WO2013067750A1 - Polyclonal antibody immunologic mass spectrometry kit of liver cancer marker - Google Patents

Abstract

Disclosed is a polyclonal antibody immunologic mass spectrometry kit of liver cancer marker and a detection method. The kit comprises a serum polypeptide polyclonal antibody and buffer solution, wherein the polyclonal antibody is fixed on a solid phase carrier, the sold phase carrier being agarose particles coating protein A or protein G, the serum polypeptide polyclonal antibody is a polyclonal antibody against synthetic polypeptide 6, and the full-length sequence of the synthetic polypeptide 6 is NLGHG HKHDR DHGHG HQ.

Description

 Hepatoma marker multi-antibody immuno-mass spectrometry kit

 The invention belongs to the field of biotechnology, and particularly relates to a multi-anti-immuno-mass spectrometry kit and a detection method thereof. Background technique

 Primary liver cancer is one of the most common cancers in the world. The five-year survival rate of early diagnosis of malignant tumors can reach 70% -95%, while the five-year survival rate of advanced tumors is only 20% -30%. Under the premise that the treatment can not be improved quickly, improving the early diagnosis rate of malignant tumors, achieving early detection and early treatment of malignant tumors is an effective means to improve the overall survival rate of patients with malignant tumors, improve the quality of life, and reduce medical expenses. Current diagnostic tools commonly used in clinical practice include physical examination, imaging examination, serum tumor marker detection, and pathological examination. Due to the small volume of the early stage of the cancer, the liver is hidden in the deep upper part of the abdomen, and the ribs are used as a barrier. It is difficult to find early by using B-ultrasound and CT scanning. In addition, the liver has a strong compensatory function and often has no clinical symptoms in the early stage. It also brings difficulties to the early diagnosis of liver cancer. Although pathological examination is a diagnostic gold standard, it is difficult to obtain materials, and most of them are invasive examinations, which are not suitable for population screening. The non-invasive and reproducible serum tumor markers have theoretically become an ideal means for population screening for malignant tumors.

Advances in serum proteomics technology, including detection of serum sample separation and scale preparation techniques, high-throughput, high-sensitivity, high-resolution protein analysis and identification techniques, bioinformatics and statistical analysis techniques, all for proteomics Malignant tumor screening, early diagnosis, disease monitoring, and prognosis have laid the technical foundation, but the tumor marker proteome discovery technology still faces many difficulties. First of all, the composition of body fluids, especially serum samples, is extremely complex. In addition to interfering substances such as organic small molecule compounds and inorganic salts, thousands of proteins and polypeptides have a wide range of dynamic fluctuations; many disease-specific proteins are low-abundance proteins; During sample processing, the target protein may be removed simultaneously while removing high abundance proteins. Secondly, it is an experimental repetitive problem. The mass spectrometry technology has the characteristics of high sensitivity and high throughput. Can provide a huge amount of information. However, many factors in the experiment may affect the results of mass spectrometry. Such as the collection, processing, preservation of the sample, the conditions of the test and even the temperature and humidity at the time of detection may have more effects. In addition, from previous studies, different mass spectrometry systems, different sample processing methods, and different bioinformatics methods have been used to treat different diseases with different results.

 The biological mass spectrometer that has emerged in recent years has been successfully applied to the diagnosis of several cancers, such as breast cancer, ovarian cancer, prostate cancer, etc., mainly using surface-enhanced laser desorption ionization.

(SELDI) technology and immunomagnetic beads technology. Petricoin et al., using the FDA/NIH clinical proteomics program, used protein chips to enrich peptides or proteins in serum, and used SELDI-TOF MS technology to generate serum peptide maps of healthy people and patients, using bioinformatics. The pattern of the composition of the serum peptide map was analyzed and the early ovarian cancer was successfully diagnosed. The results showed that all 50 patients with ovarian cancer were correctly detected, of which 18 were stage I ovarian cancer patients; 66 were correctly detected in 63 patients with benign gynecologic tumors. The sensitivity, specificity, and positive predictive measures of ovarian cancer were 100%, 95%, and 94%, respectively, which were significantly better than traditional CA125 tests, especially for the diagnosis of early stage ovarian cancer (stage I). This technique is expected to be used for early or early warning of ovarian cancer.

However, with clinical application, the researchers further improved and improved the SELDI technology, and changed the sample purification and enrichment process originally performed on the solid chip to magnetic beads, which improved the poor repeatability, detection sensitivity and specificity. Not high disadvantages. In 2006, Villanueva et al. used immunomagnetic beads and mass spectrometry to study the serum peptides of 32 cases of prostate cancer, 21 cases of breast cancer and 20 cases of bladder cancer, and found that 14, 10 and 58 can be used as prostate cancer, breast cancer and The tumor marker polypeptide of bladder cancer has a prediction accuracy of 100%. This technology can effectively enrich and analyze low molecular weight proteins/peptides in samples such as blood, and it also has the advantages that SELDI does not have, such as the sample after enrichment is easy to elute for identification. However, this system has the disadvantages of being expensive and disadvantageously popularized with SELDI-TOF-MS. Therefore, the search for serum biomarker enrichment detection technology with low cost, strong enrichment ability and high repetitiveness has become one of the important research hotspots. Summary of the invention

 In order to solve the above technical problems, the present invention relates to a multi-antibody immuno-mass spectrometry kit for liver cancer markers.

 The polyclonal antibody anti-immunoassay detection kit for liver cancer markers provided by the invention comprises a serum polypeptide polyclonal antibody and a buffer, wherein the polyclonal antibody is immobilized on a solid phase carrier, and the solid phase carrier is coated egg A ( Protein A) or protein G (protein G) agarose particles. Wherein the polyclonal antibody is an anti-synthetic peptide 6 antibody, and the full length sequence of the synthetic polypeptide 6 is: NLGHG HKHDR DHGHG HQo

 Wherein, the buffer is preferably 0.01 mol/L, pH 7.4 in PBS buffer. Agarose granules coated with protein A or protein G (Protein A)

Agarose / Protein G Agarose is a matrix for immunoprecipitation and is more cost effective than Protein A or Protein A magnetic G magnetic beads.

 The multi-antibody immuno-mass spectrometry kit provided by the present invention preferably further comprises an eluent selected from the group consisting of a 0.1 mol/l glycine-HC1 solution having a pH of 2.7 and a mixture of 70% ACN containing 0.1% TFA. Mixture of 50% ACN with 0.1% TFA or 5°/. Acetic acid.

 The preferred anti-synthetic peptide 6 antibodies of the invention are prepared as follows:

 1) using the synthetic polypeptide 6 coupled with the carrier protein KLH as an immunogen, and combining the Freund's complete adjuvant with the incomplete adjuvant to immunize the white rabbit;

 2) After 3-4 weeks, the titer of the blood in the ear vein is detected, and the titer is above 1:50000.

 3) blood is taken from the carotid artery to obtain multi-antiserum;

 4) Purification of polyclonal antibodies by affinity chromatography.

The invention also provides a preparation method of the multi-antibody immuno-mass spectrometry kit, which comprises the steps of: mixing the purified polyclonal antibody with the solid phase carrier suspension to obtain a concentration of 0.075 g/L- 0.6 g ^ L, incubate at 4 ° C for 5 minutes - 4 hours, place 1-5 min precipitation, filter, use 0.01mol / L, pH 7.4 PBS buffer 100-200μί The solid phase carrier was washed 2 to 5 times to obtain the polypeptide immunoassay kit.

 The invention also provides a method of using the kit in a serum epitope antigen, comprising the steps of:

 1) Take 15-25μ1 solid phase carrier, place in Eppendorf tube, add 1.5 g-24 g polyclonal antibody, rotate and mix at 4 °C for 5 minutes - 4 hours, place for l-5 min, remove the supernatant, use 0.01 M, pH 7.4 PBS buffer 100-200nL washing the resulting solid phase carrier precipitate 2-5 times;

 2) taking 10-40μ1 serum sample, 10-50μ PBS, mixing with the solid phase carrier after step 1), rotating and mixing at 4 ° C for 8-24h;

 3) Place for 1-5 min, remove the supernatant, and wash the pellet 2-5 times with 100-200 μ PBS; move the suspension to another clean Eppendorf tube during the last wash;

 4) Add 10-15μί eluent, mix and drain for 1-5 min;

 5) Centrifuge and take the supernatant.

 The invention also provides a method for detecting a polypeptide marker by immuno Mass Spectrometry, and the polypeptide standard and the serum polypeptide marker antigen separated by the kit of the invention are detected by MALDI-TOF-MS, and the peak value and the theoretical peak value are less than 0.3 Da, This peak is the marker peak.

The detection conditions of MALDI-TOF-MS are positive ion detection mode, the ion source acceleration voltage is 20kV, N ₂ laser, laser wavelength 337 nm, energy 2500, ion delay extraction time 390 ns, mass spectrometry signal single scan cumulative 2000 times, Using the peptide II standard kit ion peak calibration, the mass scan range is 1000-10000 Da.

 The invention selects a plurality of polypeptide markers of liver cancer, and correspondingly prepares a plurality of polyclonal antibodies. Through detection and analysis and data statistics, it is unexpectedly found that the specificity and sensitivity of one of the polyclonal antibodies are much higher than other ones, indicating This marker has a strong ability to diagnose liver diseases. Thus, an immunomass detection kit and a detection method containing the polyclonal antibody are provided.

The immuno-mass spectrometry of the present invention uses a polyclonal antibody to enhance the enrichment specificity, and at the same time, the preparation process is simplified, further reducing the cost of the immuno-mass spectrometry kit. The invention combines conventional immunoassay technology with the most advanced mass spectrometry technology to make full use of the two technologies. High specificity, high throughput, high accuracy, high sensitivity, and low false positive are the latest frontiers of diagnostic technology. DRAWINGS

 1 is a schematic diagram of a multi-antibody immuno-mass spectrometric detection method of the present invention.

 Figure 2 is a mass spectrum of a synthetic peptide 6 standard sample in an embodiment of the present invention.

 Figure 3 is a mass spectrum of a liver cancer serum sample in an embodiment of the present invention.

 Figure 4 shows the detection specificity and sensitivity of the kit of the present invention. detailed description

 The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1 Anti-synthetic peptide 6 Preparation method of polyclonal antibody

 1) The synthetic polypeptide 6 conjugated to the carrier protein KLH was used as an immunogen to immunize the white rabbit; its full length sequence is: NLGHGHKHDRDHGHGHQ (SEQ ID No. 1).

 1) using the synthetic polypeptide 6 coupled with the carrier protein KLH as an immunogen, and combining the Freund's complete adjuvant with the incomplete adjuvant to immunize the white rabbit;

 2) After 3-4 weeks, the blood was collected from the ear vein to detect the titer, and the titer reached the blood standard of 1:50000;

 3) blood is taken from the carotid artery to obtain polyclonal antibody serum;

 4) Purification of polyclonal antibodies by affinity chromatography.

Example 2 Preparation method of multi-antibody immunoassay kit

20 μl of Protein A agarose particles (Protein A Agarose, Santa Cruz), placed in a 0.2 mLEppendorf tube, 7.5 μ _§ Multi-antibody prepared in Example 1, rotated at 4 ° C (rotation speed 5 r / min), mixed After 1 hour, it was allowed to stand for 2 minutes, and the supernatant was removed, and the resulting solid phase carrier was washed 3 times with 10 (^LPBS buffer (0.01 mol/l, pH 7.4).

Example 3 Preparation method of multi-antibody immunoassay kit

Take 25 μl of Protein A agarose particles (Protein A Agarose, Santa Cruz), place in a 0.2 mLEppendorf tube, add 24 g of the polyclonal antibody prepared in Example 1, rotate at 4 ° C (rotation speed 5 r / min), mix 2 Hour, place for 3 minutes, remove the supernatant, use lOO LPBS The resulting solid phase carrier was washed 5 times with a buffer (0.01 mol/l, pH 7.4).

Example 4 Preparation method of multi-antibody immunoassay kit

 15 μl of protein G agarose particles (Protein G Agarose, Santa Cruz) were placed in a 0.2 mLEppendorf tube, and 4.5 g of the polyclonal antibody prepared in Example 1 was added, 4'C rotation (rotation speed 5 r/min), mixing 4 After standing for 3 minutes, the supernatant was removed, and the resulting solid phase carrier was washed 3 times with 100 μL of PBS buffer (0.01 mol/l, pH 7.4).

Example 5 Multi-antibody immunoassay detection method

 The flow of the polypeptide immunoassay detection method of the present invention is shown in Fig. 1. Specifically: 1) Take 20 L of Protein A agarose particles (Protein A Agarose, Santa Cruz), placed in a 0.2 mLEppendorf tube, add 7.5 g of the polyclonal antibody prepared in Example 1, and rotate at 4 ° C (rotation speed 5 r /min), mixed for 0.5 hours, placed for 2 min, the supernatant was removed, and Protein G Agarose was washed 3 times with 100 mL of PBS buffer (0.01 mol/l, pH 7.4);

2) Take 24 g of synthetic peptide 6 peptide standard (solid phase chemical synthesis, Beijing Zhongke Yaguang Biotechnology Co., Ltd.), 5 (^LPBS, mixed with washed Protein G Agarose, and mix and rotate at 4 ° C for 12 h;

 3) Place the supernatant for 3 min, remove the Protein G Agarose 3 times with 200 μί PBS; move the suspension to another clean Eppendorf tube during the last wash;

 4) Add 15 L of 5% acetic acid eluent, and mix and drain for 3 min;

 5) Centrifugation, taking the supernatant and detecting with MALDI-TOF-MS, the spectrum is shown in Figure 2. As can be seen from the map, the peak of the peptide standard is obvious, and the theoretical deviation is less than 0.1 Da.

The detection condition is positive ion detection mode, the ion source acceleration voltage is 20kV, the N ₂ laser, the laser wavelength is 337 nm, the energy is 2500, the ion ion extraction time (PIE) is 390 ns, and the mass spectrometry signal is accumulated 2,000 times in a single scan. Using the peptide II standard kit (Bruker) ion peak correction (m/z700-m/z 3500), the mass scan range is 1000~10000Da.

The results showed that the synthetic peptide 6 standard was detected by the method of the present invention, and the obtained MALDI-TOF MS mass spectrum has the characteristics of high resolution and strong specificity. Example 6 Multi-antibody immuno-mass spectrometry

 The flow of the polypeptide immunoassay detection method of the present invention is shown in Fig. 1. Specifically:

1) 15 μί of Protein G agarose particles (Protein G Agarose, Santa Cruz), placed in a 0.2 mL Eppendorf tube, 4.5 g of the polyclonal antibody prepared in Example 1, and rotated at 4 ° C (rotation speed 5 r / min) , mixed for 4 hours, placed for 3 min, the supernatant was removed, and Protein G Agarose was washed 3 times with 100 μL of PBS buffer (0.01 mol/l, pH 7.4);

 2) Take 24 g of synthetic peptide 6 peptide standard (solid phase chemical synthesis, Beijing Zhongke Yaguang Biotechnology Co., Ltd.), 5 (^L PBS, mixed with washed Protein G Agarose, and mix and rotate at 4 °C for 16 h;

 3) Place the supernatant for 2 min, remove the Protein G Agarose 3 times with 200joL PBS; move the suspension to another clean Eppendorf tube during the last wash;

 4) Add 50% ACN eluate containing 0.1% TFA, and mix and drain for 3 min;

 5) Centrifugation, taking the supernatant, and detecting with MALDI-TOF-MS, the detection spectrum showed that the peak of the peptide standard was obvious, and the theoretical deviation was less than 0.1 Da.

Among them, the detection condition is positive ion detection mode, ion source acceleration voltage is 20kV, N ₂ laser, laser wavelength 337 nm, energy 2500, ion ion extraction time (PIE) 390 ns, mass spectrometry signal single scan cumulative 2000 The peptide II standard kit ( Bruker ) ion peak correction (m/z 700-m/z 3500) was used, and the mass scanning range was 1000 to 10000 Da.

Example 7 Multi-antibody immunoassay detection method

 The flow of the polypeptide immunoassay detection method of the present invention is shown in Fig. 1. Specifically:

1) Take 20μί coated protein A agarose granules (Protein A Agarose, Santa

Cruz ), placed in a 0.2 mL Eppendorf tube, 7.5 g of the polyclonal antibody prepared in Example 1, rotated at 4 ° C (rotation speed 5 r / min), mixed for 1 hour, placed for 2 min, the supernatant was removed, buffered with 100 nL PBS Washing Protein G Agarose 3 times with liquid (0.01mol/l, pH 7.4);

2) Take 3 (^L synthetic peptide 6 peptide standard (solid phase chemical synthesis, Beijing Zhongke Yaguang Biotechnology Co., Ltd.), 5 (^L PBS, mixed with cleaned Protein G Agarose, 4 °. Rotating and mixing for 8 hours;

 3) Place the supernatant for 3 min, wash the Protein G Agarose 3 times with 200 nL PBS; move the suspension to another clean Eppendorf tube during the last wash;

 4) Add 5 L of 5% acetic acid eluent, mix and drain for 3 min;

 5) Centrifugation, taking the supernatant and detecting by MALDI-TOF-MS, the peak value of the peptide marker is less than 0.2 Da. There are fewer peaks in the spectrum, indicating a higher specificity of the target peak.

 6) Another 30 L test 100 cases of liver cancer serum and 100 normal serum were detected by MALDI-TOF-MS. The mass spectrum of liver cancer serum is shown in Figure 3. Figure 3 is similar to Figure 2, with fewer peaks, indicating a higher specificity of the target peak. Figure 4 shows the sensitivity of the method of the invention of 83.0% and specificity of 93.3%.

Among them, the detection condition is positive ion detection mode, ion source acceleration voltage is 20kV, N ₂ laser, laser wavelength 337 nm, energy 2500, ion ion extraction time (PIE) 390 ns, mass spectrometry signal single scan cumulative 2000 The peptide II standard kit ( Bruker ) ion peak correction (m/z 700-m/z 3500) was used, and the mass scan range was 1000~10000 Da. Test example 1

 In the same manner as in Example 1, to synthesize peptide 5 (NLGHG HKHER DQGHG HQ) (known liver cancer marker) (SEQ ID No. 2), synthetic peptide 6 (NLGHG HKHDR DHGHG HQ) (mutant polypeptide of peptide 5) (SEQ ID No. 1), synthetic peptide 7 (NLGHG HKHKH DQGHG HQ) (mutant polypeptide of peptide 5) (SEQ ID No. 3) and synthetic peptide 8 (NLGHG HKHKR DHGHG HQ) (mutant polypeptide of peptide 5) (SEQ ID No. 4) Obtain respective polyclonal antibodies for the immunogen. According to the method of Example 7, 100 cases of liver cancer serum and 100 normal serum were examined, and the results are shown in the following table.

Immunogenicity sensitivity specificity

Synthetic peptide 5 70.8% 85.6%

Synthetic peptide 6 83.0% 93.3% Synthetic peptide 7 50.9% 67.7% Synthetic peptide 8 41.3% 78.7%

 As can be seen from the above table, it was unexpectedly found that the specificity and sensitivity of detection of liver cancer by multi-antibody immuno-mass spectrometry established by using synthetic peptide 6 as an immunogen were significantly higher than the other three markers, even more than before the mutation. Peptide 5 is high, so the use of synthetic peptide 6 polyclonal antibody as a material composition of the kit is more conducive to the detection and diagnosis of liver cancer.

 The above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make several improvements and retouchings without departing from the technical principles of the present invention. It should also be considered as the scope of protection of the present invention. Industrial applicability

 The immunoassay detection kit and detection method of the polyclonal antibody of the invention combines the conventional immunoassay technology with the most advanced mass spectrometry test technology, and fully utilizes the two technologies with high specificity, high throughput, high accuracy and high sensitivity. The advantages of sex, low false positive, etc., have realized the detection of liver cancer markers, and have important economic value and application prospects.

Claims

Claim A kit for detecting a multi-antibody immunological mass spectrometer of a liver cancer marker, comprising: a serum polypeptide polyclonal antibody and a buffer, wherein the polyclonal antibody is immobilized on a solid phase carrier, and the solid phase carrier is coated Agarose particles of protein A or protein G, the serum polypeptide polyclonal antibody is an anti-synthetic peptide 6 polyclonal antibody, and the full length sequence of the synthetic polypeptide 6 is shown in SEQ ID No. 1.  2. The polyclonal antibody immunoassay kit according to claim 1, further comprising an eluent selected from the group consisting of 0.1 mol/l glycine-HC1 solution having a pH of 2.7, and containing 0.1% TFA. A 70% ACN mixture containing 0.1°/. TFA's 50% ACN mixture or 5% acetic acid.  The polyclonal antibody immunoassay kit according to claim 1, wherein the buffer is 0.01 M, pH 7.4 PBS buffer.  The polyclonal antibody immunoassay kit according to any one of claims 1 to 3, wherein the anti-synthetic peptide 6 polyclonal antibody is prepared according to the following steps:  1) using the synthetic polypeptide 6 coupled with the carrier protein KLH as an immunogen, and combining the Freund's complete adjuvant with the incomplete adjuvant to immunize the white rabbit;  2) After 3-4 weeks, the titer of the blood in the ear vein is detected, and the titer is above 1:50000.  3) taking blood from the carotid artery and separating the multi-antiserum;  4) Purification of polyclonal antibodies by affinity chromatography.  The method for preparing a polyclonal antibody immunoassay kit according to any one of claims 1 to 4, comprising the steps of: mixing the purified polyclonal antibody with the solid phase carrier suspension , the obtained concentration is 0.075 g ^ L-0.6 g ^ L, incubated at 4 ° C for 5 minutes - 4 hours, placed for 1-5 min precipitation, filtered, with 0.01 mol / L, pH 7.4 PBS buffer 100 - The solid phase carrier was washed 2 to 5 times at 200 μί to obtain the polypeptide immunodetection kit. 6. The use of the kit of any one of claims 1 to 4 for separating a polypeptide marker in serum A method of antigen, comprising the steps of:  1) Take 15-25μ1 solid phase carrier, place in Eppendorf tube, add 1.5 g-24 g polyclonal antibody, rotate and mix at 4 °C for 5 minutes - 4 hours, place for 1-5 min, remove the supernatant, use 0.01 M, pH 7.4 PBS buffer 100-200ML cleaning obtained solid phase carrier precipitation 2-5 times;  2) taking 10-40μ1 serum sample, 10-50μί PBS, mixing with the solid phase carrier after step 1), rotating and mixing at 4 ° C for 8-24h;  3) Place for 1-5 min, remove the supernatant, and wash the pellet 2-5 times with 100-200^iL PBS; move the suspension to another clean Eppendorf tube during the last wash;  4) Add 10-15μί eluent, mix and drain for 1-5 min;  5) Centrifuge and take the supernatant.  A method for detecting an antigenic mass spectrometric of a polypeptide marker antigen, characterized in that the polypeptide marker antigen in serum isolated according to claim 6 is subjected to MALDI-TOF-MS detection.