution to wash for 20s at a speed of 400rpm-600rpm; then placing the mixture into 20ug/ml of fluorescein-labeled human chorionic gonadotrophin beta subunit antibody solution to react for 150s-200s at a rotating speed of 400rpm-600rpm; finally placing the mixture in a sealing solution for sealing for 10 seconds at a rotating speed of 400rpm-600rpm;
Drying the coated quartz needle at 40 ℃ for 5 hours to prepare a quartz needle biosensor, and storing the quartz needle biosensor in a dry environment for later use;
in this example, the 30ug/mL anti-FITC antibody was diluted with PBS buffer, and the 20ug/mL fluorescein-labeled human chorionic gonadotrophin beta subunit antibody was diluted with a diluent comprising 3.58g/L disodium hydrogen phosphate dodecahydrate, 0.27g potassium dihydrogen phosphate, 9g/L sodium chloride, 0.2g potassium chloride, 0.05% Tween-20, 0.5% BSA, 1mg/mL Ficoll-400, 0.05% proclin 300.
In this example, in the anti-human chorionic gonadotrophin β subunit antibodies labeled with biotin in the reagent well 2 of the kit, the ratio of biotin to anti-human chorionic gonadotrophin β subunit antibodies is 8:1, a step of;
in this embodiment, the polysaccharide-labeled streptavidin fluorescent material is preferably labeled with Ficoll-400 polysucrose, and the streptavidin fluorescent material is Cy5.
In this example, the wash solution in each wash well of the kit was a PBST wash solution having a concentration of 10mM and a pH of 7.4.
In this example, the preservation solution is preferably a phosphate preservation solution having a concentration of 10 mM.
The following performance tests were performed on the human chorionic gonadotrophin beta subunit content detection kit in the preferred embodiment described above:
minimum detection limit:
human chorionic gonadotrophin beta subunit blank buffers (n=20) were tested and calculated separately(blank mean) and S (standard deviation), statistics->"concentration values, the results of which are shown in table 1 below:
table 1:
conclusion: the lowest detection limit is less than or equal to 1IU/L.
Linear range:
samples of different concentrations were mixed in proportion to 8 concentrations, each concentration point was tested 4 times (4=3), and the mean value (Yi) of the test results was calculated separately. At diluted concentration (Xi)Independent variables, a linear regression equation is obtained by taking the mean value (Yi) of the detection results as a dependent variable, and a linear regression correlation coefficient (r) 2 ) The test results and linear ranges are shown in table 2 and fig. 1 below:
table 2:
as can be seen from the above test results and fig. 1: in the range of 1IU/L to 300000IU/L, r 2 1, and no outlier, so that in the range of 1IU/L to 300000IU/L, the accuracy and consistency of the test results can be ensured, in this embodiment, 300000IU/L is set as the upper detection limit, and in other embodiments, other upper detection limits can be set according to the difference in the requirement for standard curve consistency.
Precision:
low, medium and high value samples (30 IU/L, 300IU/L, 5000 IU/L) were tested, each concentration was repeated 20 times (n=20), coefficient of Variation (CV) was calculated,
wherein, each concentration sample and the detection result are shown in the following table 3:
table 3:
wherein, CV of low concentration level is 7.5%, CV of medium concentration level is 2.9%, CV of high concentration level is 5.6%.
Accuracy:
the high and low concentrations (500 IU/L and 5000 IU/L) of the reference were each tested 3 times (n=3), the test results were noted as Xi, and the relative deviations (Bi), bi= (Xi-T)/t×100% were calculated according to the formula, respectively. The test results are shown in table 4 below:
table 4:
the accuracy deviation of the invention is less than or equal to + -10 percent
HOOK effect:
the detection limit (300000 IU/L) of human chorionic gonadotrophin beta subunits, and samples (600000 IU/L, 1200000IU/L, 1800000 IU/L) 2 times, 4 times and 6 times of the detection limit are detected, and whether the detection signal value is more than 1000pg/mL is verified. The test results are shown in table 5 below:
table 5:
| concentration of | 300000IU/L | 600000IU/L | 1200000IU/L | 1800000IU/L |
| Signal value | 24574 | 33209 | 40730 | 40177 |
Referring to Table 5 above in combination with FIG. 2, the results show that 600000IU/L, 1200000IU/L, 1800000IU/L, and detection results are all >300000IU/L, and no HD-HOOK effect is observed. With the increase of human chorionic gonadotrophin beta subunit antigen concentration, the signal value of the reaction tends to be gentle, but the signal value does not certainly have a descending trend because the washing process is added in each step of reaction of the platform.
Specificity:
other glycoprotein hormones in human samples produce cross-major indicators: follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone,
the concentration-crossing substances described in Table 6 below were measured as samples, and the signal value deviation thereof was measured to be within.+ -. 10%.
Table 6:
in conclusion, the human chorionic gonadotrophin beta subunit content detection kit has the advantages of high detection signal level and high detection sensitivity.
In accordance with the above method for detecting the content of beta subunits of human chorionic gonadotrophin, a preferred embodiment is further provided, comprising the steps of:
s1: preparing a human chorionic gonadotrophin beta subunit content detection kit;
in this example, the above-mentioned preferred embodiment of the kit for detecting the content of human chorionic gonadotrophin beta subunit is used, and it is known that the upper limit of detection of the above-mentioned kit is set to 300000IU/L;
s2: placing the calibrator in a reagent hole 1 of a kit, sequentially placing the calibrator in the reagent hole 1, a reagent hole 2 and a reagent hole 3 by using a quartz needle biosensor, reading after the first reaction is finished, and reading a first reading signal;
in this embodiment, the step S2 includes:
S2.1: placing a quartz needle biosensor into a sample in the reagent hole 1 to react for 15 seconds at a rotating speed of 1200rpm;
s2.2: washing the reacted biosensor in the washing hole 7 and the washing hole 8 for 7s at 1200rpm;
s2.3: placing the cleaned biosensor into a reagent hole 2 for reaction for 15s at a rotating speed of 1200rpm;
s2.4: washing the reacted biosensor in the washing hole 5 and the washing hole 6 for 7s at 1200rpm;
s2.5: placing the cleaned biosensor into a reagent hole 3 for reaction for 15s at a rotating speed of 1200rpm;
s2.6: placing the circularly reacted biosensor into a cleaning hole 9 for reading, reading a first reading signal, determining each point signal of a calibrator for subsequent preparation of a low-concentration section signal standard curve, and back calculating a sample to be measured on the standard curve to obtain the concentration in the range;
s3: after the quartz needle is put into the reagent box for cleaning, the quartz needle biosensor is continuously put into the reagent hole 2 and the reagent hole 3 in sequence, reading is carried out after the second reaction is completed, and a second reading signal is read;
in this embodiment, the step S3 includes:
s3.1: placing the reacted biosensor into a cleaning hole 3, and cleaning the biosensor in the cleaning hole 4 for 7s at a rotating speed of 1200rpm;
s3.2: placing the cleaned biosensor into the reagent hole 2 for reaction for 60s at 1200rpm;
S3.3: washing the reacted biosensor in the washing hole 5 and the washing hole 6 for 7s at 1200rpm;
s3.4: placing the cleaned biosensor into a reagent hole 3 for reaction for 15s at a rotating speed of 1200rpm;
s3.5: washing the reacted biosensor in the washing hole 1 and the washing hole 2 for 7s at 1200rpm;
s3.6: placing the circularly reacted biosensor into a cleaning hole 9 for reading, reading a second reading signal, determining each point signal of the calibrator for subsequent preparation of a low-concentration section signal standard curve, and back calculating a sample to be measured on the standard curve to obtain the concentration in the range;
s4: repeating the steps S2 and S3 for the calibration materials with different concentrations, and sequentially recording a first reading signal and a second reading signal of the calibration materials with each concentration;
in this example, steps S2 and S3 were performed on 12 different concentrations of human chorionic gonadotrophin β subunit antigen calibrator within the upper detection limit of the kit, and the first read signal and the second signal were recorded for 12 calibrator, respectively, wherein 12 calibrator comprises calibrator 1 (Cal 1) having human chorionic gonadotrophin β subunit antigen concentration of 300000IU/L, calibrator 2 (Cal 2) having 100000IU/L, calibrator 3 (Cal 3) having 33333IU/L, calibrator 4 (Cal 4) having 11111IU/L, calibrator 5 (Cal 5) having 3704IU/L, calibrator 6 (Cal 6) having 1235IU/L, calibrator 7 (Cal 1) having 412IU/L, calibrator 7 (Cal 7) having 137IU/L, calibrator 8 (Cal 8) having 46IU/L, calibrator 9 (Cal 9) having 15IU/L, calibrator 10 (Cal 10) having 5IU/L, calibrator 12 (Cal 10) having 5IU/L, and calibrator 12 having the following table of test results shown in table 12 (Cal 7):
Table 7:
s5: sequentially obtaining a high-concentration section signal standard curve and a low-concentration section signal standard curve according to the first reading signal and the second reading signal obtained in the step S4;
in this embodiment, the step S5 includes: and (3) according to the first reading signal and the second reading signal of the calibrator with each concentration recorded in the step S4, and preparing a plurality of first reading signals into a high-concentration section signal standard curve, wherein the high-concentration section signal standard curve and the low-concentration section signal standard curve are shown in fig. 3.
The second reading signals are made into a low-concentration section signal standard curve, one concentration in a section with good separation degree in the two standard curves is selected to be set as a decision point, in the embodiment, when the concentration value in the two standard curves is 412IU/L-1235IU/L, the separation degree is better, therefore, any concentration value can be selected between 412IU/L-1235IU/L to be used as the decision point, and in the embodiment, the decision point is set to be 1000IU/L;
s6: detecting a signal value of a sample to be detected, and back-calculating the sample to be detected by utilizing a high-concentration section signal standard curve and/or a low-concentration section signal standard curve to obtain the concentration of the sample to be detected;
In this embodiment, the step S6 includes:
s6.1: placing a sample to be detected into a reagent hole 1 of a reagent kit, sequentially placing a quartz needle biosensor into the reagent hole 1, a reagent hole 2 and a reagent hole 3, reading after finishing the first reaction, and reading a first reading signal;
in this embodiment, the step S6.1 includes:
s6.1.1: placing a quartz needle biosensor into a sample in the reagent hole 1 to react for 15 seconds at a rotating speed of 1200rpm;
s6.1.2: washing the reacted biosensor in the washing hole 7 and the washing hole 8 for 7s at 1200rpm;
s6.1.3: placing the cleaned biosensor into a reagent hole 2 for reaction for 15s at a rotating speed of 1200rpm;
s6.1.4: washing the reacted biosensor in the washing hole 5 and the washing hole 6 for 7s at 1200rpm;
s6.1.5: placing the cleaned biosensor into a reagent hole 3 for reaction for 15s at a rotating speed of 1200rpm;
s6.1.6: placing the circularly reacted biosensor into a cleaning hole 9 for reading, reading a first reading signal, and using the first reading signal of each measured calibrator for the subsequent preparation of a low-concentration section signal standard curve;
s6.2: after the quartz needle is put into the reagent box for cleaning, the quartz needle biosensor is continuously put into the reagent hole 2 and the reagent hole 3 in sequence, reading is carried out after the second reaction is completed, and a second reading signal is read;
In this embodiment, the step S6.2 includes:
s6.2.1: placing the reacted biosensor into a cleaning hole 3, and cleaning the biosensor in the cleaning hole 4 for 7s at a rotating speed of 1200rpm;
s6.2.2: placing the cleaned biosensor into the reagent hole 2 for reaction for 60s at 1200rpm;
s6.2.3: washing the reacted biosensor in the washing hole 5 and the washing hole 6 for 7s at 1200rpm;
s6.2.4: placing the cleaned biosensor into a reagent hole 3 for reaction for 15s at a rotating speed of 1200rpm;
s6.2.5: washing the reacted biosensor in the washing hole 1 and the washing hole 2 for 7s at 1200rpm;
s6.2.6: placing the circularly reacted biosensor into a cleaning hole 9 for reading, reading a second reading signal, determining each point signal of the calibrator for subsequent preparation of a low-concentration section signal standard curve, and back calculating a sample to be measured on the standard curve to obtain the concentration in the range;
s6.3: if the sample is lower than the concentration value of the resolution point, only reading the signal for the second time, and putting the sample on a low-concentration section signal standard curve for back calculation, and comparing to obtain a sample concentration value;
because the concentration value of the resolution point in the embodiment is 1000IU/L, a sample with the concentration value set between 1IU/L and 1000IU/L is put into a low-concentration section signal standard curve for back calculation;
If the sample is higher than the concentration value of the resolution point, only the first reading signal can be read, the sample is put on a signal standard curve of a high concentration section for back calculation, and the concentration value of the sample is obtained after comparison;
because the concentration value of the resolution point in the embodiment is 1000IU/L, the samples with the concentration value between 1000IU/L and 300000IU/L are put into a high-concentration section signal standard curve for back calculation;
if the sample is near the resolution point, the first reading signal and the second reading signal can be read at the same time, at the moment, the sample is respectively put into a low-concentration section signal standard curve and a high-concentration section signal standard curve to be calculated back, two sample concentration values are obtained by comparison, and an average value of the two sample concentration values is calculated to be used as the sample concentration value, wherein two concentration value endpoints of a section with good separation degree in the two standard curves can be selected as the concentration critical value of the section near the resolution point when the resolution point is selected;
in this embodiment, the separation degree is better when the concentration value is 412IU/L-1235IU/L in the two standard curves, and at this time, the difference between the first reading signal and the second reading signal is not obvious, so that when the concentration of the sample to be measured is between 412IU/L-1235IU/L, the first reading signal and the second reading signal can be read at the same time, at this time, the sample is placed on the low concentration section signal standard curve and the high concentration section signal standard curve respectively, the two sample concentration values are obtained by comparison, and the average value of the two sample concentration values is calculated as the sample concentration value.
The invention has various embodiments, and all technical schemes formed by equivalent transformation or equivalent transformation fall within the protection scope of the invention.
Claims (10)
1. The method for detecting the content of human chorionic gonadotrophin beta subunit is characterized by comprising the following steps of: the method comprises the following steps:
s1: preparing a human chorionic gonadotrophin beta subunit content detection kit;
the kit comprises a quartz needle biosensor, a reagent hole 1 for containing a sample, a reagent hole 2 containing an anti-human chorionic gonadotrophin beta subunit antibody marked with biotin, a reagent hole 3 containing a streptavidin fluorescent substance marked with polysaccharide, and a cleaning hole 1, a cleaning hole 2, a cleaning hole 3, a cleaning hole 4, a cleaning hole 5, a cleaning hole 6, a cleaning hole 7, a cleaning hole 8 and a cleaning hole 9 which are reading holes;
s2: placing the calibrator in a reagent hole 1 of a kit, sequentially placing the calibrator in the reagent hole 1, a reagent hole 2 and a reagent hole 3 by using a quartz needle biosensor, reading after the first reaction is finished, and reading a first reading signal;
s3: after the quartz needle is put into the reagent box for cleaning, the quartz needle biosensor is continuously put into the reagent hole 2 and the reagent hole 3 in sequence, reading is carried out after the second reaction is completed, and a second reading signal is read;
S4: repeating the steps S2 and S3 for the calibration materials with different concentrations, and sequentially recording a first reading signal and a second reading signal of the calibration materials with each concentration;
s5: sequentially obtaining a high-concentration section signal standard curve and a low-concentration section signal standard curve according to the first reading signal and the second reading signal obtained in the step S4;
s6: detecting a signal value of a sample to be detected, and back-calculating the sample to be detected by utilizing a high-concentration section signal standard curve and/or a low-concentration section signal standard curve to obtain the concentration of the sample to be detected;
in the step S2 and the step S3, each reaction is carried out, before the quartz needle biosensor is placed in the next reagent hole, the quartz needle biosensor is placed in a cleaning hole for cleaning.
2. The method for detecting the content of beta subunits of human chorionic gonadotrophin according to claim 1, wherein: the quartz needle is Dan Yingzhen modified by aminopropyl, and the bottom surface of the quartz needle biosensor is dried after being coated with the capture antibody.
3. The method for detecting the content of beta subunits of human chorionic gonadotrophin according to claim 1, wherein: the reaction time of the first reaction is less than the reaction time of the second reaction.
4. The method for detecting the content of beta subunits of human chorionic gonadotrophin according to claim 1, wherein: the rotation speed of the quartz needle biosensor in the first reaction is lower than that in the second reaction.
5. The method for detecting the content of beta subunits of human chorionic gonadotrophin according to claim 1, wherein: in step S4, "repeating steps S2 and S3 for different concentrations of calibrator" means that steps S2 and S3 are performed on a plurality of known different concentrations of human chorionic gonadotrophin β subunit antigen calibrator, respectively, having a concentration within the upper detection limit of the kit.
6. The method for detecting the content of beta subunits of human chorionic gonadotrophin according to claim 5, wherein: the step S5 further includes: and (3) according to the first reading signal and the second reading signal of the calibrator with each concentration recorded in the step S4, preparing a plurality of first reading signals into a high-concentration section signal standard curve, preparing a plurality of second reading signals into a low-concentration section signal standard curve, and setting decision points of the high-concentration section signal standard curve and the low-concentration section signal standard curve.
7. The method for detecting the content of beta subunits of human chorionic gonadotrophin according to claim 1, wherein: the step S2 includes:
s2.1: placing a quartz needle biosensor into a sample in the reagent hole 1 to react for 10s-20s at a rotating speed of 1000rpm-1200rpm;
s2.2: washing the reacted biosensor in the washing hole 7 for 5s-10s at 1000rpm-1200rpm;
s2.3: placing the cleaned biosensor into a reagent hole 2 for reaction for 10s-20s, wherein the rotating speed is 1000rpm-1200rpm;
s2.4: washing the reacted biosensor in the washing hole 5 for 5s-10s at 1000rpm-1200rpm;
s2.5: placing the cleaned biosensor into a reagent hole 3 for reaction for 10s-20s, wherein the rotating speed is 1000rpm-1200rpm;
s2.6: the circularly reacted biosensor is put into the washing hole 9 for reading, and the first reading signal is read.
8. The method for detecting the content of beta subunits of human chorionic gonadotrophin according to claim 1, wherein: the step S3 includes:
s3.1: placing the reacted biosensor into a cleaning hole 3, and cleaning the biosensor in the cleaning hole 4 for 5s-10s at a rotating speed of 1000rpm-1200rpm;
s3.2: placing the cleaned biosensor into a reagent hole 2 for reaction for 40s-80s, wherein the rotating speed is 1200rpm-1500rpm;
S3.3: washing the reacted biosensor in the washing hole 5 for 5s-10s at 1000rpm-1200rpm;
s3.4: placing the cleaned biosensor into a reagent hole 3 for reaction for 10s-20s, and rotating at 1000rpm-1200rpm;
s3.5: washing the reacted biosensor in the washing hole 1 and the washing hole 2 for 5s-10s at 1000rpm-1200rpm;
s3.6: the circularly reacted biosensor is put into the washing hole 9 for reading, and the second reading signal is read.
9. The method for detecting the content of beta subunit of human chorionic gonadotrophin according to claim 6, wherein: the step S6 includes:
s6.1: placing a sample to be detected into a reagent hole 1 of a reagent kit, sequentially placing a quartz needle biosensor into the reagent hole 1, a reagent hole 2 and a reagent hole 3, reading after finishing the first reaction, and reading a first reading signal;
s6.2: after the quartz needle is put into the reagent box for cleaning, the quartz needle biosensor is continuously put into the reagent hole 2 and the reagent hole 3 in sequence, reading is carried out after the second reaction is completed, and a second reading signal is read;
s6.3: if the sample is lower than the concentration value of the resolution point, only reading the signal for the second time, and putting the sample on a low-concentration section signal standard curve for back calculation, and comparing to obtain a sample concentration value;
If the sample is higher than the concentration value of the resolution point, only the first reading signal can be read, the sample is put on a signal standard curve of a high concentration section for back calculation, and the concentration value of the sample is obtained after comparison;
if the sample is near the resolution point, the first reading signal and the second reading signal can be read at the same time, and the sample is put into the low-concentration section signal standard curve and the high-concentration section signal standard curve to be calculated back, two sample concentration values are obtained by comparison, and the average value of the two sample concentration values is calculated to be used as the sample concentration value.
10. The human chorionic gonadotrophin beta subunit content detection kit is characterized in that: comprises a quartz needle biosensor, a reagent hole 1 for containing a sample, a reagent hole 2 for containing an anti-human chorionic gonadotrophin beta subunit antibody marked with biotin, a reagent hole 3 containing a streptavidin fluorescent substance marked with polysaccharide, and a cleaning hole 1, a cleaning hole 2, a cleaning hole 3, a cleaning hole 4, a cleaning hole 5, a cleaning hole 6, a cleaning hole 7, a cleaning hole 8 and a cleaning hole 9 which are reading holes;
the bottom surface of the quartz needle biosensor is dried after being coated with a capture antibody, the capture antibody comprises an anti-FITC antibody and a fluorescein-labeled human chorionic gonadotrophin beta subunit antibody, wherein the concentration ratio of the anti-FITC antibody to the fluorescein-labeled human chorionic gonadotrophin beta subunit antibody is 2:3, a step of;
In the anti-human chorionic gonadotrophin beta subunit antibody labeled with biotin in the reagent well 2, the ratio of biotin to the anti-human chorionic gonadotrophin beta subunit antibody is (5-10): 1, and the biotin-labeled anti-human chorionic gonadotrophin beta subunit antibody is diluted to 1ug/mL with a preservation solution at room temperature;
the polysaccharide-labeled streptavidin fluorescent material in the reagent well 3 was diluted to 5ug/mL with a preservative solution at room temperature.
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