CN111721938B - Sugar chain antigen 125 latex immunoturbidimetry kit - Google Patents

Sugar chain antigen 125 latex immunoturbidimetry kit Download PDF

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CN111721938B
CN111721938B CN202010237171.1A CN202010237171A CN111721938B CN 111721938 B CN111721938 B CN 111721938B CN 202010237171 A CN202010237171 A CN 202010237171A CN 111721938 B CN111721938 B CN 111721938B
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antibody
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content
calibrator
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CN111721938A (en
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李晓舟
果玮
刘瑶
刘希
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Beijing Strong Biotechnologies Inc
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Abstract

The application relates to a carbohydrate chain antigen 125 latex immunoturbidimetric kit. Specifically, the application relates to a latex immunoturbidimetry kit for detecting the content of CA125 antigen, which comprises a first reagent, a second reagent and an optional calibrator. The first reagent comprises a buffer, and the second reagent comprises polystyrene latex particles, two specific strains of CA125 antibodies, and a buffer. The kit can accurately detect CA125 in the range of <800U/mL, and has good linearity and reliable accuracy. Can be applied to the detection of CA125 antigen in clinic.

Description

Sugar chain antigen 125 latex immunoturbidimetry kit
The present application claims priority from chinese patent application 201910246941.6, filed on 29/3/2019.
Technical Field
The present application relates to the field of clinical testing. In particular to a reagent for quantitatively detecting sugar chain antigen 125 (CA 125 for short).
Background
With the advent of monoclonal technology, a large number of tumor markers emerged. The sugar chain antigen 125 (CA 125) is an ovarian cancer cell surface tumor-associated antigen discovered in 1981, and is present in coelomic epithelial cells during embryonic development. CA125 disappears after birth, but reappears in ovarian cancer cells and enters the systemic circulation. CA125 belongs to macromolecular protein complex, and the molecular weight is between 200 and 1000 KD. The CA125 antigen contains a large number of repeats. Each of the repeated sequences contains highly conserved amino acid sequences.
There are studies showing that about 80% of epithelial ovarian cancer patients have elevated levels of CA125 in their serum. In addition to ovarian cancer, certain benign gynecological diseases cause elevated CA125 detection, such as ovarian cysts, ovarian metaplasia, endometrial displacement, uterine fibroids, and cervicitis. CA125 is slightly elevated during the early pregnancy and in some benign diseases (such as acute and chronic pancreatitis, benign gastrointestinal diseases, renal failure, autoimmune diseases, etc.). Liver diseases (e.g., cirrhosis, hepatitis) cause moderate elevation of CA 125. Ascites CA125 caused by various malignant and benign diseases can be increased sharply.
The detection method of the kit for determining the content of CA125 appearing in the market mainly comprises the following steps: immunofluorescence, radioimmunoassay, enzyme-linked immunosorbent assay and chemiluminescence assay. Currently, enzyme-linked immunosorbent assay and chemiluminescence assay are more applied. The main disadvantages of the detection by enzyme-linked immunosorbent assay are that the detection process needs manual operation, the time consumption is long, and the result reproducibility is poor. The chemiluminescence method has good detection accuracy and repeatability, but has high detection cost.
According to the existing report, CA125 is determined by chemiluminescence, and detection is carried out by using two epitopes of OC125 and M11. The inventors have found that the use of such combinations of epitopes in latex turbidimetric methods results in more false positive results. Therefore, there is still a need to provide an improved kit for detecting the CA125 content in a human sample.
Disclosure of Invention
According to some embodiments of the present application, there is provided a quantification reagent for CA 125.
According to some embodiments of the present application, there is provided an assay kit comprising a first reagent, a second reagent, and optionally a calibrator;
wherein the first reagent comprises:
Figure BDA0002431377950000021
the second reagent comprises:
Figure BDA0002431377950000022
the calibrator comprises:
Figure BDA0002431377950000023
in some embodiments, the buffer is selected from: hepes buffer solution, glycine buffer solution, PBS buffer solution, boric acid buffer solution and acetic acid buffer solution.
In some embodiments, the latex particles have an average particle size of 100nm to 500nm.
In some specific embodiments, the latex particles have an average particle size of 100, 150, 200, 250, 300, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500nm.
In some embodiments, the carboxyl content is from 20 to 200 μmol/g relative to the mass of the latex particles.
In some specific embodiments, the carboxyl group content is from 30 to 180. Mu. Mol/g, relative to the mass of the latex particles.
In some embodiments, the CA125 antibody is derived from: mouse, rabbit, sheep, fowl and camel.
In some specific embodiments, the CA125 antibody is a monoclonal antibody.
In some specific embodiments, the concentration of CA125 antigen in the calibrator is 0U/mL, 50U/mL, 100U/mL, 200U/mL, 400U/mL, 800U/mL.
In some embodiments, the stabilizing agent is selected from: bovine serum albumin, ovalbumin.
According to some embodiments of the present application, the latex particle-coated CA125 monoclonal antibody is two different antibodies, each recognizing a different CA125 epitope.
In some specific embodiments, the monoclonal antibody comprises a first CA125 antibody and a second CA125 antibody, wherein the first CA125 antibody targets an OC125 epitope; and the second CA125 antibody does not target the M11 epitope.
In some specific embodiments, the first CA125 antibody targets an OC125 epitope; and the second CA125 antibody targets the OV197 epitope.
According to some embodiments of the application, the first reagent comprises:
Figure BDA0002431377950000031
the second reagent comprises:
Figure BDA0002431377950000032
optionally, the calibrator comprises:
Figure BDA0002431377950000041
wherein the CA125 antibody is a monoclonal antibody;
the particle size range of the polystyrene latex particles is 350nm.
According to some embodiments of the present application, the carboxyl group content is 30 to 180 μmol/g with respect to the mass of the polystyrene latex particles.
According to some embodiments of the application, the concentration of the CA125 antigen in the calibrator is 0U/mL, 50U/mL, 100U/mL, 200U/mL, 400U/mL, 800U/mL.
The application also provides the application of the monoclonal antibody combination in preparing a detection device.
According to some embodiments of the present application, the combination of monoclonal antibodies comprises a first CA125 antibody and a second CA125 antibody; the first CA125 antibody targets the OC125 epitope; and the second CA125 antibody does not target the M11 epitope.
In a specific embodiment, the detection device is a kit for determining the CA125 content or level.
In specific embodiments, the first CA125 antibody targets an OC125 epitope; and the second CA125 antibody targets the OV197 epitope.
Drawings
FIG. 1: results of linear experiments with the kit of the present application.
FIG. 2: the kit of the application and a commercial chemiluminescence kit have correlation results.
Detailed Description
Optimization example influence of combination of antibody epitopes on false positives
On the ISOBM TD-1Workshop platform, the epitope of CA125 is known to include B27.1, B43.13, K90, K91, K93, K94, K95, K96, K97, K100, K101, K102, OV185, OV197, OV198, M11, MA602-1, MA602-6, OC125, ZS33, ZR38, ZR45. However, the inventors have unexpectedly found that antibodies directed against the M11 epitope are the main cause of false positive results when performing antibody pairing tests.
The specific test method comprises the following steps: three antibodies directed against different epitopes were designated antibody M11, antibody OC125 and antibody OV197, respectively. The three antibodies are combined in pairs, and the detection results of the samples are compared, so that the two combinations containing the antibody M11 have obvious high sample detection values.
TABLE 1 false Positive test
Figure BDA0002431377950000051
Examples
EXAMPLE 1 preparation of the first reagent
1. The first reagent (1) was formulated with the following composition:
Figure BDA0002431377950000052
2. alternatively, the first reagent (2) is formulated according to the following composition:
Figure BDA0002431377950000061
EXAMPLE 2 preparation of latex particles
1. The first method comprises the following steps:
1. providing polystyrene latex particles:
taking latex particles with the average particle size of 350nm, adding the latex particles into 135mL Hepes pH6.5 buffer solution to obtain particle suspension with the latex particle concentration of 2.0% in terms of w/v;
2. providing a cross-linking agent:
dissolving 30mg of carbodiimide in 8mL of deionized water to prepare a carbodiimide aqueous solution for later use;
3. activated polystyrene latex particles:
adding a carbodiimide solution into the particle suspension, and reacting and activating at 45 ℃ for 0.5h to obtain activated particles;
4. providing an antibody:
100mg of CA125 antibody (two antibodies are coated separately) is added into 100mL Hepes pH8.5 buffer solution, so that the final concentration of the antibody is 1.00mg/mL;
5. and (3) crosslinking reaction:
mixing the activated particles with a CA125 antibody solution, and uniformly mixing for 2 hours at 37 ℃ in a shaking table;
6. and (3) sealing:
adding 80mL of blocking solution (containing 2% w/v Tween 20, 2% w/v BSA, glycine buffer, pH 7.4) and reacting at room temperature (20-25 deg.C) for 3h;
7. and (4) terminating:
centrifugation at 14000rpm for 40 minutes at 4 ℃;
8. cleaning:
the particles were resuspended in 1000mL Hepes pH7.5 buffer and washed by centrifugation.
2. The second method comprises the following steps:
according to the first method, the only difference is that the final antibody concentration of step 4 is 3.00mg/mL.
EXAMPLE 3 preparation of the second reagent
1. Adding the granules prepared in the first method of example 2 into the solution (containing 0.5% w/v BSA, 0.5% 3 50mM glycine buffer pH 7.5) and obtaining the second reagent (1) after ultrasonic resuspension. Wherein: antibody-coated gelMilk particle concentration equivalent to 0.2% w/v; the concentration of antibody corresponds to 100. Mu.g/mL.
2. The particles prepared in the second method of example 2 were added to the solution (containing 2.5% w/v BSA, 2.5% w/v NaN) 3 500mM glycine buffer pH 7.5) to obtain the second reagent (2) after ultrasonic resuspension. Wherein: the concentration of antibody-coated latex particles corresponds to 0.2% w/v; the concentration of antibody corresponds to 300. Mu.g/mL.
EXAMPLE 4 preparation of calibrator
1. Calibrator (1) was prepared according to the following composition:
Figure BDA0002431377950000071
2. alternatively, calibrator (2) was formulated as follows:
Figure BDA0002431377950000072
example 5 preparation of kits of the present application
Kit a was assembled from the first reagent (1) of example 1, the second reagent (1) of example 3, and the calibrator (1) prepared in example 4.
Kit B was assembled from the first reagent (2) of example 1, the second reagent (2) of example 3, and the calibrator (2) prepared in example 4.
Test example
Test example 1 Linear Range test
One high value serum sample was diluted with deionized water at a ratio of 1/40, 1/20, 1/10, 2/10, 3/10 \8230; 10/10, and the measurement was repeated twice for each concentration using kit A, and the linearity was examined by calculating the correlation coefficient (see Table 2, FIG. 1). The results show that the regression equation is:
y＝794.21x+4.6995，R 2 ＝0.9991。
TABLE 2 Linear results for kit A
Measured value Theoretical value Relative deviation of
1 794.6 768.9 3.34％
0.9 719.2 692.5 3.85％
0.8 635.2 616.1 3.11％
0.7 560.3 539.6 3.82％
0.6 504.0 463.2 8.80％
0.5 401.4 386.8 3.76％
0.4 314.5 310.4 1.33％
0.3 242.5 234.0 3.63％
0.2 154.3 157.5 -2.06％
0.1 89.3 81.1 10.08％
0.05 43.4 42.9 1.14％
0.025 25.6 23.8 7.54％
Test example 2 correlation test
More than 50 samples (not less than 20 abnormal samples) are detected by using the kit A and the Roche electrochemiluminescence CA125 detection kit, and the detection results of the two kits are recorded (see table 3 and figure 2).
And calculating the correlation coefficient of the experimental result by using a linear regression method. The correlation equation is y =0.9541x +2.6957 2 =0.9844. The result shows that the kit has accurate measured value and good correlation with the luminescent reagent, and can be used for detecting the clinical CA 125.
TABLE 3 correlation test
Figure BDA0002431377950000081
Figure BDA0002431377950000091

Claims (5)

1. A kit for determining CA125 content or level, comprising:
a first reagent for the first time and a second reagent for the second time,
a second reagent for the second reagent, wherein the second reagent is a reagent for the second reagent,
wherein,
the first reagent comprises:
Figure FDA0003811832300000011
the second reagent comprises:
Figure FDA0003811832300000012
the CA125 antibody is derived from: mouse, rabbit, sheep, fowl, camel;
the CA125 antibody is a monoclonal antibody;
the CA125 antibody comprises a first CA125 antibody and a second CA125 antibody;
the first CA125 antibody targets an OC125 epitope; and the second CA125 antibody targets the OV197 epitope;
the polystyrene latex particles have an average particle size in the range of 250nm to 400nm;
the carboxyl group content is 30 to 180. Mu. Mol/g relative to the mass of the polystyrene latex particles.
2. The kit for determining the content or level of CA125 according to claim 1, further comprising a calibrator and/or a quality control.
3. The kit of claim 2 for determining the CA125 content or level, the calibrator comprising:
Figure FDA0003811832300000021
the buffer is selected from one or a combination of the following: hepes buffer solution, glycine buffer solution, PBS buffer solution, boric acid buffer solution and acetic acid buffer solution;
the stabilizer is selected from: bovine serum albumin, ovalbumin.
4. The kit of claim 3 for determining the CA125 content or level, the calibrator comprising:
Figure FDA0003811832300000022
5. the kit of claim 4 for determining the content or level of CA125, wherein the concentration of CA125 in the calibrator is 0U/mL, 50U/mL, 100U/mL, 200U/mL, 400U/mL, 800U/mL.
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