CN114675019B - Kit for detecting insulin receptor extracellular domain antibody - Google Patents
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Abstract
The invention discloses a kit for detecting an insulin receptor extracellular domain antibody, and belongs to the technical field of biological medicines. The kit comprises: radioactivity I 125 Labeled insulin receptor extracellular domain protein, TBST buffer solution, protein A agarose and protein G agarose; the radioactivity I 125 The marked extracellular domain protein of the insulin receptor is Na-I 125 The protein is obtained by marking the insulin receptor extracellular domain protein, and the amino acid sequence of the insulin receptor extracellular domain protein is shown as SEQ ID NO. 1. The invention firstly provides the detection of the insulin receptor extracellular domain antibody, and takes the complex space structure of the extracellular domain different from the intracellular domain into consideration, and utilizes I 125 The method for marking the protein effectively reserves the complete disulfide bond of the extracellular domain protein, maintains the complex spatial structure of the extracellular domain protein and successfully establishes the detection of the insulin receptor extracellular domain antibody.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a kit for detecting an insulin receptor extracellular domain antibody.
Background
Type B Insulin Resistance Syndrome (TBIRS) is a clinical syndrome due to the production of autoantibodies against the insulin receptor in vivo, with severe hyperglycemia and insulin resistance being the major clinical features of the disease. The diagnosis of insulin resistance syndrome type B relies mainly on both clinical and immunological aspects, immunodiagnosis, i.e. the detection of insulin receptor antibodies. Insulin receptor antibody positivity is the standard for confirmation of diagnosis. Due to the limitation of experimental conditions, the detection of the insulin receptor antibody is not widely developed in clinic, and no mature kit exists, so that the research of most cases still depends on clinical diagnosis at present.
Insulin receptor antibodiesThe detection of (a) has lacked an efficient, accurate method in the past. One of the earlier detection methods that has been used to indirectly predict the presence of insulin receptor antibodies by competitive binding assays using insulin is time consuming and is only suitable for analysis of small samples and is currently performed in only a few laboratories worldwide. In 2008, use S appeared 35 The labeled insulin receptor is combined with a sample (Ping Zhou et al 2008), and the detection of an insulin receptor antibody is carried out by an immunoprecipitation method, but the method uses the full-length protein of the insulin receptor for labeling, and the insulin receptor protein as a transmembrane protein has hydrophobicity, is inevitably aggregated in the translation process, and influences the translation efficiency and the stability of an experiment; in addition, the extracellular domain of the insulin receptor protein is rich in disulfide bonds, and is not suitable for protein expression by the method, so that the disulfide bonds are deleted, and the formation of the original space structure of the whole protein is influenced.
Disclosure of Invention
The invention aims to provide a kit for detecting an insulin receptor extracellular domain antibody, which is characterized in that an antigen protein is obtained by constructing an insulin receptor protein extracellular domain plasmid and expressing the insulin receptor protein extracellular domain plasmid, and the antigen protein is obtained by I 125 After radioactive labeling, the detection of the insulin receptor extracellular domain antibody is realized.
In order to achieve the purpose, the invention adopts the following technical scheme:
a kit for detecting an insulin receptor extracellular domain antibody, comprising: radioactivity I 125 Labeled insulin receptor extracellular domain protein, TBST buffer solution, protein A agarose and protein G agarose;
the radioactivity I 125 The marked extracellular domain protein of the insulin receptor is Na-I 125 The protein is obtained by marking the insulin receptor extracellular domain protein, and the amino acid sequence of the insulin receptor extracellular domain protein is shown as SEQ ID NO. 1.
Further, the method adopts Na-I 125 The method is used for marking the extracellular domain protein of the insulin receptor and comprises the following specific processes: mixing Na-I 125 Adding into phosphate buffer, adding insulin receptor extracellular domain proteinAnd chloramine-T, then adding reducing agent sodium metabisulfite, and carrying out column chromatography to obtain the marked protein.
The invention constructs a plasmid containing an extracellular domain (consisting of an a chain and a part of a b chain) of an insulin receptor protein, obtains an antigen protein INRab through cell transfection, and then carries out I 125 The INRab after being labeled by the radioactive label still has good biological activity and can carry enough radioactive signals. Based on the improvement, the invention successfully establishes a first detection kit for the insulin receptor extracellular domain antibody INRabA.
Drawings
Fig. 1 is a graph of radiation readings for INRabA detection at different simulated sample addition levels.
Fig. 2 shows the results of the rbairraba test in healthy population.
Fig. 3 shows the distribution of INRabA in non-T1 DM, T1DM and healthy population, respectively.
Detailed Description
The detection of the insulin receptor antibody has extremely high reference significance for clinically diagnosing the B-type insulin resistance syndrome. In view of the low efficiency and instability of the detection method adopting the full-length protein in the past, the invention firstly evaluates the overall structure of the insulin receptor protein, selects and independently splits out the extracellular domain (consisting of an a chain and a part of a b chain) of the insulin receptor protein, and after avoiding a transmembrane hydrophobic region, the extracellular domain has good hydrophilicity and is convenient for the subsequent reaction. Due to the particularity that the extracellular domain is rich in disulfide bonds, S is reported in the past 35 Radioligand methods are not suitable for expression of this partial structure. Thus, the present invention is in Applicant's possession I 125 On the detection platform by the radioligand method, the integrity of all disulfide bonds of the structural domain is reserved, and the step I of the extracellular segment protein is completed 125 The marked insulin receptor extracellular domain protein INRab still has good biological activity and can carry enough radioactive signals. Based on the improvement, the invention successfully establishes a first detection kit for the antibody (INRabA) of the insulin receptor extracellular domain protein INRab.
The principle of the kit is to synthesize the insulin receptorExtracellular domain protein, marker I 125 And (3) placing the isotope in a liquid phase environment for combination, combining the insulin receptor extracellular domain antibody in the serum with the isotope in the liquid phase environment to form a polymer, grabbing the polymer by agarose and fixing the polymer on a PVDF flat plate, and calculating the amount of the antibody in the serum through the radioactive signal on the combined antigen protein.
The invention summarizes the experience and the deficiency of the past detection of the insulin receptor antibody on the basis of the existing radioligand detection method, combines the transmembrane structural characteristics of the insulin receptor protein, and firstly disassembles the insulin receptor into two parts, one part is an extracellular section, and the other part is an intracellular section, so that the disassembles effectively solves the adverse effect of protein aggregation caused by insolubility of water in a transmembrane region in the past detection, and also effectively distinguishes the antibodies aiming at different sites.
The invention is described in further detail below with reference to the figures and the specific examples, which should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.
Example 1
1. Experimental materials
1. Sample source: INRabA test positive quality control samples were obtained from purchased commercial antibodies against INRab intracellular fragment protein. Negative control serum samples were taken from healthy volunteers without family history of diabetes. 533 cases of Diabetic (DM) serum, with type 1 diabetes (T1 DM 270 cases) and non-type 1 diabetes (non-T1 DM 263 cases). 345 healthy people from the recruited population [ age (28.6 ± 4.6) years; 176 male and 169 female cases ]; the glucose tolerance test (OGTT) is fasting and normal in blood sugar for 2 hours, eliminates chronic and endocrine diseases such as heart, brain, liver, kidney and the like, and has no family history of diabetes and autoimmune disease history. All subjects signed informed consent.
2. Main reagents and instruments: na- 125 I (NEZ 033L, perkinElmer), protein A Sepharose PA (17-5280-02, GE); protein G sepharose PG (17061805,GE); 96-well PVDF Millipore filter plate (3504, corning); scintillation fluid (Microscint-20, perkin-Elmer); TBST buffer (Tris-Base 2.424g, naCl 8.70g, tween-20.5mL, distilled water to 1000mL, pH7.4); antigen buffer (TBST buffer plus bovine serum albumin 0.25mg/250 mL); INRabA (ab 44914, abcam); sephadex G-50 (17004301, GE); inorganic salts were purchased from Sigma. CO 2 2 Incubator (Thermo), nanODROP2000 (Thermo), β Counter liquid scintillation Counter (2450 Microplate Counter, perkin-Elmer).
2. Experimental methods
1. Construction of INRab plasmid
The INRab plasmid sequence includes a signal peptide + a chain + part b chain.
2. Transfecting cells, purifying the antigen protein
And (3) culturing 293F cells in a logarithmic growth phase, wherein the activity is more than 95%, adding the incubated plasmids into the cells, performing suspension culture at 37 ℃, counting the cells on the 4 th day, observing the cell state and the death rate, and harvesting the cells. The cell pellet was collected at 12000rpm for 15min, the cell supernatant was discarded, and the pellet was used for downstream purification. After the cells are broken, a mixture of a sample and a Ni-IDA-Sepharose CL-6B affinity chromatographic column after incubation is slowly added into a purification empty column by using a low-pressure chromatographic system. The collected protein solution was added to a dialysis bag and dialyzed overnight against 50mM Tris containing 300mM NaCl, pH8.0, to obtain purified antigen protein.
The amino acid sequence of the protein (SEQ ID NO. 1) is as follows:
MATGGRRGAAAAPLLVAVAALLLGAAGHLYPGEVCPGMDIRNNLTRLHELENCSVIEGHLQILLMFKTRPEDFRDLSFPKLIMITDYLLLFRVYGLESLKDLFPNLTVIRGSRLFFNYALVIFEMVHLKELGLYNLMNITRGSVRIEKNNELCYLATIDWSRILDSVEDNYIVLNKDDNEECGDICPGTAKGKTNCPATVINGQFVERCWTHSHCQKVCPTICKSHGCTAEGLCCHSECLGNCSQPDDPTKCVACRNFYLDGRCVETCPPPYYHFQDWRCVNFSFCQDLHHKCKNSRRQGCHQYVIHNNKCIPECPSGYTMNSSNLLCTPCLGPCPKVCHLLEGEKTIDSVTSAQELRGCTVINGSLIINIRGGNNLAAELEANLGLIEEISGYLKIRRSYALVSLSFFRKLRLIRGETLEIGNYSFYALDNQNLRQLWDWSKHNLTITQGKLFFHYNPKLCLSEIHKMEEVSGTKGRQERNDIALKTNGDQASCENELLKFSYIRTSFDKILLRWEPYWPPDFRDLLGFMLFYKEAPYQNVTEFDGQDACGSNSWTVVDIDPPLRSNDPKSQNHPGWLMRGLKPWTQYAIFVKTLVTFSDERRTYGAKSDIIYVQTDATNPSVPLDPISVSNSSSQIILKWKPPSDPNGNITHYLVFWERQAEDSELFELDYCLKGLKLPSRTWSPPFESEDSQKHNQSEYEDSAGECCSCPKTDSQILKELEESSFRKTFEDYLHNVVFVPRPSRKRRSLGDVGNVTVAVPTVAAFPNTSSTSVPTSPEEHRPFEKVVNKESLVISGLRHFTGYRIELQACNQDTPEERCSVAAYVSARTMPEAKADDIVGPVTHEIFENNVVHLMWQEPKEPNGLIVLYEVSYRRYGDEELHLCVSRKHFALERGCRLRGLSPGNYSVRIRATSLAGNGSWTEPTYFYVTDYLDVPSNIAK。
3. antigen protein labeling isotope
Na-I of 3mCi 125 The volume was 20. Mu.L, 80. Mu.L of 0.2M phosphate buffer PB was added, and 50. Mu.L of the antigen protein solution was added. 20. Mu.g/. Mu.L of chloramine-T (dissolved in 50mM PB) was added and shaken for 1.5min. Adding 40 μ G/20 μ L of reducing agent sodium metabisulfite (dissolved in 50mM PB solution), shaking for 2min, passing through column Sephadex G-50, eluting with 70mM barbitone solution, and collecting labeled antigen protein solution.
4. Binding and detection of a test sample to an antigenic protein
Adding 8 mu L of sample serum or simulation sample into each hole, wherein each sample and quality control serum are double-hole, taking a proper amount of labeled antigen, diluting the labeled antigen to 20000CPM/60 mu L by using 6ml of TBST buffer solution, adding 60 mu L of diluted INRab antigen into each hole, wherein the CPM value of each hole is required to be more than or equal to 20000, uniformly mixing the labeled antigen and the serum, oscillating for 1 hour, and standing overnight in a refrigerator at 4 ℃. PVDF plate, 150 u L TBST/hole, 4 degrees in the refrigerator overnight. The next day, the PVDF plate was decanted, 25. Mu.L of protein A/G mixed agarose (62.5% PA and 20% PG formulated as 4:1) was added to each well, 50. Mu.L of the mixed solution was sequentially removed from each well of the 96-well plate and transferred to a 96-well PVDF filter plate, mixed well in a refrigerator at 4 ℃ for 1h to precipitate the antigen-antibody complex, the mixture was removed by vacuum pump, 200. Mu.L of TBST buffer was added to each well of the PVDF filter plate to wash the precipitate, the liquid was removed by vacuum pump to leave the precipitate, 150. Mu.L of buffer was added to wash the mixture 7 times, the mixture was oven dried, 60. Mu.L of scintillation fluid was added to each well, the mixture was counted on a 96 Kong Counter, and 1min was counted per well.
The results were calculated as follows:
and the radiation Index (Index) = (specimen serum CPM-negative quality control CPM)/(positive quality control CPM-negative quality control CPM).
All data are counted by SPASS26 software, and all measurement data are in accordance with normal distribution so as to use average +/-standard deviation
The comparison among groups is shown by adopting a t test, a one-factor analysis of variance and an analysis of variance trend test. P<0.05 is significant in difference and has statistical significance.
3. Results of the experiment
Validity judgment of INRab antigen protein carrying radioactive signal for capturing INRabA
The purchased INRabA was used as a mock sample, incubated with INRab antigen with a radioactive signal, and captured. According to the experimental method described above, radiation readings at different simulated sample volumes were measured.
As shown in fig. 1, the radioactivity readings decreased with decreasing loading of the simulated samples, with a trend of very significant with analysis of variance trend test P < 0.0001. Therefore, the INRab antigen can effectively capture INRabA, and the radioactive signal carried by the INRab antigen changes along with the change of the content of the antibody.
In the subsequent experiments, the purchased antibody was used as the positive ginseng, and 0.2ug of antibody and CPM about 4693 as the sample amount of the positive ginseng were selected according to the signal-to-noise ratio (S/N) >15 (S/N: CPM value of different INRabA addition amounts/CPM value of the negative ginseng) and the cost, as shown in Table 1.
TABLE 1 Signal to noise ratio (S/N) at different INRabA addition levels
Normal human threshold determination for INRabA assay
The RBAINRabA of 115 cases of healthy human serum is measured, the radiation index is calculated, 99 percent of percentile points are taken as a threshold, the calculated positive threshold is 0.125, and the positive judgment standard is not less than 0.125, as shown in figure 2.
Intra-batch to batch variation of INRabA assay
Selection of 3 sera from normal humans and patients based on low, medium, high INRabA index was performed 5 times each (n = 5) in each of the batches and between the batches, and the Coefficient of Variation (CV) between the batches is shown in table 2.
TABLE 2 Intra-batch variation of INRabA assay
The result shows that the intra-batch CV of the RBA INRabA detection index is 4.81-8.83%, the inter-batch CV is 6.69-12.70%, and the repeatability is 100% according to the positive and negative results.
Distribution of INRabA in different populations
The percentage of INRabA in T1DM, non-T1 DM and healthy population was 3.7% (10/270), 0.38% (1/263) and 0.87% (2/230), respectively, and the difference was very significant when P was <0.01 by one-way anova. As shown in fig. 3.
The insulin receptor extracellular domain protein INRab of the firmware can effectively carry radioactive signals, and can effectively identify INRabA by a radioligand method, thereby filling the blank of INRabA detection.
Sequence listing
<110> Jiangsu province national hospital (the first subsidiary hospital of Nanjing medical university)
<120> kit for detecting insulin receptor extracellular domain antibody
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Val Lys Thr Leu Val Thr Phe Ser Asp Glu Arg Arg Thr Tyr Gly Ala
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Ile Arg Ala Thr Ser Leu Ala Gly Asn Gly Ser Trp Thr Glu Pro Thr
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Claims (5)
1. A kit for detecting an insulin receptor extracellular domain antibody is characterized in that: the method comprises the following steps: radioactivity I 125 Labeled insulin receptor extracellular domain protein, TBST buffer solution, protein A agarose and protein G agarose;
the radioactivity I 125 The marked extracellular domain protein of the insulin receptor is Na-I 125 Marking the insulin receptor extracellular domain protein;
the insulin receptor extracellular domain protein is an extracellular domain of the insulin receptor protein which is independently split, the extracellular domain of the insulin receptor protein consists of an a chain and a part of a b chain, a transmembrane hydrophobic region is avoided, and the insulin receptor extracellular domain protein has good hydrophilicity;
the amino acid sequence of the insulin receptor extracellular domain protein is shown in SEQ ID NO. 1.
2. The kit of claim 1, wherein: the above mentioned is Na-I 125 The method is used for marking the extracellular domain protein of the insulin receptor and comprises the following specific processes: mixing Na-I 125 Adding the mixture into a phosphate buffer solution, adding insulin receptor extracellular domain protein and chloramine-T, adding a reducing agent sodium metabisulfite, and carrying out column chromatography to obtain the marked protein.
3. The kit of claim 1, wherein: positive and negative controls are also included.
4. The kit of claim 3, wherein: the negative control is human serum.
5. Use of the kit of claim 1 for the preparation of a diagnostic agent for insulin resistance syndrome type B.
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| WO2018014091A1 (en) * | 2016-07-22 | 2018-01-25 | University Of Utah Research Foundation | Insulin analogs |
| CN110536899A (en) * | 2017-03-23 | 2019-12-03 | 韩美药品株式会社 | There is the insulin analog compound and application thereof of reduced affinity to insulin receptor |
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| AU2007262666B2 (en) * | 2006-06-22 | 2013-09-12 | Walter And Eliza Hall Institute Of Medical Research | Structure of the insulin receptor ectodomain |
| US8399407B2 (en) * | 2009-09-17 | 2013-03-19 | Case Western Reserve University | Non-standard insulin analogues |
| US10995129B2 (en) * | 2011-07-13 | 2021-05-04 | Case Western Reserve University | Non-standard insulin analogues |
| US11186623B2 (en) * | 2019-12-24 | 2021-11-30 | Akston Bioscience Corporation | Ultra-long acting insulin-Fc fusion proteins and methods of use |
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| US4888414A (en) * | 1987-04-09 | 1989-12-19 | Board Of Regents, University Of Texas System | Insulin-binding peptides and uses thereof |
| WO2018014091A1 (en) * | 2016-07-22 | 2018-01-25 | University Of Utah Research Foundation | Insulin analogs |
| CN110536899A (en) * | 2017-03-23 | 2019-12-03 | 韩美药品株式会社 | There is the insulin analog compound and application thereof of reduced affinity to insulin receptor |
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