CN117624351A - Anti-human phosphorylated tau181 rabbit monoclonal antibody and application thereof - Google Patents
Anti-human phosphorylated tau181 rabbit monoclonal antibody and application thereof Download PDFInfo
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- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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Abstract
The application belongs to the technical field of immunodetection, and discloses an anti-human phosphorylated tau181 rabbit monoclonal antibody and application thereof. The anti-human phosphorylated tau181 rabbit monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein LCDR1 of the light chain variable region comprises an amino acid sequence shown as SEQ ID NO.3, LCDR2 comprises an amino acid sequence shown as SEQ ID NO.4, and LCDR3 comprises an amino acid sequence shown as SEQ ID NO. 5; the HCDR1 of the heavy chain variable region comprises an amino acid sequence shown as SEQ ID NO.6, HCDR2 comprises an amino acid sequence shown as SEQ ID NO.7, and HCDR3 comprises an amino acid sequence shown as SEQ ID NO. 8. The rabbit monoclonal antibody can be combined with human phosphorylated tau181 protein with high specificity, and can be applied to an immunoassay kit prepared by a double-antibody sandwich ELISA or a chemiluminescence method, wherein the double-antibody sandwich ELISA detects human phosphorylated tau181 standard antigen with detection sensitivity of less than 75pg/ml, and the detection sensitivity of 50pg/ml can be achieved by detecting human phosphorylated tau181 standard antigen by a magnetic particle chemiluminescence method.
Description
Technical Field
The application relates to the technical field of immunodetection, in particular to an anti-human phosphorylated tau181 rabbit monoclonal antibody and application thereof.
Background
Alzheimer's disease (Alzheimer disease, AD) is a hidden, progressive neurodegenerative disorder that is manifested clinically by neuropsychiatric symptoms such as progressive memory impairment, cognitive dysfunction, personality changes, and language impairment, and whose typical histopathological changes are deposition of beta Amyloid (Abeta) that leads to senile plaques, loss of neurofibrillary tangles, neurons, and synapses due to abnormal phosphorylation of tau protein. AD not only severely affects the social, professional and life functions of the patient, but also brings great mental stress and heavy economic burden to the patient's home and society.
Tau protein is a protein encoded by chromosome 17 of human body, is widely present in organisms, especially nervous system, is a main component composing microtubules, and has functions of promoting microtubule assembly and maintaining microtubule stability. In the course of AD onset, microtubule structure changes, the integrity of microtubule structure is destroyed, dissociation of tubulin is promoted, and stability is destroyed. While tau protein bound to microtubules is also changed, hyperphosphorylated tau protein loses its biological activity in whole or in part, not only is binding force of the tau protein to the microtubules reduced, but also competes with normal microtubules to bind to the microtubules, thereby depolymerizing the microtubules, leading to degeneration of neurons starting from the protruding ends of the neurons, and finally leading to death of neurons and occurrence of AD.
According to the difference of phosphate sites, the P-Tau mainly comprises P-Tau181, P-Tau231, P-Tau217 and the like, and the detection of the content of the P-Tau181 in serum has higher clinical value for diagnosing AD. In the diagnosis and treatment guidelines for Alzheimer's disease in China (2020 edition), it is pointed out that in the conventional examination flow of AD differential diagnosis, blood routine, biochemical and serological examinations should be recommended, and in the blood examination, the P-Tau181 concentration can be used as a biomarker for distinguishing AD from non-AD dementia.
At present, ELISA detection kit is mostly adopted to detect Tau protein phosphorylation, namely, monoclonal antibody which specifically recognizes P-Tau181 is needed, in the prior art, anti-human phosphorylated Tau181 mouse monoclonal antibody and detection method are available, but no report on rabbit monoclonal antibody and detection method exists, so that anti-human phosphorylated Tau181 rabbit monoclonal antibody and detection method which have good specificity and high affinity for blood P-Tau181 are needed to be realized, and the anti-human phosphorylated Tau181 rabbit monoclonal antibody and detection method are used for screening and diagnosing Alzheimer disease in the whole clinical process.
Disclosure of Invention
In order to overcome the technical problems, the application provides an anti-human phosphorylated tau181 rabbit monoclonal antibody with good specificity and high affinity, and application of the anti-human phosphorylated tau181 rabbit monoclonal antibody in an immunodetection kit prepared by a double antibody sandwich ELISA or a chemiluminescence method, and provides auxiliary diagnosis for Alzheimer's disease related to human phosphorylated tau 181.
In a first aspect, the present application provides a rabbit monoclonal antibody against human phosphorylated tau181, comprising a light chain variable region and a heavy chain variable region.
The light chain variable region comprises LCDR1-3, wherein LCDR1 comprises an amino acid sequence shown as SEQ ID NO.3, LCDR2 comprises an amino acid sequence shown as SEQ ID NO.4, and LCDR3 comprises an amino acid sequence shown as SEQ ID NO. 5;
the heavy chain variable region comprises HCDR1-3, the HCDR1 comprises an amino acid sequence as shown in SEQ ID NO.6, the HCDR2 comprises an amino acid sequence as shown in SEQ ID NO.7, and the HCDR3 comprises an amino acid sequence as shown in SEQ ID NO. 8.
Alternatively, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.1, VL of 106 amino acids in full length, FR of 23, 17, 36 and 10 amino acids in the 4 domains, LCDR of 9, 3 and 8 amino acids in the 3 domains, LCDR1, LCDR2 and LCDR3 of 24aa-32aa,50aa-52aa and 89aa-96aa, respectively, and the amino acid sequences are: QASQNVYNN (SEQ ID NO. 3), EAS (SEQ ID NO. 4), AGGYSRTS (SEQ ID NO. 5).
The heavy chain variable region comprises an amino acid sequence shown as SEQ ID NO.2, wherein the total length of VH is 113 amino acids, the amino acid numbers of 4 structural domains of FR are respectively 24, 17, 38 and 11, the amino acid numbers of 3 structural domains of HCDR are respectively 8, 7 and 12, HCDR1, HCDR2 and HCDR3 are respectively 25aa-32aa,50aa-56aa and 95aa-102aa, and the amino acid sequences are respectively GFPLSNNYA (SEQ ID NO. 6), IGSSGST (SEQ ID NO. 7) and ARSYPGYS (SEQ ID NO. 8).
In a second aspect, the present application also provides an isolated polynucleotide encoding the rabbit monoclonal antibody of the first aspect against human phosphorylated tau 181.
In a third aspect, the present application also provides a recombinant expression vector comprising a polynucleotide according to the second aspect of the present application.
In a fourth aspect, the present application also provides a host cell comprising the recombinant expression vector of the third aspect, preferably the host cell is a prokaryotic cell or a eukaryotic cell.
In a fifth aspect, the present application also provides an immunoassay product for detecting human phosphorylated tau181, the immunoassay product comprising a rabbit monoclonal antibody of the first aspect of the present application.
Optionally, the immunodetection product is a detection kit, a chip or a detection test paper.
In a sixth aspect, the application also provides the use of the rabbit monoclonal antibody of the first aspect in the preparation of an immunoassay product for detecting human phosphorylated tau181 protein.
Optionally, the immunodetection product is a detection kit, a chip or a detection test paper.
Optionally, the detection kit is a double-antibody sandwich ELISA detection kit or a chemiluminescent immunoassay kit.
Compared with the prior art, the rabbit monoclonal antibody of the anti-human phosphorylated tau181 can be combined with the human phosphorylated tau181 protein in a high specificity and has high affinity, and the affinity constant Ka can reach 5.9 multiplied by 10 8 L/mol. The rabbit monoclonal antibody for resisting human phosphorylated tau181 can also be prepared into various immunodetection kits for detecting phosphorylated tau181, and particularly can be applied to immunodetection kits prepared by a double antibody sandwich ELISA or a chemiluminescence method. The double-antibody sandwich ELISA detects the human phosphorylated tau181 standard antigen, and the detection sensitivity is less than 75pg/ml; the detection sensitivity of the magnetic particle chemiluminescence method for detecting the human phosphorylated tau181 standard antigen can reach 50pg/ml at the lowest.
Drawings
FIG. 1 is an electrophoretogram of full-length amplification products of heavy and light chains of the rabbit monoclonal antibody OTIR4C1, M is a DNA molecular weight Marker.
FIG. 2 shows the detection of human phosphorylated tau181 polypeptide, non-phosphorylated tau181 polypeptide, human phosphorylated tau181 full-length protein and non-phosphorylated full-length tau protein samples by indirect sandwich ELISA, with the ordinate indicating the OD values of the detection.
FIG. 3 shows the detection of human phosphorylated tau181 standard antigen by double antibody sandwich ELISA, the abscissa shows the concentration (ng/ml) of human phosphorylated tau181 standard antigen and the ordinate shows the OD value of the detection. R of standard curve 2 =0.9994, linear detection range 0-10000pg/mL, and the sample concentration calculation formula is deduced: y=0.0003x+0.0703.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally performed under conventional conditions, those described in the laboratory manual, or those suggested by the manufacturer.
Example 1 preparation of anti-human phosphorylated tau181 rabbit monoclonal antibody
1) Preparation of human phosphorylated tau181 immunogen
The immunogenic polypeptide is designed according to the 1-441aa sequence of tau protein (NP 005901.2) (2N 4R variant), the polypeptide sequence is 160-200aa, the purity of the polypeptide is over 90%, and the purity requirement for preparing monoclonal antibody is met. The polypeptides were synthesized by peptide biochemistry limited in a third party.
2) Immunization of animals
The synthesized human phosphorylated tau181 polypeptide is emulsified by complete Freund's adjuvant in a volume ratio of 1:1, about 2kg of New Zealand white rabbits are immunized by subcutaneous injection, the immunization dose is 800 mug/animal, the immunization is carried out for the second time after two weeks, the incomplete Freund's adjuvant is emulsified in a volume ratio of 1:1, and the immunization dose is 400 mug/animal. Tail blood is taken after two times of immunization, and serum titer is measured by ELISA method gradient dilution; and judging whether to collect PBMCs or continue immunization according to the result by taking OD450 at ELISA titer 128000 as a standard and selecting rabbits with highest antibody titers for collecting the PBMCs.
3) PBMCs separation, specific B cell separation, cloning recombination
Fixing rabbit on operation table, removing hair from heart, sterilizing skin with alcohol, selecting the most obvious heart beat, puncturing with 50ml syringe, injecting blood into syringe after needle, rapidly removing needle after obtaining required blood volume, transferring whole blood in syringe into sterile 50ml tube, mixing with equal amount of PBS, slowly adding dropwise above lymphocyte separating liquid, centrifuging at room temperature 400×g for 30min, and separating liquid level into four layers from top to bottom: the rabbit PBMCs are obtained by carefully sucking the mononuclear cell layer and washing and removing the platelet and lymphocyte separating liquid by PBS.
Antigen-specific B cells were further sorted from rabbit PBMCs for culture, and the supernatant of the cultured B cells was screened for positive clones using antigen-coated ELISA plates. The positive cloned cells are collected, lysed, RNA is extracted and reversely transcribed into cDNA, the natural paired rabbit monoclonal antibody light chain and heavy chain full-length sequences are amplified from the cDNA corresponding to the positive clone, a rabbit monoclonal antibody expression vector is constructed by a cloning recombination method, the sequences are determined by sequencing, and the amplified full-length PCR product results are shown in figure 1.
4) Preparation and purification of monoclonal antibodies
To obtain a plurality of rabbit monoclonal antibodies recognizing human phosphorylated tau181 protein, heavy chain and light chain genes of the rabbit monoclonal antibodies are loaded on an expression vector, KEK293 cells are transfected by plasmids, and the recombinant rabbit monoclonal antibodies recognizing human phosphorylated tau181 protein are obtained from culture supernatants after 120-144 hours of transfection. Collecting cell suspension, centrifuging to obtain supernatant, and performing antibody purification by affinity chromatography. The concentration of the purified monoclonal antibody was measured by BCA method, and then sub-packaged and lyophilized to give the purified antibody designated as rabbit monoclonal antibody otor 4C1.
Example 2 identification of anti-human phosphorylated tau181 rabbit monoclonal antibody OTIR4C1
1) Specificity identification of Rabbit monoclonal antibodies
Indirect ELSA assay was used. Coating ELISA plate with human phosphorylated tau181 polypeptide, non-phosphorylated tau181 polypeptide, human phosphorylated tau181 full-length protein and non-phosphorylated full-length tau protein, sealing ELISA plate with PBST containing 1% BSA at 4deg.C overnight at concentration of 1 μg/ml, adding 10 4 The purified rabbit monoclonal antibody OTIR4C1 subjected to double dilution is reacted for 50min at 37 ℃, PBST is washed for 3 times, HRP-goat anti-rabbit Ig secondary antibody is added, the reaction is carried out for 50min at 37 ℃, PBST is washed for 5 times, TMB is added for color development for 10min, stop solution is added, and A450 is measured by an enzyme-labeled instrument.
FIG. 2 shows that both the non-phosphorylated tau181 polypeptide and the non-phosphorylated full-length tau protein react negatively with the rabbit monoclonal antibody OTIR4C1, with OD450 less than 0.1; the human phosphorylated tau181 polypeptide and the human phosphorylated tau181 full-length protein react positively with the OTIR4C1 antibody, and OD450 is larger than 1, which indicates that the anti-human phosphorylated tau181 rabbit monoclonal antibody OTIR4C1 specifically recognizes a 181 threonine (T) phosphorylation site epitope in tau protein.
2) Affinity constant determination of rabbit monoclonal antibodies
Affinity constants (Ka) were determined by non-competitive ELISA.
Coating: diluting antigen with carbonate buffer solution to concentration of 1, 0.5, 0.1 and 0.05 mug/mL, respectively coating the antigen with 96-well ELISA plates according to 100 mug/well, and incubating for 24 hours at 4 ℃;
closing: the plates were washed 4 times with PBST, BSA solution was added at 200. Mu.L/well and incubated for 2h at 37 ℃;
adding monoclonal antibody: plates were washed 4 times with PBST, and mab OTIR4C1 was diluted with carbonate buffer at a starting doubling ratio of 100. Mu.g/mL, 100. Mu.L was added to each well and incubated for 2h at 37 ℃;
adding enzyme-labeled secondary antibodies: washing the plate with PBST for 4 times, adding 100 mu L of L10000 times diluted goat anti-rabbit Ig secondary antibody at 37 ℃ into each hole, and standing for 30min;
color development and termination: washing the plate with PBST for 4 times, adding 100 mu L of substrate color development liquid into each hole, and carrying out light-shielding reaction for 15min at 37 ℃; 50. Mu.L of 1.0mol/L H was added to each well 2 SO 4 A stop solution for stopping the reaction;
and (3) detection: the absorbance at a wavelength of 450nm (A450 nm) was measured.
Drawing an S-shaped curve by taking the logarithm of the concentration of the antibody as the abscissa and the OD value as the ordinate, and calculating the affinity constant Ka=5.9×10 of the anti-human phosphorylated tau181 rabbit monoclonal antibody OTIR4C1 8 L/mol。
3) Antibody pairing
To select the optimal combination of coated and detection antibodies, three purified rabbit monoclonal antibodies, OTIR5A6, OTIR5F1 and OTIR4℃ 1, were coated on the ELISA plate, respectively, overnight at 4 ℃. The enzyme label plate is taken out the next day, PBST is washed once, 1% BSA solution is blocked for 2 hours at 37 ℃, and PBST is washed 3 times; 100 μl of human phosphorylated tau181 full-length protein was added to each well at a concentration of 20ng/ml and incubated for 1 hour at 37 ℃; after the incubation, the ELISA plate was removed, washed 3 times with PBST, and HRP-labeled murine monoclonal antibody OTI9A5 was added as a detection antibody, and incubated at 37℃for 1 hour. PBST was washed 5 times, TMB substrate was added, and color development was performed at 37℃for 10min. After removal, stop solution was added and OD450 readings were measured on an microplate reader. The optimal rabbit monoclonal antibody pair is selected according to the OD value of the sample and the background value of the negative control, and the pairing screening result is shown in Table 1.
Table 1 results of screening of antibody pairing experiments
It can be seen that the antibody OTIR4C1 referred to in this application was the best coated antibody, and murine monoclonal antibody OTI9A5 was the best detection antibody.
Example 3 analysis of the variable region Gene and amino acid sequence of the rabbit monoclonal antibody OTIR4C1 recombinant plasmid of the OTIR4C1 antibody was used as a DNA template, and the light chain variable region and heavy chain variable region sequencing primers were designed based on the 5' -end vector sequences of the light chain and heavy chain on the template, and sequenced using a sequencer ABI 3730. The nucleotide sequence of the variable region of the light chain and the heavy chain of the rabbit monoclonal antibody OTIR4C1 is obtained through sequencing.
The nucleotide sequences of the light chain variable region and the heavy chain variable region are respectively subjected to sequencing result data analysis by using IMGT/V-QUEST analysis software on http:// www.imgt.org through the Internet, the amino acid sequence of the light chain variable region of the rabbit monoclonal antibody OTIR4C1 is shown as SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2. VL is 106 amino acids in total, the number of amino acids in 4 domains of FR is 23, 17, 36 and 10, the number of amino acids in3 domains of LCDR is 9, 3 and 8, and the regions of LCDR1, LCDR2 and LCDR3 are 24aa-32aa,50aa-52aa and 89aa-96aa, respectively, and the amino acid sequences thereof are: QASQNVYNN (SEQ ID NO. 3), EAS (SEQ ID NO. 4), AGGYSRTS (SEQ ID NO. 5); the total length of VH is 113 amino acids, the number of 4 structural domain amino acids of FR is 24, 17, 38 and 11 respectively, the number of 3 structural domain amino acids of HCDR is 8, 7 and 12 respectively, HCDR1, HCDR2 and HCDR3 are 25aa-32aa,50aa-56aa and 95aa-102aa respectively, and the amino acid sequences are GFPLSNNYA (SEQ ID NO. 6), IGSSGST (SEQ ID NO. 7) and ARSYPGYS (SEQ ID NO. 8) respectively.
Example 4 anti-human phosphorylated tau181 rabbit monoclonal antibody OTIR4C1 is used for preparing double antibody sandwich ELISA detection kit the detection technology based on ELISA double antibody sandwich principle, and human phosphorylated tau181 detection kit is prepared for clinical auxiliary diagnosis of diseases related to human phosphorylated tau 181.
1. Kit composition
1. Laths coated with OTIR4C1 antibodies: diluting the antibody to 1 mug/ml with PBS buffer, coating onto a microplate, incubating overnight at 4 ℃ with 100 mug/well, washing 3 times with PBST, and spin-drying; blocking with PBS containing 1% BSA, 5% sucrose and 0.05% proclin300, reacting at 37deg.C for 2 hr, discarding the blocking solution in the hole, and drying; and (3) placing the coating plate in a baking oven at 37 ℃ for 2 hours to finish coating, sealing by an aluminum foil bag, and storing at 4 ℃ for standby.
2. Reagent preparation
2.1 preparation of enzyme conjugate the simple sodium periodate method is adopted to label anti-human non-phosphorylated tau protein mouse monoclonal antibody OTI9A5, the working concentration is titrated, 1:20 concentration solution is prepared, the dilution solution is PBS containing 1% BSA, 5% glycerol and 0.05% Proclin300, and filtering and sterilizing are carried out.
2.2 washing buffer was conventional PBST at pH7.4 containing 0.05% Proclin300 and was formulated as a 20-fold concentrate.
2.3 substrate solution (color developing solution) of enzyme one-component TMB (purchased from market).
2.4 sample dilutions contained 1% BSA, 0.05% Proclin300 in PBS, and were sterilized by filtration.
2.5 stop solution 10ml HCl (36-38%) was added to 110ml distilled water and the mixing was slowly performed to prepare 1N HCl.
2.6 Standard full Length human phosphorylated tau181 protein, samples were diluted to 20 μg/ml with PBS containing 1% BSA, 5% sucrose, 10% glycerol, 0.05% Proclin300 as dilution, filtered, sterilized, and aseptically packaged.
2. Detecting the operation key points
1. In order to ensure the accuracy of the detection result, it is suggested that both the standard and the sample are provided with double-hole measurement. Standard curves are needed for each detection.
2. If the content of the substance to be detected in the sample is too high, the sample is diluted by the sample diluent to ensure that the sample accords with the detection range of the kit, and finally, the sample is multiplied by the corresponding dilution multiple during calculation.
3. Sample adding: during sample addition, a disposable clean suction head is required to be used, so that cross contamination is avoided. The sample should be added as slowly as possible to avoid foaming, and the sample is added at the bottom of the ELISA plate hole without adding the sample along the hole wall.
4. Incubation: in order to prevent sample evaporation or contamination, the ELISA plate must be covered and attached during incubation, and the ELISA plate should be prevented from being in a dry state during the experiment. During the incubation process, whether the temperature of the incubator is constant at 37 ℃ or not should be observed at any time, and the temperature should be adjusted in time. During the incubation, the incubator is not easily opened too many times to avoid affecting the temperature balance.
5. Washing: the washing process is very important, and insufficient washing is prone to false positives.
6. Color development: in order to ensure the accuracy of the experimental result, the stop solution should be added as soon as possible after the substrate reaction time. The color development can be observed at intervals after the substrate solution is added to control the reaction time (e.g., at intervals of 10 minutes). When the front 3-4 holes of the standard product are visible to naked eyes and have obvious gradient blue, and the color development of the rear 3-4 holes is not obvious, stopping solution can be added to stop the reaction, and the blue color immediately turns yellow. The order of addition of the stop solution should be as similar as possible to the order of addition of the substrate solution.
7. The substrate solution should be bluish or colorless and must be discarded if the color becomes severely darkened. The substrate solution is easy to be polluted and is required to be preserved properly in dark place.
3. Determination of standard human phosphorylated tau181 protein different dilutions standard curve
The prepared double-antibody sandwich ELISA detection kit for detecting the human phosphorylated tau181 protein is taken out from a refrigerator at 4 ℃ and balanced to the room temperature. The standard was diluted from 20. Mu.g/ml to 10000pg/ml with PBS to prepare 9 series of concentration standard substances of 0, 75, 150, 300, 600, 1200, 2500, 5000, 10000pg/ml, 100. Mu.l of the standard substance was added to each well for incubation according to the above detection method, then the liquid was discarded, an HRP-labeled detection antibody working solution was added for incubation, the liquid in the well was discarded, a substrate solution was added to each well after spin-drying, a stop solution was added to each well after development in the absence of light, and the optical density OD value of each well was measured sequentially at 450nm with an enzyme-labeled instrument within 5 minutes after termination of the reaction to make a standard curve, and the results are shown in Table 2.
Table 2 double antibody sandwich ELISA method for detecting human phosphorylated tau181 standard substance
A standard curve for human phosphorylated tau181 assay was generated according to Table 2, see FIG. 3. R of standard curve 2 =0.9994, linear detection range 0-10000pg/mL, and the sample concentration calculation formula is deduced: y=0.0003x+0.0703, and the detection sensitivity is less than 75pg/ml. Example 5 anti-human phosphorylated tau181 rabbit monoclonal antibody otor 4C1 was used for magnetic particle chemiluminescent immunoassay.
1. Detection principle and method
Adopts a magnetic particle chemiluminescence immunoassay technology based on the principle of a double-antibody sandwich method. Biotin marks a rabbit monoclonal antibody OTIR4C1 of anti-human phosphorylated tau181, the biotinylated OTIR4C1 is fixed with SA magnetic beads, ALP is coupled with a non-phosphorylated tau protein mouse monoclonal antibody OTI9A5, and meanwhile, the full-length protein of the human phosphorylated tau181, an ALP substrate and corresponding buffer components are placed on a Kestelmi full-automatic magnetic particle chemiluminescence instrument, and an instrument program is set for detection. And judging that the result is positive according to the fact that the signal to noise ratio of the luminescence value is larger than 2.0. The magnitude of the luminescence value reflects the amount of bound enzyme-labeled antibody, which is proportional to the concentration of human phosphorylated tau181 in the sample. Drawing a standard curve according to the measured luminous value of the standard substance, and obtaining the concentration value of the human phosphorylated tau181 in the sample to be measured from the standard curve.
2. Magnetic particle chemiluminescence detection kit for detecting human phosphorylated tau181
SA magnetic bead binding biotin-OTIR 4C1: taking 50 μl of magnetic beads into a 0.5ml centrifuge tube, placing on a magnetic rack, and removing supernatant after 1 min; washing the beads 3 times with 0.5ml of antibody dilution; adding a certain amount of biotin-labeled OTIR4C1, and mixing at room temperature for 60min; after magnetic separation, the suspension was resuspended in magnetic preservation buffer at a working concentration of 0.5mg/ml.
OTI9A5 coupled ALP: the antibody OTI9A5 was first reduced using 2-IT; and secondly, ALP-SMCC intermediate formation is carried out, and finally, ALP-SMCC is coupled with a reducing antibody. After the coupling, the mixture was diluted to the working concentration using ALP preservation buffer.
3. The wash buffer was the same as in example 4.
4. Chemiluminescent developer is purchased from lovir organisms.
5. The sample dilutions contained 1% BSA, 0.05% Proclin300 in PBST, and were sterilized by filtration.
6. Standard full-length human phosphorylated tau181 full-length protein, a sample is diluted to 5 mug/ml by using PBS containing 1% BSA, 5% sucrose, 10% glycerol and 0.05% Proclin300 as a diluent, filtered, sterilized and aseptically packaged.
3. Detection of human phosphorylated tau181 standard antigen
The human phosphorylated tau181 standard antigen was subjected to gradient dilution of 0pg/ml, 50pg/ml, 100pg/ml, 200pg/ml, and 500pg/ml, respectively, and 5 gradient diluted antigens were detected by the above detection method using the OTIR4C1 antibody as the coating antibody and the OTI9A5 antibody as the detection antibody, and the detection results are shown in Table 3.
TABLE 3 detection of human phosphorylated tau181 Standard antigen by magnetic microparticle chemiluminescence
The rabbit monoclonal antibody is used for a magnetic particle chemiluminescence immunoassay reagent according to the judgment that the signal-to-noise ratio is greater than 2.0, the lowest sensitivity is 50pg/ml, and the linear range is 0-500pg/ml.
In conclusion, the anti-human phosphorylated tau181 rabbit monoclonal antibody is applied to an immunoassay kit prepared by a double-antibody sandwich ELISA or a chemiluminescence method, the double-antibody sandwich ELISA detects phosphorylated tau181 standard antigen, the detection sensitivity can reach 75pg/ml, and the magnetic particle chemiluminescence method detects phosphorylated tau181 standard antigen, and the detection sensitivity can reach 50pg/ml.
The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.
Claims (10)
1. A rabbit monoclonal antibody against human phosphorylated tau181, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1-3, wherein LCDR1 comprises an amino acid sequence shown as SEQ ID No.3, wherein LCDR2 comprises an amino acid sequence shown as SEQ ID No.4, and wherein LCDR3 comprises an amino acid sequence shown as SEQ ID No. 5; the heavy chain variable region comprises HCDR1-3, the HCDR1 comprises an amino acid sequence as shown in SEQ ID NO.6, the HCDR2 comprises an amino acid sequence as shown in SEQ ID NO.7, and the HCDR3 comprises an amino acid sequence as shown in SEQ ID NO. 8.
2. The rabbit monoclonal antibody of claim 1, wherein the light chain variable region comprises an amino acid sequence set forth in SEQ ID No.1 and the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID No. 2.
3. An isolated polynucleotide encoding the rabbit monoclonal antibody of any one of claims 1-2.
4. A recombinant expression vector comprising the polynucleotide of claim 3.
5. A host cell comprising the recombinant expression vector of claim 4, wherein the host cell is a prokaryotic cell or a eukaryotic cell.
6. An immunoassay product of human phosphorylated tau181, comprising the rabbit monoclonal antibody of claim 1 or 2.
7. The immunoassay product of claim 6, wherein the immunoassay product is a test kit, chip or test strip.
8. Use of a rabbit monoclonal antibody according to claim 1 or 2 for the preparation of an immunoassay product for detecting human phosphorylated tau181 protein.
9. The use according to claim 8, wherein the immunodetection product is a detection kit, a chip or a detection strip.
10. The use according to claim 9, wherein the detection kit is a double antibody sandwich ELISA detection kit or a chemiluminescent immunoassay kit.
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