CN118085080A - Monoclonal antibody composition for detecting neurodegenerative diseases, and preparation method and application thereof - Google Patents
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Abstract
The application provides a monoclonal antibody composition for detecting neurodegenerative diseases, a preparation method and application thereof. The monoclonal antibody composition includes one or more of the following combinations: (1) CDNF protein monoclonal antibody 1 and CDNF protein monoclonal antibody 2; (2) BDNF protein monoclonal antibody 1 and BDNF protein monoclonal antibody 2; and (3) GDNF protein monoclonal antibody 1 and GDNF protein monoclonal antibody 2. The monoclonal antibody composition provided by the application can quantitatively detect the concentration of CDNF, BDNF and GDNF protein in a sample to be detected. The monoclonal antibody composition is used for detecting neurodegenerative diseases and has the advantages of high sensitivity, high selectivity, high accuracy and the like.
Description
Technical Field
The application relates to the technical field of biology, in particular to a monoclonal antibody composition for detecting neurodegenerative diseases, and a preparation method and application thereof.
Background
Neurodegenerative diseases (Neurodegenerative Disease, NDD) refer to gradual loss of neuronal structure and function, leading to symptoms such as cognitive dysfunction and dementia. Common neurodegenerative diseases include Alzheimer's Disease (AD), parkinson's Disease (PD), huntington's Disease (HD), and freezing syndrome (ALS), and the neurodegenerative diseases are often accompanied by symptoms such as sleep disorder, depression, and anxiety.
As the average life span of humans increases, the prevalence of neurodegenerative diseases increases year by year, and thus it is important to accurately detect and predict neurodegenerative diseases. Studies have shown that biomarkers of amyloid-beta (Abeta 42), total tau (T-tau) protein, and phosphorylated tau (P-tau) protein in cerebrospinal fluid can be used to aid in the diagnosis of neurodegenerative diseases. However, the above biomarkers can only be detected abnormal after the disease.
Disclosure of Invention
Based on the above, one or more embodiments of the present application provide a monoclonal antibody composition for detecting neurodegenerative diseases, and a preparation method and application thereof. The technical proposal comprises:
according to a first aspect of embodiments of the present application there is provided a monoclonal antibody composition comprising one or more of the following combinations:
(1) CDNF protein monoclonal antibody 1 and CDNF protein monoclonal antibody 2;
(2) BDNF protein monoclonal antibody 1 and BDNF protein monoclonal antibody 2; and
(3) GDNF protein monoclonal antibody 1 and GDNF protein monoclonal antibody 2;
Wherein the CDNF protein monoclonal antibody 1 comprises a VL-CDR1 with an amino acid sequence shown as SEQ ID NO. 1, a VL-CDR2 with an amino acid sequence shown as SEQ ID NO. 2, a VL-CDR3 with an amino acid sequence shown as SEQ ID NO. 3, a VH-CDR1 with an amino acid sequence shown as SEQ ID NO. 4, a VH-CDR2 with an amino acid sequence shown as SEQ ID NO. 5 and a VH-CDR3 with an amino acid sequence shown as SEQ ID NO. 6;
The CDNF protein monoclonal antibody 2 comprises VL-CDR1 with an amino acid sequence shown as SEQ ID NO. 7, VL-CDR2 with an amino acid sequence shown as SEQ ID NO. 8, VL-CDR3 with an amino acid sequence shown as SEQ ID NO. 9, VH-CDR1 with an amino acid sequence shown as SEQ ID NO. 10, VH-CDR2 with an amino acid sequence shown as SEQ ID NO. 11 and VH-CDR3 with an amino acid sequence shown as SEQ ID NO. 12;
The BDNF protein monoclonal antibody 1 comprises a VL-CDR1 with an amino acid sequence shown as SEQ ID NO. 13, a VL-CDR2 with an amino acid sequence shown as SEQ ID NO. 14, a VL-CDR3 with an amino acid sequence shown as SEQ ID NO. 15, a VH-CDR1 with an amino acid sequence shown as SEQ ID NO. 16, a VH-CDR2 with an amino acid sequence shown as SEQ ID NO. 17 and a VH-CDR3 with an amino acid sequence shown as SEQ ID NO. 18;
The BDNF protein monoclonal antibody 2 comprises VL-CDR1 with an amino acid sequence shown as SEQ ID NO. 19, VL-CDR2 with an amino acid sequence shown as SEQ ID NO. 20, VL-CDR3 with an amino acid sequence shown as SEQ ID NO. 21, VH-CDR1 with an amino acid sequence shown as SEQ ID NO. 22, VH-CDR2 with an amino acid sequence shown as SEQ ID NO. 23 and VH-CDR3 with an amino acid sequence shown as SEQ ID NO. 24;
The GDNF protein monoclonal antibody 1 comprises VL-CDR1 with an amino acid sequence shown as SEQ ID NO. 25, VL-CDR2 with an amino acid sequence shown as SEQ ID NO. 26, VL-CDR3 with an amino acid sequence shown as SEQ ID NO. 27, VH-CDR1 with an amino acid sequence shown as SEQ ID NO. 28, VH-CDR2 with an amino acid sequence shown as SEQ ID NO. 129 and VH-CDR3 with an amino acid sequence shown as SEQ ID NO. 30;
The GDNF protein monoclonal antibody 2 comprises VL-CDR1 with an amino acid sequence shown as SEQ ID NO. 31, VL-CDR2 with an amino acid sequence shown as SEQ ID NO. 32, VL-CDR3 with an amino acid sequence shown as SEQ ID NO. 33, VH-CDR1 with an amino acid sequence shown as SEQ ID NO. 34, VH-CDR2 with an amino acid sequence shown as SEQ ID NO. 35 and VH-CDR3 with an amino acid sequence shown as SEQ ID NO. 36.
Compared with the prior art, the application has the following beneficial effects:
The application provides a monoclonal antibody composition capable of quantitatively detecting CDNF, BDNF and GDNF protein concentration in a sample to be detected. The monoclonal antibody composition is used for detecting neurodegenerative diseases, and has the advantages of high selectivity, strong specificity, high accuracy and the like.
Drawings
FIG. 1 is a diagram showing the detection of monoclonal antibodies by electrophoresis obtained by the screening in example 1 of the present application.
Detailed Description
The detailed description of the present application will be provided to make the above objects, features and advantages of the present application more obvious and understandable. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. The present application may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit of the application, whereby the application is not limited to the specific embodiments disclosed below.
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 application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present application are commercially available or may be prepared by existing methods.
The technical characteristics described in an open mode in the application comprise a closed technical scheme composed of the listed characteristics and also comprise an open technical scheme containing the listed characteristics.
In a first aspect of the application, there is provided a monoclonal antibody composition comprising one or more of the following combinations:
(1) CDNF protein monoclonal antibody 1 and CDNF protein monoclonal antibody 2;
(2) BDNF protein monoclonal antibody 1 and BDNF protein monoclonal antibody 2; and
(3) GDNF protein monoclonal antibody 1 and GDNF protein monoclonal antibody 2.
Brain dopamine neurotrophic factor (CDNF) is an important neuroprotective factor that plays a critical role in the development and functional maintenance of the nervous system. Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease affecting the motor neurons of the spinal cord, brainstem and motor cortex, leading to paralysis and eventually death within 3-5 years after symptoms appear. Detecting CDNF levels in human blood can predict to some extent the risk of developing freezing syndrome and assess the disease progression or therapeutic efficacy of freezing syndrome patients.
Brain-derived neurotrophic factor (brain derived neurotrophic factor, BDNF) is a protein synthesized in the brain, plays an important role in survival, differentiation, growth and development of neurons in the development process of the central nervous system, can prevent injury and death of neurons, improve pathological states of neurons, promote biological effects such as regeneration and differentiation of injured neurons, and is also necessary for the neurons of the mature central and peripheral nervous systems to maintain survival and normal physiological functions. BDNF has been found to be associated with various neurological and psychiatric conditions, for example, lower serum BDNF levels in depressed patients than normal controls and is inversely related to the extent of depression, the course of the disease, which may be associated with longer course, apoptosis, reduced volume of the patient's nerve cells, and more severe decline in their ability to take up neurotrophic factors.
Glial cell line-derived neurotrophic factor (GLIALCELLLINE-derived neurotrophic factor, GDNF) is a neurotrophic factor that promotes survival of different neuronal subsets at different stages of development of the central and peripheral nervous systems, and plays a vital role in promoting motor neuron survival and axon growth. The binding of GDNF to its receptor triggers several intracellular signaling pathways that play a role in promoting the development, survival and maintenance of neuronal-neuronal and neuronal-target tissue interactions. The synthesis and modulation of GDNF has been shown to play a role in many diseases, aging, and may also improve neurodegenerative diseases such as Parkinson's Disease (PD). Detecting GDNF content in human blood can predict Parkinson's disease to some extent, and evaluate the extent of development or therapeutic effect of Parkinson's disease.
In some of these embodiments, CDNF protein monoclonal antibody 1 comprises VL-CDR1 (light chain variable region CDR 1) having an amino acid sequence as shown in SEQ ID NO. 1, VL-CDR2 (light chain variable region CDR 2) having an amino acid sequence as shown in SEQ ID NO.2, VL-CDR3 (light chain variable region CDR 3) having an amino acid sequence as shown in SEQ ID NO. 3, VH-CDR1 (heavy chain variable region CDR 1) having an amino acid sequence as shown in SEQ ID NO. 4, VH-CDR2 (heavy chain variable region CDR 2) having an amino acid sequence as shown in SEQ ID NO. 5, and VH-CDR3 (heavy chain variable region CDR 3) having an amino acid sequence as shown in SEQ ID NO. 6.
In some embodiments, CDNF protein monoclonal antibody 1 includes a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO. 37, and a light chain variable region having an amino acid sequence set forth in SEQ ID NO. 38.
In some of these embodiments, CDNF protein monoclonal antibody 2 includes VL-CDR1 having an amino acid sequence shown in SEQ ID NO. 7, VL-CDR2 having an amino acid sequence shown in SEQ ID NO. 8, VL-CDR3 having an amino acid sequence shown in SEQ ID NO. 9, VH-CDR1 having an amino acid sequence shown in SEQ ID NO. 10, VH-CDR2 having an amino acid sequence shown in SEQ ID NO. 11, and VH-CDR3 having an amino acid sequence shown in SEQ ID NO. 12.
In some embodiments, CDNF protein monoclonal antibody 2 includes a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO. 39, and a light chain variable region having an amino acid sequence set forth in SEQ ID NO. 40.
In some of these embodiments, BDNF protein monoclonal antibody 1 includes VL-CDR1 having an amino acid sequence shown as SEQ ID NO. 13, VL-CDR2 having an amino acid sequence shown as SEQ ID NO. 14, VL-CDR3 having an amino acid sequence shown as SEQ ID NO. 15, VH-CDR1 having an amino acid sequence shown as SEQ ID NO. 16, VH-CDR2 having an amino acid sequence shown as SEQ ID NO. 17, and VH-CDR3 having an amino acid sequence shown as SEQ ID NO. 18.
In some embodiments, BDNF protein monoclonal antibody 1 includes a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 41 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 42.
In some of these embodiments, BDNF protein monoclonal antibody 2 includes VL-CDR1 having an amino acid sequence shown as SEQ ID NO. 19, VL-CDR2 having an amino acid sequence shown as SEQ ID NO. 20, VL-CDR3 having an amino acid sequence shown as SEQ ID NO. 21, VH-CDR1 having an amino acid sequence shown as SEQ ID NO. 22, VH-CDR2 having an amino acid sequence shown as SEQ ID NO. 23, and VH-CDR3 having an amino acid sequence shown as SEQ ID NO. 24.
In some embodiments, BDNF protein monoclonal antibody 2 includes a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 43 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 44.
In some of these embodiments, GDNF protein monoclonal antibody 1 comprises VL-CDR1 having an amino acid sequence as shown in SEQ ID NO. 25, VL-CDR2 having an amino acid sequence as shown in SEQ ID NO. 26, VL-CDR3 having an amino acid sequence as shown in SEQ ID NO. 27, VH-CDR1 having an amino acid sequence as shown in SEQ ID NO. 28, VH-CDR2 having an amino acid sequence as shown in SEQ ID NO. 129, and VH-CDR3 having an amino acid sequence as shown in SEQ ID NO. 30.
In some embodiments, GDNF protein monoclonal antibody 1 includes a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO. 45 and a light chain variable region having an amino acid sequence set forth in SEQ ID NO. 46.
In some of these embodiments, GDNF protein monoclonal antibody 2 comprises VL-CDR1 having an amino acid sequence as shown in SEQ ID NO. 31, VL-CDR2 having an amino acid sequence as shown in SEQ ID NO. 32, VL-CDR3 having an amino acid sequence as shown in SEQ ID NO. 33, VH-CDR1 having an amino acid sequence as shown in SEQ ID NO. 34, VH-CDR2 having an amino acid sequence as shown in SEQ ID NO. 35, and VH-CDR3 having an amino acid sequence as shown in SEQ ID NO. 36.
In some embodiments, GDNF protein monoclonal antibody 2 includes a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO. 47, and a light chain variable region having an amino acid sequence set forth in SEQ ID NO. 48.
Specifically, the amino acid sequences shown in SEQ ID NO. 1 to SEQ ID NO. 48 are shown in Table 1.
The monoclonal antibody composition provided by the application can detect whether the concentration of CDNF protein, BDNF protein and GDNF protein in a sample to be detected is abnormal or not in the early stage of disease. Further, the antibody composition of the application can be used for quantitatively detecting the concentration of CDNF protein, BDNF protein and GDNF protein, thereby being used for evaluating or diagnosing neurodegenerative diseases.
The monoclonal antibodies obtained by screening have the advantages of high titer, high affinity, high specificity and the like.
In a second aspect, the application provides the use of a monoclonal antibody composition as described above in the preparation of a product for detecting neurodegenerative diseases.
In a third aspect of the application, a kit for detecting neurodegenerative diseases is provided, comprising the monoclonal antibody composition described above.
In some embodiments, the kit includes a capture antibody and a detection antibody.
In some embodiments, the kit further comprises a solid support and a fluorescent label; wherein the solid phase carrier is bound to the capture antibody and the fluorescent label is bound to the detection antibody.
In some embodiments, one of the CDNF protein monoclonal antibody 1 and CDNF protein monoclonal antibody 2 is linked to a solid support as a capture antibody, and the other is labeled with a fluorescent label and then used as a detection antibody.
In some embodiments, one of the BDNF protein monoclonal antibody 1 and BDNF protein monoclonal antibody 2 is linked to a solid support as a capture antibody and the other is labeled with a fluorescent label and then used as a detection antibody.
In some embodiments, one of the GDNF protein monoclonal antibody 1 and GDNF protein monoclonal antibody 2 is linked to a solid support as a capture antibody and the other is labeled with a fluorescent label as a detection antibody.
In some embodiments, the solid support comprises magnetic particles.
In some embodiments, the fluorescent label comprises an acridinium ester.
In a fourth aspect of the application, there is provided a nucleic acid encoding a monoclonal antibody composition as described above.
In some embodiments, the nucleic acid comprises a nucleic acid encoding an antibody variable region having an amino acid sequence as set forth in one or more of SEQ ID NOS: 1-36.
In a fifth aspect of the application, there is provided a recombinant vector comprising a nucleic acid as described above.
In a sixth aspect of the application there is provided a host cell comprising a recombinant vector as described above.
In some embodiments, the host cell is a CHO cell, a COS cell, an NSO cell, a HeLa cell, a BHK cell, or a HEK293 cell. Of course, in other embodiments, the host cell is not limited to the above, but may be other cells.
In a seventh aspect of the present application, there is provided a hybridoma cell secreting the monoclonal antibody described above.
In an eighth aspect of the present application, there is provided a method for producing a monoclonal antibody composition.
In some embodiments, the above preparation method comprises the steps of: culturing the above host cells, collecting cell culture solution, and preparing monoclonal antibody composition.
In some embodiments, the above preparation method comprises the steps of: injecting hybridoma cells into the abdominal cavity of a mouse, collecting ascites, and separating and purifying to prepare a monoclonal antibody composition.
In a ninth aspect of the present application, there is provided a method for detecting a neurodegenerative disease, comprising steps S10 to 20.
Step S10: the concentration of the target protein in the sample to be detected is detected by adopting the kit.
Step S20: and analyzing whether the sample to be detected is a potential patient of the neurodegenerative disease according to the detection result.
In some embodiments, in step S10, the sample to be tested is a blood sample.
In some of these embodiments, in step S10, the protein of interest comprises one or more of CDNF protein, BDNF protein, and GDNF protein.
In some embodiments, in step S20, the concentration of CDNF protein in the sample to be tested is less than 200pg/mL, and the sample to be tested is a potential patient for neurodegenerative disease.
In some embodiments, in step S20, if the concentration of BDNF protein in the sample to be tested is less than 230pg/mL, the sample to be tested is a potential patient for neurodegenerative disease.
In some embodiments, in step S20, if the concentration of GDNF protein in the sample is less than 300pg/mL, the sample is a potential patient for neurodegenerative disease.
The present application will be further described with reference to specific examples and comparative examples, which should not be construed as limiting the scope of the application. The materials used in the following examples were all commercially available, unless otherwise specified, the equipment used, and the processes involved, unless otherwise specified, were all routinely selected by those skilled in the art.
Example 1:
1. preparation of monoclonal antibodies
(1) Respectively mixing CDNF recombinant protein (purchased from Newton organism), BDNF recombinant protein (purchased from Wuhan Huamei organism) and GDNF recombinant protein (purchased from Kirschner biotechnology) with adjuvants, emulsifying, and immunizing mice. Spleen cells were isolated 3 days after the last challenge immunization.
The step of immunizing an animal comprises:
a) The first immunization is subcutaneous immunization; each mouse was selected from 6 injection sites, each injected with 40 μl of adjuvant-mixed antigen (1:1 mix).
B) The first boost was performed 3 weeks after the first immunization, and 20 μl of antigen mixed with adjuvant was injected into the abdominal cavity of the mice (1: 1) mixing; the second boost was performed 2 weeks after the first boost in the same manner as the first; the second boost was followed by a third boost 2 weeks later in the same manner as the first.
C) The final challenge immunization was performed 3 days after the third boost, and 20 μl of antigen mixed with physiological saline was injected into the abdominal cavity of the mice (1: 1) mixing).
(2) The myeloma cell line is adapted to culture by using a culture medium containing 8-azaguanine before fusion, and mouse abdominal cavity cells are selected as feeder cells.
(3) Myeloma cells and spleen cells were prepared according to 1:3, then adding polyethylene glycol after loosening the cells to fuse the cells, standing for 60 seconds, adding DMEM culture medium to stop, then standing in a water bath at 37 ℃ for 10 minutes, centrifuging, re-suspending by HAT culture medium, and placing in a culture plate hole for culture.
(4) 6 Positive cell strains are obtained by screening by adopting a limiting dilution method, namely hybridoma cells capable of secreting CDNF monoclonal antibody 1, CDNF monoclonal antibody 2, BDNF monoclonal antibody 1, BDNF monoclonal antibody 2, GDNF monoclonal antibody 1 and GDNF monoclonal antibody 2; and (5) performing expansion culture and freezing.
(5) First, the mice were intraperitoneally injected with liquid paraffin, and after one week, the six hybridoma cells were inoculated into the intraperitoneally of the mice, respectively. After one week of inoculation, obvious ascites can be produced, and 5-10 mL of monoclonal antibody-enriched ascites can be collected for each mouse.
(6) Purifying the antibody by an affinity purification method to obtain the following six purified monoclonal antibodies: CDNF monoclonal antibody 1, CDNF monoclonal antibody 2, BDNF monoclonal antibody 1, BDNF monoclonal antibody 2, GDNF monoclonal antibody 1, and GDNF monoclonal antibody 2.
(7) The six monoclonal antibodies were all of the IgG type as determined by the Elisa sandwich method. The molecular weight of the six monoclonal antibodies is about 150kD, the purity is higher, and the six monoclonal antibodies are free of impurity proteins (as shown in figure 1, the 1 st hole to the 7 th hole in figure 1 are respectively CDNF monoclonal antibody 1, CDNF monoclonal antibody 2, marker, BDNF monoclonal antibody 1, BDNF monoclonal antibody 2, GDNF monoclonal antibody 1 and GDNF monoclonal antibody 2) by adopting an electrophoresis method.
(8) The total RNA of the above hybridoma cells was extracted separately, and then the total RNA was reverse transcribed into cDNA. The cDNAs were amplified by PCR, and the amplified products were sent to Bio Inc. for sequencing, and the amino acid sequences of the variable regions of the six monoclonal antibodies were analyzed. The specific results are shown in Table 1.
TABLE 1
2. Monoclonal antibody Performance test
(1) Potency test
The titers of the CDNF monoclonal antibody 1, the CDNF monoclonal antibody 2, the BDNF monoclonal antibody 1, the BDNF monoclonal antibody 2, the GDNF monoclonal antibody 1 and the GDNF monoclonal antibody 2 are respectively tested by an indirect Elisa method; the method comprises the following specific steps:
a) Coating antigen: taking a new ELISA plate, adding a coating solution (CB buffer solution (carbonate-bicarbonate buffer solution) with concentration of 0.02M, pH and value of 9.6 containing 10ng/mL CDNF/BDNF/GDNF antigen, sealing with a preservative film, and placing in a refrigerator at 4 ℃ overnight;
b) Taking out the ELISA plate in a), discarding the supernatant, washing 3 times with PBST solution, adding PBST solution containing 1% BSA (the sample adding amount is 200 mu L/hole), and incubating at 37 ℃ for 1h;
c) Taking out the ELISA plate in b), discarding the supernatant, washing 3 times with PBST solution, adding gradient concentration antibody solution (PBS solution with concentration of 2mg/mL and concentration of 7.4 is used for carrying out double dilution with PBS solution with concentration of 0.02M, pH value of 7.4 according to 1:10, 1:100, 1:1000, 1:10000, 1:100000, 1:1000000, 1:10000000 and 1:100000000), blank control is PBS solution with concentration of 0.02: 0.02M, pH value of 7.4, and placing the mixture at 37 ℃ for incubation for 1h; two replicates were set for each sample;
d) Taking out the ELISA plate in c), discarding the supernatant, washing 3 times with PBST solution, adding diluted HBR-labeled IgG secondary antibody (diluted 1:5000 with PBST solution containing 1% BSA), adding 100 μl/well, and incubating at 37deg.C for 1 hr;
e) The ELISA plate in d) was removed, the supernatant was discarded, and after washing 4 times with PBST solution, substrate solution (10 mL of 10 mg/mL 3,3', 5' -Tetramethylbiphenyl (TMB) dissolved in 10. Mu.L dimethyl sulfoxide in PBS buffer with a concentration of 0.1M, pH and a value of 6.0, as prepared, was added to the solution, and the solution was incubated at 37℃for 10 minutes in the absence of light, with an amount of 100. Mu.L/well, and 15. Mu.L of 30% H 2O2).
F) The microplate in e) was removed, a reaction stop solution (H 2SO4 at a concentration of 2M) was added thereto in an amount of 50. Mu.L/well, and the absorbance at each well of 450nm was measured by placing the plate in a microplate reader. The average of the results of the titer determinations in the two replicates is shown in table 2.
TABLE 2
The results show that the titers of CDNF monoclonal antibody 1, CDNF monoclonal antibody 2, BDNF monoclonal antibody 1, BDNF monoclonal antibody 2, GDNF monoclonal antibody 1 and GDNF monoclonal antibody 2 can reach 1:1000000.
(2) Affinity test
Respectively testing the affinity of the CDNF monoclonal antibody 1, the CDNF monoclonal antibody 2, the BDNF monoclonal antibody 1, the BDNF monoclonal antibody 2, the GDNF monoclonal antibody 1 and the GDNF monoclonal antibody 2 by an indirect Elisa method; the specific procedure is similar to the method of potency testing in (1), except that:
The initial concentration of antibody in step c) was 0.5 μg/mL, with dilution ratios of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512.
The average of the results of the affinity assays performed in duplicate is shown in Table 3.
TABLE 3 Table 3
The results show that CDNF monoclonal antibody 1, CDNF monoclonal antibody 2, BDNF monoclonal antibody 1 and BDNF monoclonal antibody
2. The titer of the GDNF monoclonal antibody 1 and the GDNF monoclonal antibody 2 is more than 0.001ng/mL, and the GDNF monoclonal antibody have no obvious cross reaction with each other, and the GDNF monoclonal antibody has the characteristics of high affinity and high specificity.
3. Preparation of the kit
Methods for identifying and capturing target molecules using two different antibody molecules. First, an antibody molecule binds to a target molecule to form an antigen-antibody complex. Then, another antibody molecule binds to the complex to form a double antibody sandwich complex (antibody-antigen-antibody complex). Finally, by detecting the signal of the diabody sandwich complex, the presence and concentration of the target molecule can be determined.
(1) And (3) coating magnetic beads: activating functional groups on the surfaces of magnetic particles by adopting 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) with the concentration of 50mg/mL and N-hydroxysuccinimide (NHS) with the concentration of 50mg/mL, then cleaning magnetic beads, adding a CDNF monoclonal antibody 1, combining the antibody and the magnetic beads through hydrophobic acting force and covalent connection, then cleaning the magnetic beads, adding a magnetic bead sealing liquid containing 10% BSA to seal the magnetic beads, cleaning the magnetic beads, and diluting and preserving the magnetic beads by using a magnetic bead preservation liquid containing new-born calf serum, glycerol, a surfactant and a preservative to obtain the CDNF monoclonal antibody 1 coated with the magnetic particles for later use.
Similarly, magnetic particle coated BDNF monoclonal antibody 1 and magnetic particle coated GDNF monoclonal antibody 1 were prepared.
(2) Acridinium ester labeling: and mixing the CDNF monoclonal antibody 2 with carbonate buffer solution and acridinium ester, incubating for 3 hours at 25 ℃, blocking with blocking solution containing lysine after the incubation is completed, desalting the solution by using a desalting column after the blocking is completed, and finally preserving the desalted acridinium ester-labeled CDNF monoclonal antibody 2 by using acridinium ester preservation solution containing BSA, glycerol and preservative.
Similarly, acridinium ester-labeled BDNF monoclonal antibody 2 and acridinium ester-labeled GDNF monoclonal antibody 2 were prepared.
(3) Detection of CDNF protein concentration based on double antibody sandwich method
A) Using the magnetic particle coated CDNF monoclonal antibody 1 prepared in step (1) and the acridinium ester labeled CDNF monoclonal antibody 2 prepared in step (2), CDNF concentration levels of 200 healthy population samples under 30 years old obtained from clinical institutions were detected (unit: pg/mL), the results are shown in Table 4, and the CDNF reference range for healthy people is determined. The sample detection results are normally distributed, and the lower limit of the distribution range (X+1.96 s) of 95% is determined as a reference value, namely the reference value is: 200pg/mL.
TABLE 4 Table 4
B) The magnetic particle coated CDNF monoclonal antibody 1 prepared in the step (1) and the acridinium ester marked CDNF monoclonal antibody 2 prepared in the step (2) are adopted to detect 4 clinical samples of patients suffering from the acquired progressive freezing disease, and the results are shown in the table 5 (the "+" indicates that the detection result can accurately reflect the state of the patient). The results show that the monoclonal antibody composition can be used for quantitatively detecting patients suffering from the progressive freezing disease, and the accuracy is 100%.
TABLE 5
| Numbering device | Results (pg/mL) | Remarks |
| 1 | 64.93 | + |
| 2 | 77.24 | + |
| 3 | 47.95 | + |
| 4 | 58.56 | + |
(4) BDNF protein concentration detection based on double antibody sandwich method
A) The BDNF concentration levels of 200 healthy human samples under 30 years old obtained from clinical institutions were tested using the magnetic particle coated BDNF monoclonal antibody 1 prepared in step (1) and the acridinium ester labeled BDNF monoclonal antibody 2 prepared in step (2) (unit: pg/mL), the results are shown in table 6, and the BDNF reference range for healthy people is determined. The sample detection results are normally distributed, and the lower limit of the distribution range (X+1.96 s) of 95% is determined as a reference value, namely the reference value is: 230pg/mL.
TABLE 6
B) The detection of 20 samples of patients with sleep disorder was performed using the magnetic particle coated BDNF monoclonal antibody 1 prepared in step (1) and the acridinium ester labeled BDNF monoclonal antibody 2 prepared in step (2), and the results are shown in Table 7 ("+" indicates that the detection result can accurately reflect the patient state, and "-" indicates that the detection result cannot accurately reflect the patient state). The results show that the detection accuracy of the monoclonal antibody composition can reach 70% for patients suffering from the progressive freezing disease.
TABLE 7
| Numbering device | Results (pg/mL) | Remarks | Numbering device | Results (pg/mL) | Remarks |
| 1 | 161.44 | + | 11 | 266.73 | - |
| 2 | 276.98 | - | 12 | 146.53 | + |
| 3 | 195.18 | + | 13 | 112.00 | + |
| 4 | 202.07 | + | 14 | 100.68 | + |
| 5 | 256.36 | - | 15 | 156.40 | + |
| 6 | 232.20 | - | 16 | 268.73 | - |
| 7 | 150.09 | + | 17 | 168.69 | + |
| 8 | 197.86 | + | 18 | 99.23 | + |
| 9 | 268.12 | - | 19 | 162.47 | + |
| 10 | 164.91 | + | 20 | 101.92 | + |
C) 15 tinnitus patient samples were tested using the magnetic particle coated BDNF monoclonal antibody 1 prepared in step (1) and the acridinium ester labeled BDNF monoclonal antibody 2 prepared in step (2), and the results are shown in Table 8 ("+" indicates that the test results can accurately reflect patient status). The results show that the accuracy of detection of tinnitus patients by adopting the monoclonal antibody composition can reach 100%.
TABLE 8
D) The detection of 20 patient samples suffering from anxiety by using the magnetic particle coated BDNF monoclonal antibody 1 prepared in the step (1) and the acridinium ester labeled BDNF monoclonal antibody 2 prepared in the step (2) is shown in Table 9 ("+" indicates that the detection result can accurately reflect the patient state, and "-" indicates that the detection result cannot accurately reflect the patient state). The results show that the accuracy of detection of patients with focus concern can reach 75% by adopting the monoclonal antibody composition.
TABLE 9
| Numbering device | Results (pg/mL) | Remarks | Numbering device | Results (pg/mL) | Remarks |
| 1 | 218.04 | + | 11 | 134.23 | + |
| 2 | 160.08 | + | 12 | 207.99 | + |
| 3 | 176.90 | + | 13 | 194.05 | + |
| 4 | 259.05 | - | 14 | 191.32 | + |
| 5 | 184.58 | + | 15 | 228.12 | + |
| 6 | 108.61 | + | 16 | 211.67 | + |
| 7 | 119.30 | + | 17 | 240.20 | - |
| 8 | 215.46 | + | 18 | 226.30 | + |
| 9 | 281.31 | - | 19 | 133.01 | + |
| 10 | 244.37 | - | 20 | 286.03 | - |
E) The magnetic particle coated BDNF monoclonal antibody 1 prepared in the step (1) and the acridinium ester marked BDNF monoclonal antibody 2 prepared in the step (2) are adopted to detect 6 cases of patients suffering from depression, and the results are shown in table 10 (the "+" indicates that the detection results can accurately reflect the state of the patients). The results show that the accuracy of detection of depression patients by adopting the monoclonal antibody composition can reach 100%.
Table 10
| Numbering device | Results (pg/mL) | Remarks |
| 1 | 50.05 | + |
| 2 | 63.60 | + |
| 3 | 78.60 | + |
| 4 | 83.12 | + |
| 5 | 55.42 | + |
| 6 | 86.62 | + |
(5) GDNF protein concentration detection based on double antibody sandwich method
A) Using the magnetic particle coated GDNF monoclonal antibody 1 prepared in step (1) and the acridinium ester labeled GDNF monoclonal antibody 2 prepared in step (2), GDNF concentration levels of 200 healthy human samples under 30 years old obtained from clinical institutions were detected (unit: pg/mL), the results are shown in Table 11, and the GDNF reference range for healthy people is determined. The sample detection results are normally distributed, and the lower limit of the distribution range (X+1.96 s) of 95% is determined as a reference value, namely the reference value is: not less than 300pg/mL.
TABLE 11
B) 10 clinically collected patient samples of parkinson's disease were tested using the magnetic particle coated GDNF monoclonal antibody 1 prepared in step (1) and the acridinium ester labeled GDNF monoclonal antibody 2 prepared in step (2), and the results are shown in table 12 ("+" indicates that the test results can accurately reflect the patient status). The result shows that the monoclonal antibody composition can be used for quantitatively detecting the parkinsonism, and the accuracy is 100%.
Table 12
| Numbering device | Results (pg/mL) | Remarks | Numbering device | Results (pg/mL) | Remarks |
| 1 | 123.88 | + | 6 | 177.20 | + |
| 2 | 147.08 | + | 7 | 154.28 | + |
| 3 | 164.07 | + | 8 | 215.39 | + |
| 4 | 144.89 | + | 9 | 141.43 | + |
| 5 | 160.79 | + | 10 | 197.48 | + |
Taken together, the monoclonal antibody composition of the application can be used for detecting neurodegenerative diseases, in particular, freezing syndrome, anxiety, tinnitus, insomnia, parkinsonism and the like; and the accuracy of the detection result is high. The monoclonal antibody composition and the detection kit fill the blank of the reagent for detecting diseases such as depression, anxiety, sleep disorder and the like at present.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of the application should be assessed as that of the appended claims.
Claims (10)
1. A monoclonal antibody composition comprising one or more of the following combinations:
(1) CDNF protein monoclonal antibody 1 and CDNF protein monoclonal antibody 2;
(2) BDNF protein monoclonal antibody 1 and BDNF protein monoclonal antibody 2; and
(3) GDNF protein monoclonal antibody 1 and GDNF protein monoclonal antibody 2;
Wherein the CDNF protein monoclonal antibody 1 comprises a VL-CDR1 with an amino acid sequence shown as SEQ ID NO. 1, a VL-CDR2 with an amino acid sequence shown as SEQ ID NO. 2, a VL-CDR3 with an amino acid sequence shown as SEQ ID NO. 3, a VH-CDR1 with an amino acid sequence shown as SEQ ID NO. 4, a VH-CDR2 with an amino acid sequence shown as SEQ ID NO. 5 and a VH-CDR3 with an amino acid sequence shown as SEQ ID NO. 6;
The CDNF protein monoclonal antibody 2 comprises VL-CDR1 with an amino acid sequence shown as SEQ ID NO. 7, VL-CDR2 with an amino acid sequence shown as SEQ ID NO. 8, VL-CDR3 with an amino acid sequence shown as SEQ ID NO. 9, VH-CDR1 with an amino acid sequence shown as SEQ ID NO. 10, VH-CDR2 with an amino acid sequence shown as SEQ ID NO. 11 and VH-CDR3 with an amino acid sequence shown as SEQ ID NO. 12;
The BDNF protein monoclonal antibody 1 comprises a VL-CDR1 with an amino acid sequence shown as SEQ ID NO. 13, a VL-CDR2 with an amino acid sequence shown as SEQ ID NO. 14, a VL-CDR3 with an amino acid sequence shown as SEQ ID NO. 15, a VH-CDR1 with an amino acid sequence shown as SEQ ID NO. 16, a VH-CDR2 with an amino acid sequence shown as SEQ ID NO. 17 and a VH-CDR3 with an amino acid sequence shown as SEQ ID NO. 18;
The BDNF protein monoclonal antibody 2 comprises VL-CDR1 with an amino acid sequence shown as SEQ ID NO. 19, VL-CDR2 with an amino acid sequence shown as SEQ ID NO. 20, VL-CDR3 with an amino acid sequence shown as SEQ ID NO. 21, VH-CDR1 with an amino acid sequence shown as SEQ ID NO. 22, VH-CDR2 with an amino acid sequence shown as SEQ ID NO. 23 and VH-CDR3 with an amino acid sequence shown as SEQ ID NO. 24;
The GDNF protein monoclonal antibody 1 comprises VL-CDR1 with an amino acid sequence shown as SEQ ID NO. 25, VL-CDR2 with an amino acid sequence shown as SEQ ID NO. 26, VL-CDR3 with an amino acid sequence shown as SEQ ID NO. 27, VH-CDR1 with an amino acid sequence shown as SEQ ID NO. 28, VH-CDR2 with an amino acid sequence shown as SEQ ID NO. 129 and VH-CDR3 with an amino acid sequence shown as SEQ ID NO. 30;
The GDNF protein monoclonal antibody 2 comprises VL-CDR1 with an amino acid sequence shown as SEQ ID NO. 31, VL-CDR2 with an amino acid sequence shown as SEQ ID NO. 32, VL-CDR3 with an amino acid sequence shown as SEQ ID NO. 33, VH-CDR1 with an amino acid sequence shown as SEQ ID NO. 34, VH-CDR2 with an amino acid sequence shown as SEQ ID NO. 35 and VH-CDR3 with an amino acid sequence shown as SEQ ID NO. 36.
2. The monoclonal antibody composition according to claim 1, wherein one or more of the following conditions are met:
(1) The CDNF protein monoclonal antibody 1 comprises a light chain variable region with an amino acid sequence shown as SEQ ID NO. 37 and a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 38;
(2) The CDNF protein monoclonal antibody 2 comprises a light chain variable region with an amino acid sequence shown as SEQ ID NO. 39 and a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 40;
(3) The BDNF protein monoclonal antibody 1 comprises a light chain variable region with an amino acid sequence shown as SEQ ID NO. 41 and a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 42;
(4) The BDNF protein monoclonal antibody 2 comprises a light chain variable region with an amino acid sequence shown as SEQ ID NO. 43 and a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 44;
(5) The GDNF protein monoclonal antibody 1 comprises a light chain variable region with an amino acid sequence shown as SEQ ID NO. 45 and a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 46; and
(6) The GDNF protein monoclonal antibody 2 comprises a light chain variable region with an amino acid sequence shown as SEQ ID NO. 47 and a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 48.
3. Use of a monoclonal antibody composition according to any one of claims 1-2 for the preparation of a product for detecting neurodegenerative diseases.
4. A kit for detecting a neurodegenerative disease, comprising the monoclonal antibody composition of any one of claims 1-2.
5. The kit for detecting a neurodegenerative disease according to claim 4, wherein the kit comprises a capture antibody and a detection antibody, and one or more of the following conditions are satisfied:
(1) One of the CDNF protein monoclonal antibody 1 and the CDNF protein monoclonal antibody 2 is connected with a solid phase to be used as a capture antibody, and the other is used as a detection antibody after being marked with a fluorescent marker;
(2) One of the BDNF protein monoclonal antibody 1 and the BDNF protein monoclonal antibody 2 is connected with a solid phase carrier to be used as a capture antibody, and the other one is labeled with a fluorescent marker to be used as a detection antibody;
(3) And one of the GDNF protein monoclonal antibody 1 and the GDNF protein monoclonal antibody 2 is connected with a solid phase carrier to be used as a capture antibody, and the other is labeled with a fluorescent marker to be used as a detection antibody.
6. The kit for detecting a neurodegenerative disease according to claim 5, wherein one or more of the following conditions are satisfied:
(1) The solid support comprises magnetic particles;
(2) The fluorescent label comprises an acridinium ester.
7. A nucleic acid encoding the monoclonal antibody composition of claim 1.
8. A recombinant vector comprising the nucleic acid of claim 7.
9. A host cell comprising the recombinant vector of claim 8.
10. A method of preparing a monoclonal antibody composition comprising the steps of:
culturing the host cell of claim 9 to produce the monoclonal antibody composition.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107206078A (en) * | 2014-12-19 | 2017-09-26 | 上海易乐生物技术有限公司 | Specifically bind the application of the binding molecule of BDNF precursor protein |
| CN110831960A (en) * | 2017-05-04 | 2020-02-21 | 赫尔辛基大学 | C-terminal CDNF and MANF fragments, pharmaceutical compositions containing them and uses thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107206078A (en) * | 2014-12-19 | 2017-09-26 | 上海易乐生物技术有限公司 | Specifically bind the application of the binding molecule of BDNF precursor protein |
| CN110831960A (en) * | 2017-05-04 | 2020-02-21 | 赫尔辛基大学 | C-terminal CDNF and MANF fragments, pharmaceutical compositions containing them and uses thereof |
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