CN116593720A - Calibration quality control product for antigen monomer detection and application thereof - Google Patents
Calibration quality control product for antigen monomer detection and application thereof Download PDFInfo
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Classifications
-
- 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
-
- 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
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
-
- 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
- G01N2333/575—Hormones
- G01N2333/5756—Prolactin
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of biology, and particularly relates to a calibration quality control product for antigen monomer detection and application thereof. The invention uses the multi-state antigen as a calibration quality control product, and simply realizes the stable quality control of monomer detection in the composite antigen in a natural state. And by utilizing the two-time linear classification of the interaction of the specific proteins, the distinction of the monomer and the polymer is achieved, the interference can be better eliminated, and the false positive result is avoided. The invention has important significance for the immunodetection of the monomer in the composite antigen in a natural state.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a calibration quality control product for antigen monomer detection.
Background
Detection methods based on immune reactions (antibody-antigen interactions) in clinical assays are widely used in the qualitative/quantitative detection of biomarkers of the chemical nature of proteins or small molecule organics. Such as antibody latex turbidimetry, enzyme-linked immunity, chemiluminescence, colloidal gold color development, and the like. This broad class of detection methods, when reading single measurement information (turbidity, light value, absorbance, fluorescence intensity, etc.), is essentially a linear classification based on specific protein interactions. The object to be measured represented by protein molecules has multi-dimensional molecular characteristics, all the characteristics of the object to be measured cannot be read from a classification set of the object to be measured by one-dimensional information, and confusion caused by characteristic loss is unavoidable in the classification set.
The methodological limitation described above is also the combined measurement of a mixture of protein molecules of different polymerization states or complexes. For example, FDP project is measured for fibrinogen degradation products, which are composed of fragments such as X, Y, D, and D-dimer and gamma-dimer, fragments A, B, C, H, complex I, complex II and complex III, etc. [ http:// dx.doi.org/10.1016/j.str.2016.08.009]. The FDP project is intended to achieve quantification of FDP mixtures; whereas the D-dimer project achieves quantification of a subclass thereof by selecting epitopes with higher D-dimer specificity. Free prostate specific antigen (fPSA) and total prostate specific antigen (tPSA), as the name implies, the subjects have intersections. Similar examples are not enumerated in medical immunodetection. In some projects, the combined measurement of a mixture of protein molecules of different polymerization states causes measurement interference with the target analyte.
Disclosure of Invention
In order to solve the problem of measurement interference of a target object to be measured caused by combined measurement of protein molecule mixtures in different polymerization states, the invention achieves the aim of detecting the polymerization states of the object to be measured through the design of an immunoreaction measurement method, and can effectively determine the real content of the object to be measured in a monomer form.
The specific implementation is as follows:
a calibration quality control for antigen monomer detection, comprising a multimeric antigen comprising the antigen monomer.
The invention relates to a conjugate of a monomer antigen and a protein, wherein the conjugate contains more than one monomer antigen, the protein is a substance with a certain space structure formed by the twisting folding of a polypeptide chain formed by amino acid in a dehydration condensation mode, and the protein can be an antigen or an antibody.
Further, the concentration of the multimeric antigen is 0.1ng to 1mg.
The concentration range varies widely, and a person skilled in the art can select a proper concentration according to the detection item and the kind of the multimeric antigen.
Further, the multimeric antigen is selected from the group consisting of antigen monomers, antigen monomer-IgG antibodies, and macromolecular complexes at different polymerization levels.
The antigen monomers include PRL, TSH, FSH, FDA and D-Dimer.
Further, the macromolecular complexes comprise complexes of an antigen monomer with a carrier protein, preferably selected from BSA or casein.
Further, the antigen monomer is 1 or more.
Further, the calibration quality control further comprises a buffer, a surfactant, a stabilizing protein and a preservative.
Further, the buffer solution is selected from Tris buffer solution, phosphate buffer solution or MES buffer solution, and the concentration is 10-100mmol/L; the surfactant is selected from triton X-100, tween 20, tween 80 or NP40, and the concentration is 1-15g/L; the stabilizing protein is BSA or casein, and the concentration is 0.1% -2.0%; the preservative is Proclin@300 or sodium azide, and the concentration is 0.4-1.2g/L.
In another aspect, the invention discloses a kit for detecting an antigen monomer, which comprises the calibration quality control.
Further, the kit is a chemiluminescent detection kit.
The present invention is not particularly limited to buffers, surfactants, stabilizing proteins and preservatives, and one skilled in the art can select an appropriate kind depending on the items to be tested.
In another aspect, the invention discloses a method for detecting an antigen monomer, comprising the steps of:
(4) Detecting the total amount T of the antigen to be detected by using antibodies recognizing different epitopes of the antigen to be detected;
(5) Detecting the polymerization state content M of the antigen to be detected by using an antibody for recognizing a single epitope of the antigen to be detected;
(6) Obtaining the content of the antigen monomer by using T-M;
the method further comprises a quality control process, wherein the quality control process is arranged in any process in the antigen monomer detection method, and the quality control process is calibrated by using the calibration quality control product.
The antigen to be detected is an antigen existing in a natural state, and comprises an antigen monomer and an antigen polymerization state, wherein the antigen polymerization state comprises the following components: non (low) physiologically active multimers generated by abnormal binding or adsorption of antigen monomers themselves; a non (low) physiologically active aggregate mixture is produced upon binding of a plurality of antigens to human autoimmune antibodies.
Further, the antibodies in the step (1) are a first antibody and a second antibody, and the sequences of the first antibody and the second antibody are different.
Further, the antibody in the step (2) is the first antibody, the second antibody or a third antibody, and the sequence of the third antibody is different from the sequences of the first antibody and the second antibody.
The beneficial effects are that:
for the detection of the composite antigen in a natural state, the invention uses the multimeric antigen as a calibration quality control product, and simply realizes the stable quality control of the monomer detection.
The invention is based on the two-time linear classification of the interaction of specific proteins, so as to distinguish the monomers and the polymers, better eliminate interference and avoid false positive results.
Compared with the method for analyzing the polymerization degree of the prolactin by adding screening characterization means such as a polyethylene glycol sedimentation method, an exclusion chromatography method and the like, the method is completely established on an immune measurement platform, and no extra equipment and labor investment are needed. Meanwhile, the method is integrated on a single kit, and can be used at a user end without generating deviation in user experience with the existing luminescence method determination kit.
Drawings
FIG. 1 shows the comparison of the detection effect of the method (1A) according to the invention with that of a common double antibody sandwich (1B).
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Preparation example: preparation of multimeric antigens
The preparation method of the multimeric antigen used in the invention specifically comprises the following steps:
(1) Desalting: taking antigen monomer and protein, desalting by using a chromatographic desalting column, and determining the protein concentration by using an ultraviolet spectrophotometer.
(2) Activating: diluting or concentrating antigen monomer to 3.0mg/mL, adding 4-way according to the mass of coupled protein
(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester, standing and reacting for 20 minutes; the protein was diluted or concentrated to 4.0mg/mL, and 2-iodo-L-tyrosine was added thereto based on the mass of the coupled protein, and allowed to stand for reaction for 20 minutes.
(3) Desalting: the activated antigen monomer and protein are desalted using a chromatographic desalting column, respectively.
(4) Ligation reaction: the mass ratio of the connection reaction is that the antigen monomer: protein=1:3, the linker concentration was diluted or concentrated to 0.5mg/mL, mixed well and allowed to stand for reaction for 12 hours.
(5) Purification of the linker: and (3) desalting and concentrating the reaction solution to the volume required by purification, and purifying, eluting and collecting the peak with the maximum molecular weight, namely the antigen monomer-protein coupled complex.
(6) Assigning a value to the antigen monomer-protein coupled complex, and proportionally mixing the antigen monomer-protein coupled complex with the antigen monomer according to the calibration customization requirement to prepare the multimeric antigen.
Example 1 comparison of multimeric antigen quality control with Normal quality control
This example is given as an example of the detection of Prolactin (PRL)
The combined measurement of a mixture of protein molecules of different polymerization states causes measurement interference of the target analyte. With the performance on PRL being more prominent. PRL is a polypeptide hormone secreted primarily by pituitary prolactin cells, also known as prolactin, and has a structure consisting of 4 antiparallel alpha helices. The prolactin has four types of composite molecular structures in circulating blood: small molecule prolactin, glycosylated prolactin, multimeric prolactin and macromolecular prolactin. The small-molecule lactating essence consists of 199 amino acids, has a relative molecular mass of 23kDa and has highest biological activity and immunological activity; dimeric prolactin is not physiologically active; multimeric biological immune activity is low; the macromolecular prolactin is a complex polymer of the prolactin and autoimmune globulin, and has low physiological activity. When the detection of the prolactin is carried out, the identification of the polymerization state of the prolactin is a very necessary diagnosis and treatment link, and is also a difficult point of diagnosis and treatment of the hyperprolactinemia. At present, macrolactin identification is carried out clinically by adopting a polyethylene glycol sedimentation method based on immunodetection. The principle of the method is that the rough quantification of PRL compound with higher molecular weight is achieved by utilizing different adsorption levels of polyethylene glycol to protein molecules with different sizes, and the proportion of high molecular weight prolactin in a sample is judged. The method is complex in operation and the quantitative accuracy is influenced by the severity of experimental conditions. Due to the heterogeneity of different samples, the molecular weight distribution and the surface potential of PRL in different polymerization states are different, and the polyethylene glycol sedimentation method cannot calculate the content of the monomer PRL in the sample stably and accurately.
The current method for improving the anti-macrolactin interference capability of PRL reagent in the same-row products mainly comprises screening epitope with more monomer specificity as recognition site of the antibody, namely, site of PRL easy to be embedded in self-polymerization or antibody polymerization besides adding blocker to block false positive result caused by non-ideal connection participated by human autoimmune antibody. The method relies on the biotechnology capability of the strong antibody raw material end of manufacturers, and in the raw material screening of most immunodiagnosis, a conserved sequence is selected as a recognition epitope to prevent missed detection caused by mutation. However, to discriminate heterogeneously by a single classification, it is imperative to select epitopes with prominent monomeric character. Specifically, the binding site that is prone to self-aggregation or to cause autoimmunity is likely to be a high frequency site where mutation or the like occurs. In addition, because of the diversity of the macromolecular forms of the PRL, the macromolecular interference phenomenon of the PRL cannot be completely eliminated by the method, and the index of the macrolactin level in the to-be-detected substance, which has potential diagnosis and scientific research reference values, cannot be given out.
Therefore, the quality control product and the detection method of the present invention are verified by PRL in this embodiment.
The preparation method comprises the following steps:
A. preparation of multimeric PRL antigens
The multimeric PRL antigen used in this example was selected from the group consisting of PRL-BSA conjugate complexes and PRL antigen monomers in a particular state of polymerization.
The PRL-BSA coupling complex is prepared by the following steps:
(1) Desalting: PRL antigen monomer and carrier protein BSA were taken, desalted using a chromatographic desalting column, and protein concentration was determined using an ultraviolet spectrophotometer, respectively.
(2) Activating: diluting or concentrating PRL antigen monomer to 3.0mg/mL, adding according to the mass of coupled PRL antigen
46ug/mg of 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester, and standing for reaction for 20 minutes;
the carrier protein BSA was diluted or concentrated to 4.0mg/mL, and 12ug/mg of 2-iodo-L-tyrosine was added based on the mass of the coupled BSA, followed by a reaction at rest for 20 minutes.
(3) Desalting: the activated PRL antigen monomer and carrier protein BSA were desalted using a chromatographic desalting column, respectively.
(4) Ligation reaction: the mass ratio of the ligation reaction is PRL antigen: bsa=1:3, the total protein concentration was diluted or concentrated to 0.5mg/mL, mixed well and allowed to stand for reaction for 12 hours.
(5) Purification of the linker: and (3) desalting and concentrating the reaction solution to the volume required by purification, and purifying, eluting and collecting the peak with the maximum molecular weight, namely the PRL-BSA coupling complex.
(6) Assigning a value to the PRL-BSA coupling complex, and proportionally mixing the PRL-BSA coupling complex with PRL antigen monomers according to the calibration and customization requirements to prepare the multi-state PRL antigen.
B. Preparation of magnetic particle chemiluminescence kit
TABLE 1 kit components of the invention
The multimeric PRL antigen used in this example is a PRL-BSA conjugate
Comparative example: the multimeric PRL antigens in the calibration quality control were exchanged for PRL monomeric antigens in table 1.
C. Detection of
The detection instrument used was EXI1800, a midrange Gibbs Biotechnology Co.
The detection comprises the following specific steps:
(1) And (3) performing calibration assignment by using a calibration quality control product: mixing 80 mu LR2 with 20 mu LMB and 10 mu L of calibration quality control, and calibrating a reaction main curve of the T reaction; the reaction master curve of the M reaction was calibrated by mixing 80 μ L R1 with 20 μLMB and 10 μL of calibration quality control. The calibration reaction patterns are the same as the test of the test object reaction, respectively.
(2) Mixing 80 mu L R and 20 mu L MB with 10 mu L of a sample (serum or plasma) to be tested, wherein total lactating hormone (tPRL) in the sample is fully reacted with a primary PRL antibody marked by biotin and a secondary PRL antibody marked by alkaline phosphatase to form an immune complex, and connecting the immune complex to the surface of streptavidin magnetic particles through biotin-streptavidin; by washing the magnetic particles, unreacted substances and other substances are removed. Adding chemiluminescent substrate solution, performing enzymatic reaction with alkaline phosphatase to generate photons, and measuring relative luminescence intensity (RLU); and obtaining a final detection result through a calibration curve of the detector, and recording a reading T.
(3) Mixing 80 mu L R and 20 mu L MB with 10 mu L of a sample to be tested, fully reacting polymeric prolactin (mPRL) in the sample with a biotin-labeled PRL primary antibody and an alkaline phosphatase-labeled PRL primary antibody to form an immune complex, and connecting the immune complex to the surface of streptavidin magnetic particles through biotin-streptavidin; by washing the magnetic particles, unreacted substances and other substances are removed. Adding chemiluminescent substrate solution, performing enzymatic reaction with alkaline phosphatase to generate photons, and measuring relative luminescence intensity (RLU); and obtaining a final detection result through a calibration curve of the detector, and recording a reading M.
(4) The monomer values were calculated according to the assignment of T, M: prl=t-M.
The results are shown in the following table:
TABLE 2 detection results of the kit of the present invention
Table 3 comparative kit test results
As can be seen from tables 2 and 3, the measurement of the PRL of the monomer in the solution was hardly performed by using the non-polymerized monomer antigen as the calibration quality control, i.e., the detection of the monomer was not performed by using a conventional detection of a certain antigen, and the detection of the monomer was not performed by using a certain antigen as the quality control in the detection of the antigen having a multimeric state. The PRL-BSA conjugate (i.e., multimeric PRL antigen) of the invention can be used as a calibration quality control to detect the value of the mPRL,
and as the concentration of tPRL increases, the concentration of mPRL increases, so that the value of monomeric PRL can be calculated from the values of tPRL and mPRL.
D. Species for changing multimeric PRL antigens
Detection method and multimeric PRL antigen As described above, the same sample is measured
TABLE 4 multimeric PRL antigen is PRL-Casein coupled
TABLE 5 multimeric PRL antigen is PRL-murine IgG conjugated
TABLE 6 multimeric PRL antigen PRL self-coupling
From tables 4-6 above, it can be seen that changing the type of multimeric PRL antigen relative to the monomeric PRL allows for an effective response signal in the detection of the PRL, thus allowing for an effective calibration of the PRL and ultimately detection of the monomer.
Example 2 method of the present example effect verification
This example uses the comparison method and the inventive test method of example 1 to compare with the reference test method, respectively. The comparative example method is as follows:
PRL was detected using a conventional double antibody sandwich method using the same antibody starting material (PRL primary antibody, PRL secondary antibody). The detection instrument used was EXI1800, a midrange Gibbs Biotechnology Co.
The method comprises the following specific steps:
(1) And (3) performing calibration assignment by using a conventional monomer PRL calibration quality control product, and calibrating a reaction main curve. The calibration reaction patterns are the same as the test of the test object reaction, respectively.
(2) Mixing 60 mu L R2 and 60 mu L R1 with 10 mu L of a sample (serum or plasma) to be tested, wherein the Prolactin (PRL) in the sample is fully reacted with a biotin-labeled PRL primary antibody and an alkaline phosphatase-labeled PRL secondary antibody to form an immune complex; subsequently 20. Mu.L MB was added and the immunocomplexes were attached to the streptavidin magnetic particle surface by biotin-streptavidin reaction. By washing the magnetic particles, unreacted substances and other substances are removed. The chemiluminescent substrate solution was added and an enzymatic reaction was performed with alkaline phosphatase to generate photons, and the relative luminescence intensity (RLU) was measured.
(3) And obtaining a final detection result through a calibration curve of the detector.
The reference detection method comprises the following steps:
the reference detection method adopts the detection method of the current lactating monomer in serum, and is specifically as follows:
(1) Adding an appropriate amount of serum sample into an equal volume of 25% polyethylene glycol 6000 solution, and mixing for 5 minutes at room temperature by vortex; centrifugation was carried out at 7500rpm for 10 minutes, and the supernatant was collected.
(2) Supernatant PRL concentration testing was performed on a matched immunoassay analyzer using the eleecsys PRL II (relatively less exposure of the recognition epitope to the polymerized state) from the reference reagent instructions. And multiplying the obtained detection result by a dilution factor of 2 to obtain the result of detecting the monomeric prolactin in the serum by the reference detection method.
Comparative example the results of the comparative reference detection method are shown in fig. 1B, and fig. 1B shows the results of the comparison using the conventional double antibody sandwich method and the reference detection method. It can be seen that there are more samples of the comparative test results that are higher than the reference detection method due to interference of the various polymerization states of the PRL, resulting in false positives in the PRL assay.
The results of the comparative reference detection method of example 1 are shown in FIG. 1A. It can be seen that the test results of this example are superior to those of the comparative example. Under the condition that the used measured antibody pair is unchanged, the method of the embodiment can better identify the interference of the megalin, avoid the false positive phenomenon in the PRL measurement and has better detection result.
EXAMPLE 3 replacement of detection antigen
In this example, the TSH project was detected in the same manner as in example 1, and the preparation method of the multimeric TSH antigen was as follows: the preparation method of the multimeric antigen.
Table 7TSH detection kit
In this embodiment, the multimeric TSH antigen is TSH-casein coupled, and the detection results are shown in table 8 below, which shows that the quality control product and the detection method of the present invention are also suitable for TSH detection.
TABLE 8TSH detection results
Claims (10)
1. The calibration quality control material for detecting the antigen monomer is characterized by comprising a multimeric antigen, wherein the multimeric antigen contains the antigen monomer.
2. The calibration quality control of claim 1 wherein the multimeric antigen is selected from the group consisting of antigen monomers, antigen monomer-IgG antibodies, and macromolecular complexes at different levels of polymerization; the macromolecular complex is a complex of an antigen monomer and carrier protein, and preferably, the antigen monomer is more than 1; preferably, the carrier protein is selected from BSA or casein.
3. The calibration quality control of claim 1 or 2 wherein the antigen monomer comprises PRL, TSH, FSH, FDA or D-Dimer.
4. The calibration quality control of claim 1 or 2 further comprising a buffer, a surfactant, a stabilizing protein, and a preservative.
5. The calibration quality control of claim 4 wherein the buffer is selected from Tris buffer, phosphate buffer or MES buffer at a concentration of 10-100mmol/L; the surfactant is selected from triton X-100, tween 20, tween 80 or NP40, and the concentration is 1-15g/L; the stabilizing protein is BSA or casein, and the concentration is 0.1% -2.0%; the preservative is Proclin@300 or sodium azide, and the concentration is 0.4-1.2g/L.
6. A kit for the detection of an antigen monomer, comprising the calibration quality control of any one of claims 1-4.
7. The kit of claim 6, wherein the kit is a chemiluminescent detection kit.
8. A method for detecting an antigen monomer, comprising the steps of:
(1) Detecting the total amount T of the antigen to be detected by using antibodies recognizing different epitopes of the antigen to be detected;
(2) Detecting the polymerization state content M of the antigen to be detected by using an antibody for recognizing a single epitope of the antigen to be detected;
(3) Obtaining the content of the antigen monomer by using T-M;
the method further comprises a quality control process, wherein the quality control process is arranged in any process in the antigen monomer detection method, and the quality control process is calibrated by using the calibration quality control product according to any one of claims 1-5.
9. The method of claim 8, wherein the antibody of step (1) comprises a first antibody and a second antibody, wherein the first and second antibodies differ in sequence.
10. The method of claim 9, wherein the antibody in step (2) is the first antibody, the second antibody, or a third antibody, the third antibody having a sequence different from the sequences of the first antibody and the second antibody.
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