阅读说明：本技术 百日咳毒素产品和百白破疫苗中活性蛋白的测定方法 (Determination method of active protein in pertussis toxin product and pertussis vaccine ) 是由 龙珍 卫辰 李月琪 马霄 姚劲挺 冀峰 李长坤 骆鹏 王丽婵 黄涛宏 于 2018-07-03 设计创作，主要内容包括：本发明公开了一种百日咳毒素产品和百白破疫苗中活性蛋白的定性、定量测定方法。采用高效液相色谱串联质谱方法,建立了一种高通量、高选择性、高灵敏度的百日咳毒素产品和百白破疫苗活性蛋白百日咳毒素、丝状血凝素、黏附素、菌毛蛋白和腺苷酸环化酶毒素的定量方法。本方法首次从复杂的疫苗基质中筛查出可用于各活性疫苗蛋白定性和定量分析的特征肽段,这些肽段与其它所报道的蛋白肽段不同,不能通过蛋白检索库获得,也不能通过这些疫苗蛋白的氨基酸序列简单得出。可实现不同生产厂家、不同批次百日咳毒素产品和百白破疫苗中百日咳毒素5种亚基、丝状血凝素、黏附素、菌毛蛋白和腺苷酸环化酶毒素的同时定量。(The invention discloses a qualitative and quantitative determination method for active protein in pertussis toxin products and pertussis vaccines. A high-flux, high-selectivity and high-sensitivity quantitative method for pertussis toxin products and pertussis vaccine active proteins pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin is established by adopting a high performance liquid chromatography-tandem mass spectrometry method. The method screens characteristic peptide fragments which can be used for qualitative and quantitative analysis of each active vaccine protein from a complex vaccine matrix for the first time, and the peptide fragments are different from other reported protein peptide fragments and cannot be obtained through a protein search library or simply obtained through amino acid sequences of the vaccine proteins. Can realize the simultaneous quantification of 5 subunits of pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin in pertussis toxin products and pertussis vaccines of different manufacturers and batches.)

1. The qualitative identification method of 5 subunits of pertussis toxin in the pertussis toxin product is characterized by comprising the following steps:

(1) carrying out enzymolysis pretreatment on a pertussis toxin product to obtain an enzymolysis liquid;

(2) respectively carrying out high performance liquid chromatography tandem mass spectrometry detection on the pertussis toxin standard solution and the enzymatic hydrolysate obtained in the step (1), and qualitatively identifying by ion-to-nucleus ratio and retention time of a characteristic peptide segment;

the pertussis toxin S1 subunit characteristic peptide segment is: SCQVGSSNSAFVSTSSSR, ILAGALATYQSEYLAHR, ANPNPYTSR, SVASIVGTLVR, respectively;

the pertussis toxin S2 subunit characteristic peptide segment is: GSGDLQEYLR, NTGQPATDHYYSNVTATR, LLSSTNSR, DGTPGGAFDLK, respectively;

the pertussis toxin S3 subunit characteristic peptide segment is: DAPPGAGFIYR, TGQPAADHYYSK, LLASTNSR, GNAELQTYLR, respectively;

the pertussis toxin S4 subunit characteristic peptide segment is: LGAAASSPDAHVPFCFGK, QLTFEGKPALELIR, AVFMQQRPLR, RPGSSPMEVMLR, respectively;

the pertussis toxin S5 subunit characteristic peptide segment is: IYSPADVAGLPTHLYK, IYSPADVAGLPTHLYK, NFTVQELALK are provided.

2. The method for quantitatively detecting 5 subunits of pertussis toxin in a pertussis toxin product is characterized by comprising the following steps of:

(1) carrying out enzymolysis pretreatment on a pertussis toxin product to obtain an enzymolysis liquid, and fixing the volume of the enzymolysis liquid to obtain a liquid to be detected;

(2) performing high performance liquid chromatography tandem mass spectrometry detection on the enzymatic hydrolysate obtained in the step (1), obtaining the content of each subunit of pertussis toxin in the liquid to be detected according to the chromatographic peak area and the standard working curve of the characteristic peptide segment of each subunit of pertussis toxin, and obtaining the content of each subunit in the pertussis toxin product through the conversion of the volume of the liquid to be detected and the volume of the pertussis toxin product; the chromatographic peak area of each subunit characteristic peptide segment is the sum of the chromatographic peak areas of any three or more characteristic peptide segments;

the pertussis toxin S1 subunit characteristic peptide segment is: SCQVGSSNSAFVSTSSSR, ILAGALATYQSEYLAHR, ANPNPYTSR, SVASIVGTLVR, respectively;

the pertussis toxin S2 subunit characteristic peptide segment is: GSGDLQEYLR, NTGQPATDHYYSNVTATR, LLSSTNSR, DGTPGGAFDLK, respectively;

the pertussis toxin S3 subunit characteristic peptide segment is: DAPPGAGFIYR, TGQPAADHYYSK, LLASTNSR, GNAELQTYLR, respectively;

the pertussis toxin S4 subunit characteristic peptide segment is: LGAAASSPDAHVPFCFGK, QLTFEGKPALELIR, AVFMQQRPLR, RPGSSPMEVMLR, respectively;

the pertussis toxin S5 subunit characteristic peptide segment is: IYSPADVAGLPTHLYK, IYSPADVAGLPTHLYK, NFTVQELALK are provided.

3. The quantitative detection method of 5 subunits of pertussis toxin in the pertussis vaccine is characterized by comprising the following steps:

(1) taking the diphtheria-pertussis-tetanus vaccine for enzymolysis pretreatment to obtain an enzymolysis liquid, and fixing the volume of the enzymolysis liquid to obtain a liquid to be detected;

(2) performing high performance liquid chromatography tandem mass spectrometry detection on the enzymatic hydrolysate obtained in the step (1), obtaining the content of each subunit of pertussis toxin in the liquid to be detected according to the chromatographic peak area and the standard working curve of the characteristic peptide segment of each subunit of pertussis toxin, and obtaining the content of each subunit in the pertussis vaccine through the conversion of the volume of the liquid to be detected and the volume of the pertussis vaccine; the chromatographic peak area of each subunit characteristic peptide segment is the sum of the chromatographic peak areas of any three or more characteristic peptide segments;

the pertussis toxin S1 subunit characteristic peptide segment is: SCQVGSSNSAFVSTSSSR, ILAGALATYQSEYLAHR, ANPNPYTSR, SVASIVGTLVR, respectively;

the pertussis toxin S2 subunit characteristic peptide segment is: GSGDLQEYLR, NTGQPATDHYYSNVTATR, LLSSTNSR, DGTPGGAFDLK, respectively;

the pertussis toxin S3 subunit characteristic peptide segment is: DAPPGAGFIYR, TGQPAADHYYSK, LLASTNSR, GNAELQTYLR, respectively;

the pertussis toxin S4 subunit characteristic peptide segment is: LGAAASSPDAHVPFCFGK, QLTFEGKPALELIR, AVFMQQRPLR, RPGSSPMEVMLR, respectively;

the pertussis toxin S5 subunit characteristic peptide segment is: IYSPADVAGLPTHLYK, IYSPADVAGLPTHLYK, NFTVQELALK are provided.

4. The quantitative detection method of the filamentous hemagglutinin in the diphtheria-pertussis-tetanus vaccine is characterized by comprising the following steps of:

(1) taking the diphtheria-pertussis-tetanus vaccine for enzymolysis pretreatment to obtain an enzymolysis liquid, and fixing the volume of the enzymolysis liquid to obtain a liquid to be detected;

(2) performing high performance liquid chromatography tandem mass spectrometry detection on the enzymatic hydrolysate obtained in the step (1), obtaining the content of the filamentous hemagglutinin in the liquid to be detected according to the chromatographic peak area and the standard working curve of the characteristic peptide segment of the filamentous hemagglutinin, and obtaining the content of the filamentous hemagglutinin in the pertussis vaccine through the conversion of the volume of the liquid to be detected and the volume of the pertussis vaccine;

the characteristic peptide fragments of the filamentous hemagglutinin are as follows: ATPIAAGAR, LTAAVALLNK, AALIEAGGNAR, TPLPVSLTALDNR, respectively;

the chromatographic peak area is the sum of the peak areas of characteristic peptide sections of any three or more filamentous hemagglutinin.

5. The quantitative detection method of the adhesin in the diphtheria-pertussis-tetanus vaccine is characterized by comprising the following steps:

(1) taking the diphtheria-pertussis-tetanus vaccine for enzymolysis pretreatment to obtain an enzymolysis liquid, and fixing the volume of the enzymolysis liquid to obtain a liquid to be detected;

(2) performing high performance liquid chromatography tandem mass spectrometry detection on the enzymolysis liquid obtained in the step (1), obtaining the content of the adhesin in the liquid to be detected according to the chromatographic peak area and the standard working curve of the characteristic peptide segment of the adhesin, and obtaining the content of the adhesin in the diphtheria-pertussis-tetanus vaccine through the conversion of the volume of the liquid to be detected and the volume of the diphtheria-pertussis-tetanus vaccine;

the characteristic peptide segment of the adhesin is as follows: TASGTTIK, FLGTVTVK, VLPEPVK, GANVTVQR;

the chromatographic peak area is the sum of the peak areas of the characteristic peptide sections of any three or more adhesins.

6. The quantitative detection method of pilin in the diphtheria-pertussis-tetanus vaccine is characterized by comprising the following steps of:

(1) taking the diphtheria-pertussis-tetanus vaccine for enzymolysis pretreatment to obtain an enzymolysis liquid, and fixing the volume of the enzymolysis liquid to obtain a liquid to be detected;

(2) performing high performance liquid chromatography tandem mass spectrometry detection on the enzymatic hydrolysate obtained in the step (1), obtaining the content of pilin in the liquid to be detected according to the chromatographic peak area and the standard working curve of the characteristic peptide segment of the pilin, and obtaining the content of pilin in the diphtheria-pertussis-tetanus vaccine through the conversion of the volume of the liquid to be detected and the volume of the diphtheria-pertussis-tetanus vaccine;

the characteristic peptide segments of the pilin are as follows: VVQLPK, TPFIIK, AYFEPGTTDYSTGTDLR;

the chromatographic peak area is the sum of the peak areas of the characteristic peptide sections of the three pilin proteins.

7. The quantitative detection method of adenylate cyclase toxin in the diphtheria-pertussis-tetanus vaccine is characterized by comprising the following steps of:

(1) taking the diphtheria-pertussis-tetanus vaccine for enzymolysis pretreatment to obtain an enzymolysis liquid, and fixing the volume of the enzymolysis liquid to obtain a liquid to be detected;

(2) performing high performance liquid chromatography tandem mass spectrometry detection on the enzymolysis liquid obtained in the step (1), obtaining the content of the adenylate cyclase toxin in the liquid to be detected according to the chromatographic peak area of the characteristic peptide segment of the adenylate cyclase toxin and a standard working curve, and obtaining the content of the adenylate cyclase toxin in the diphtheria-pertussis-tetanus vaccine through conversion of the volume of the liquid to be detected and the volume of the diphtheria-pertussis-tetanus vaccine;

the characteristic peptide segments of the adenylate cyclase toxin are as follows: SAVGTEAR, SVDVFVDR, ITGDAQANVLR, GVGFLSLGK, ILGTDDALTVDWYR;

the chromatographic peak area is the sum of the peak areas of any three or more characteristic peptide sections of the adenylate cyclase toxin.

8. The method for simultaneously and quantitatively detecting 5 subunits of pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin in the pertussis vaccine is characterized by comprising the following steps of:

(1) taking the diphtheria-pertussis-tetanus vaccine for enzymolysis pretreatment to obtain an enzymolysis liquid, and fixing the volume of the enzymolysis liquid to obtain a liquid to be detected;

(2) performing high performance liquid chromatography tandem mass spectrometry detection on the enzymatic hydrolysate obtained in the step (1), obtaining the contents of 5 subunits of pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin in the solution to be detected according to the chromatographic peak area and the standard working curve of each characteristic peptide segment of the pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin, and obtaining the contents of 5 subunits of pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin in the pertussis vaccine through conversion of the volume of the solution to be detected and the volume of the pertussis vaccine;

the pertussis toxin S1 subunit characteristic peptide segment is: SCQVGSSNSAFVSTSSSR, ILAGALATYQSEYLAHR, ANPNPYTSR, SVASIVGTLVR, respectively;

the pertussis toxin S2 subunit characteristic peptide segment is: GSGDLQEYLR, NTGQPATDHYYSNVTATR, LLSSTNSR, DGTPGGAFDLK, respectively;

the pertussis toxin S3 subunit characteristic peptide segment is: DAPPGAGFIYR, TGQPAADHYYSK, LLASTNSR, GNAELQTYLR, respectively;

the pertussis toxin S4 subunit characteristic peptide segment is: LGAAASSPDAHVPFCFGK, QLTFEGKPALELIR, AVFMQQRPLR, RPGSSPMEVMLR, respectively;

the pertussis toxin S5 subunit characteristic peptide segment is: IYSPADVAGLPTHLYK, IYSPADVAGLPTHLYK (3 charges), NFTVQELALK;

the characteristic peptide fragments of the filamentous hemagglutinin are as follows: ATPIAAGAR, LTAAVALLNK, AALIEAGGNAR, TPLPVSLTALDNR, respectively;

the characteristic peptide segment of the adhesin is as follows: TASGTTIK, FLGTVTVK, VLPEPVK, GANVTVQR;

the characteristic peptide segments of the pilin are as follows: VVQLPK, TPFIIK, AYFEPGTTDYSTGTDLR;

the characteristic peptide segments of the adenylate cyclase toxin are as follows: SAVGTEAR, SVDVFVDR, ITGDAQANVLR, GVGFLSLGK, ILGTDDALTVDWYR;

the chromatographic peak areas of the characteristic peptide segments of the 5 subunits of pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin are the sum of the peak areas of any three or more characteristic peptide segments in the corresponding protein characteristic peptide segments.

9. The detection method according to any one of claims 1 to 8, wherein the pretreatment method is: adding pertussis toxin product or diphtheria pertussis vaccine into ammonium bicarbonate solution; adding protein denaturant solution, and incubating at high temperature; adding a disulfide bond breaking reagent solution and an ammonium bicarbonate solution, and reacting at a high temperature; cooling to room temperature, adding iodoacetamide solution, and reacting at room temperature in a dark place; adding a trypsin solution, reacting overnight, and adding an acidic solution; and (4) performing centrifugal treatment to obtain an enzymolysis liquid.

10. The detection method according to any one of claims 1 to 8, wherein the conditions for the HPLC-MS are as follows:

chromatographic conditions are as follows: using C18 chromatographic column or C8 chromatographic column; the column temperature is 30 ℃; mobile phase a-aqueous ion exchanger solution; mobile phase B-mixed liquid of ion exchanger and organic solvent or mixed liquid of ion exchanger, organic solvent and water; gradient is 0-8min, 5% B-40% B; 8-8.1min, 40% B-100% B; 8.1-10min, 100% B; 10-10.1min, 100% B-5% B; 10.1-15min, 5% B; flow rate: 0.2-0.5 mL/min;

mass spectrum conditions: an ion source: ESI + mode; mass spectrometry: a triple quadrupole mass spectrometer; flow rate of the atomizer: 3L/min; flow rate of heater: 10L/min; interface temperature: 200 ℃; DL temperature: 235 ℃; heating module temperature: 400 ℃; flow rate of drying gas: 10L/min; interface voltage: 3 kV.

Technical Field

The invention particularly relates to a qualitative and quantitative determination method for active proteins Pertussis Toxin (PT) five subunits (S1-S5), filamentous hemagglutinin (FAH), adhesin (PRN), pilin (FIM) and Adenylate Cyclase Toxin (ACT) in a pertussis toxin product and a pertussis vaccine. The invention belongs to the technical field of vaccine quality evaluation.

Background

Pertussis is an acute respiratory infectious disease caused by bordetella pertussis, and is clinically characterized by a typical paroxysmal and spasmodic cough with gradually-increased cough accompanied by a deep chicken cry-like inspiratory roar with a disease course of 2-3 months, so that the pertussis is called as pertussis. In order to prevent pertussis disease, pertussis toxin products and a pertussis-tetanus-diphtheria (pertussis-diphtheria) combination vaccine were developed in succession by the chinese and world health organizations. Since the immunization was widely performed, the incidence of the disease has been greatly reduced. Pertussis Toxin (PT), Filamentous hemagglutinin (FAH), adhesin (PRN), pilin (fimbrial), and Adenylate Cyclase Toxin (ACT) are the major active proteins of pertussis toxin products and pertussis vaccines. The content determination of the components is important for the quality evaluation of the pertussis toxin product and the diphtheria-pertussis vaccine. At present, no matter Chinese pharmacopoeia or other national pharmacopoeias in the world, the method for measuring the content of pertussis toxin, filamentous hemagglutinin, bordetella pertussis adenylate cyclase toxin, adhesin and pilin is adopted. The deletion of the content determination method causes the quality evaluation of pertussis toxin products and pertussis vaccines to cause troubles. The content of functional proteins in pertussis toxin products and pertussis vaccines is reported in the literature by ELISA. However, the method is greatly influenced by the composition of a sample matrix, test conditions and the quality of an antibody, so that the repeatability of the method is poor. In addition, the method is difficult to interpret the causes of the differences affecting the quality of the vaccine, and thus cannot provide guidance and improvement advice for vaccine production.

The stability and accuracy of the evaluation method directly influence the quality safety of the vaccine. Therefore, establishing a stable, accurate and anti-interference method for qualitative and quantitative analysis of pertussis toxin, filamentous hemagglutinin, adhesin (PRN), pilin and adenylate cyclase toxin, which can be used for vaccine production guidance, is particularly important for quality evaluation of pertussis toxin products and diphtheria-pertussis vaccines.

Disclosure of Invention

The invention aims to improve the current situation that the methods for measuring the contents of Pertussis Toxin (PT), Filamentous hemagglutinin (FAH), adhesin (PRN), fimbrial protein (FIM) and Adenylate Cyclase Toxin (ACT) are lacked, overcome the problem of poor repeatability of the vaccine toxin tested by an ELISA method, provide the content measuring method of the components, and be used for measuring the contents of the proteins in pertussis toxin products and pertussis vaccines and evaluating the integrity of pertussis toxin subunits.

In order to achieve the purpose, the invention adopts the following technical scheme:

a qualitative identification method of 5 subunits of pertussis toxin in a pertussis toxin product comprises the following steps:

(1) carrying out enzymolysis pretreatment on a pertussis toxin product to obtain an enzymolysis liquid;

(2) and (3) respectively carrying out high performance liquid chromatography tandem mass spectrometry detection on the pertussis toxin standard solution and the enzymatic hydrolysate obtained in the step (1), and qualitatively identifying through ion-to-nucleus ratio (m/z) and retention time of the characteristic peptide segment.

The quantitative detection method of 5 subunits of pertussis toxin in the pertussis toxin product comprises the following steps:

(1) carrying out enzymolysis pretreatment on a pertussis toxin product to obtain an enzymolysis liquid, and fixing the volume of the enzymolysis liquid to obtain a liquid to be detected;

(2) and (2) performing high performance liquid chromatography tandem mass spectrometry detection on the enzymatic hydrolysate obtained in the step (1), obtaining the content of each subunit of the pertussis toxin in the liquid to be detected according to the chromatographic peak area and the standard working curve of the characteristic peptide segment of each subunit of the pertussis toxin, and obtaining the content of each subunit in the pertussis toxin product through conversion of the volume of the liquid to be detected and the volume of the pertussis toxin product.

The qualitative detection method of 5 subunits of pertussis toxin in the pertussis vaccine comprises the following steps:

(1) carrying out enzymolysis pretreatment on the diphtheria-pertussis-tetanus vaccine to obtain an enzymolysis liquid;

(2) and (3) respectively carrying out high performance liquid chromatography tandem mass spectrometry detection on the pertussis toxin standard solution and the enzymatic hydrolysate obtained in the step (1), and qualitatively identifying through ion-to-nucleus ratio (m/z) and retention time of the characteristic peptide segment.

The quantitative detection method of 5 subunits of pertussis toxin in the pertussis vaccine comprises the following steps:

(1) taking the diphtheria-pertussis-tetanus vaccine for enzymolysis pretreatment to obtain an enzymolysis liquid, and fixing the volume of the enzymolysis liquid to obtain a liquid to be detected;

(2) and (2) performing high performance liquid chromatography tandem mass spectrometry detection on the enzymatic hydrolysate obtained in the step (1), obtaining the content of each subunit of pertussis toxin in the liquid to be detected according to the chromatographic peak area and the standard working curve of the characteristic peptide segment of each subunit of pertussis toxin, and obtaining the content of each subunit in the pertussis vaccine through conversion of the volume of the liquid to be detected and the volume of the pertussis vaccine.

The qualitative detection method of the filamentous hemagglutinin in the diphtheria-pertussis-tetanus vaccine comprises the following steps:

(1) carrying out enzymolysis pretreatment on the diphtheria-pertussis-tetanus vaccine to obtain an enzymolysis liquid;

(2) and (2) respectively carrying out high performance liquid chromatography tandem mass spectrometry detection on the filamentous hemagglutinin standard solution and the enzymolysis solution obtained in the step (1), and qualitatively identifying through ion-to-nucleus ratio (m/z) and retention time of the characteristic peptide fragment.

The quantitative detection method of the filamentous hemagglutinin in the diphtheria-pertussis-tetanus vaccine comprises the following steps:

(1) taking the diphtheria-pertussis-tetanus vaccine for enzymolysis pretreatment to obtain an enzymolysis liquid, and fixing the volume of the enzymolysis liquid to obtain a liquid to be detected;

(2) and (2) performing high performance liquid chromatography tandem mass spectrometry detection on the enzymatic hydrolysate obtained in the step (1), obtaining the content of the filamentous hemagglutinin in the solution to be detected according to the chromatographic peak area and the standard working curve of the characteristic peptide segment of the filamentous hemagglutinin, and obtaining the content of the filamentous hemagglutinin in the pertussis vaccine through conversion of the volume of the solution to be detected and the volume of the pertussis vaccine.

The qualitative detection method of the adhesin in the diphtheria-pertussis-tetanus vaccine comprises the following steps:

(1) carrying out enzymolysis pretreatment on the diphtheria-pertussis-tetanus vaccine to obtain an enzymolysis liquid;

(2) and (3) respectively carrying out high performance liquid chromatography tandem mass spectrometry detection on the adhesin standard substance solution and the enzymolysis solution obtained in the step (1), and qualitatively identifying through ion-to-nucleus ratio (m/z) and retention time of the characteristic peptide section.

The quantitative detection method of the adhesin in the diphtheria-pertussis-tetanus vaccine comprises the following steps:

(1) taking the diphtheria-pertussis-tetanus vaccine for enzymolysis pretreatment to obtain an enzymolysis liquid, and fixing the volume of the enzymolysis liquid to obtain a liquid to be detected;

(2) and (2) performing high performance liquid chromatography tandem mass spectrometry detection on the enzymolysis liquid obtained in the step (1), obtaining the content of the adhesin in the liquid to be detected according to the chromatographic peak area and the standard working curve of the characteristic peptide segment of the adhesin, and obtaining the content of the adhesin in the diphtheria-pertussis-tetanus vaccine through conversion of the volume of the liquid to be detected and the volume of the diphtheria-pertussis-tetanus vaccine.

The qualitative detection method of pilin in the diphtheria-pertussis-tetanus vaccine comprises the following steps:

(1) carrying out enzymolysis pretreatment on the diphtheria-pertussis-tetanus vaccine to obtain an enzymolysis liquid;

(2) and (2) respectively carrying out high performance liquid chromatography tandem mass spectrometry detection on the pilin standard substance solution and the enzymolysis solution obtained in the step (1), and qualitatively identifying through ion-to-nucleus ratio (m/z) and retention time of the characteristic peptide segment.

The quantitative detection method of pilin in the diphtheria-pertussis-tetanus vaccine comprises the following steps:

(1) taking the diphtheria-pertussis-tetanus vaccine for enzymolysis pretreatment to obtain an enzymolysis liquid, and fixing the volume of the enzymolysis liquid to obtain a liquid to be detected;

(2) and (2) carrying out high performance liquid chromatography tandem mass spectrometry detection on the enzymatic hydrolysate obtained in the step (1), obtaining the content of pilin in the liquid to be detected according to the chromatographic peak area and the standard working curve of the characteristic peptide segment of the pilin, and obtaining the content of pilin in the diphtheria-pertussis-tetanus vaccine through the conversion of the volume of the liquid to be detected and the volume of the diphtheria-pertussis-tetanus vaccine.

The qualitative detection method of adenylate cyclase toxin in the diphtheria-pertussis-tetanus vaccine comprises the following steps:

(1) carrying out enzymolysis pretreatment on the diphtheria-pertussis-tetanus vaccine to obtain an enzymolysis liquid;

(2) and (2) respectively carrying out high performance liquid chromatography tandem mass spectrometry detection on the adenylate cyclase toxin standard substance solution and the enzymolysis solution obtained in the step (1), and qualitatively identifying through an ion-to-nucleus ratio (m/z) and the retention time of the characteristic peptide segment.

The quantitative detection method of adenylate cyclase toxin in the diphtheria-pertussis-tetanus vaccine comprises the following steps:

(1) taking the diphtheria-pertussis-tetanus vaccine for enzymolysis pretreatment to obtain an enzymolysis liquid, and fixing the volume of the enzymolysis liquid to obtain a liquid to be detected;

(2) and (2) carrying out high performance liquid chromatography tandem mass spectrometry detection on the enzymatic hydrolysate obtained in the step (1), obtaining the content of the adenylate cyclase toxin in the liquid to be detected according to the chromatographic peak area of the characteristic peptide segment of the adenylate cyclase toxin and a standard working curve, and obtaining the content of the adenylate cyclase toxin in the diphtheria-pertussis-tetanus vaccine through conversion of the volume of the liquid to be detected and the volume of the diphtheria-pertussis-tetanus vaccine.

The method for qualitatively detecting 5 subunits of pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin in the pertussis vaccine simultaneously comprises the following steps:

(1) carrying out enzymolysis pretreatment on the diphtheria-pertussis-tetanus vaccine to obtain an enzymolysis liquid;

(2) and (2) respectively carrying out high performance liquid chromatography tandem mass spectrometry on the pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin standard solution and the enzymatic hydrolysate obtained in the step (1), and qualitatively identifying through ion-to-nucleus ratio (m/z) and retention time of characteristic peptide segments of each component to be detected.

The method for simultaneously and quantitatively detecting 5 subunits of pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin in the pertussis vaccine is characterized by comprising the following steps of:

(1) taking the diphtheria-pertussis-tetanus vaccine for enzymolysis pretreatment to obtain an enzymolysis liquid, and fixing the volume of the enzymolysis liquid to obtain a liquid to be detected;

(2) and (2) performing high performance liquid chromatography tandem mass spectrometry detection on the enzymatic hydrolysate obtained in the step (1), obtaining the contents of the 5 subunits of pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin in the solution to be detected according to the chromatographic peak area and the standard working curve of each characteristic peptide segment of the 5 subunits of pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin, and obtaining the contents of the 5 subunits of pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin in the pertussis vaccine through conversion of the volume of the solution to be detected and the volume of the pertussis vaccine.

The characteristic peptide fragments of each test substance are as follows:

the pertussis toxin S1 subunit characteristic peptide segment is: SCQVGSSNSAFVSTSSSR, ILAGALATYQSEYLAHR, ANPNPYTSR, SVASIVGTLVR, respectively;

the pertussis toxin S2 subunit characteristic peptide segment is: GSGDLQEYLR, NTGQPATDHYYSNVTATR, LLSSTNSR, DGTPGGAFDLK, respectively;

the pertussis toxin S3 subunit characteristic peptide segment is: DAPPGAGFIYR, TGQPAADHYYSK, LLASTNSR, GNAELQTYLR, respectively;

the pertussis toxin S4 subunit characteristic peptide segment is: LGAAASSPDAHVPFCFGK, QLTFEGKPALELIR, AVFMQQRPLR, RPGSSPMEVMLR, respectively;

the pertussis toxin S5 subunit characteristic peptide segment is: IYSPADVAGLPTHLYK, IYSPADVAGLPTHLYK (3 charges), NFTVQELALK;

the characteristic peptide fragments of the filamentous hemagglutinin are as follows: ATPIAAGAR, LTAAVALLNK, AALIEAGGNAR, TPLPVSLTALDNR, respectively;

the characteristic peptide segment of the adhesin is as follows: TASGTTIK, FLGTVTVK, VLPEPVK, GANVTVQR;

the characteristic peptide segments of the pilin are as follows: VVQLPK, TPFIIK, AYFEPGTTDYSTGTDLR;

the characteristic peptide segments of the adenylate cyclase toxin are as follows: SAVGTEAR, SVDVFVVDR, ITGDAQANVLR, GVGFLSLGK, ILGTDDALTVDYR.

The above peptide fragments are not specifically illustrated, and all carry 2 charges in the ESI + ion source.

Preferably, in the qualitative detection, the retention time of any three or more characteristic peptide segments of the component to be detected is compared with the retention time of the characteristic peptide segment corresponding to the standard substance for qualitative identification.

Preferably, the chromatographic peak area of each characteristic peptide segment of the 5 subunits of pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin is the sum of the peak areas of at least three characteristic peptide segments. The peak area of the chromatographic peak of the pertussis toxin S1 characteristic peptide segment is the sum of the peak areas of any three or more characteristic peptide segments of the pertussis toxin S1 characteristic peptide segment. The peak area of the chromatographic peak of the pertussis toxin S2 characteristic peptide segment is the sum of the peak areas of any three or more characteristic peptide segments in the pertussis toxin S2 characteristic peptide segment. The peak area of the chromatographic peak of the pertussis toxin S3 characteristic peptide segment is the sum of the peak areas of any three or more characteristic peptide segments in the pertussis toxin S3 characteristic peptide segment. The peak area of the chromatographic peak of the pertussis toxin S4 characteristic peptide segment is the sum of the peak areas of any three or more characteristic peptide segments in the pertussis toxin S4 characteristic peptide segment. The peak area of the chromatographic peak of the pertussis toxin S5 characteristic peptide segment is the sum of the peak areas of three characteristic peptide segments of pertussis toxin S5. The peak area of the chromatographic peak of the characteristic peptide segment of the filamentous hemagglutinin is the sum of the peak areas of any three or more characteristic peptide segments of the filamentous hemagglutinin. The peak area of the chromatographic peak of the characteristic peptide segment of the adhesin is the sum of the peak areas of any three or more characteristic peptide segments of the adhesin. The peak area of the chromatographic peak of the pilin characteristic peptide segment is the sum of the peak areas of the three characteristic peptide segments of the pilin. The peak area of the chromatographic peak of the characteristic peptide segment of the adenylate cyclase toxin is the sum of the peak areas of any three or more characteristic peptide segments of the adenylate cyclase toxin.

Preferably, the pretreatment method comprises the following steps: adding pertussis toxin product or diphtheria pertussis vaccine into ammonium bicarbonate solution; adding protein denaturant solution, and incubating at high temperature; adding a disulfide bond breaking reagent solution and an ammonium bicarbonate solution, and reacting at a high temperature; cooling to room temperature, adding iodoacetamide solution, and reacting at room temperature in a dark place; adding a trypsin solution, reacting overnight, and adding an acidic solution; and (4) performing centrifugal treatment to obtain an enzymolysis liquid.

Preferably, the conditions for the high performance liquid chromatography tandem mass spectrometry detection are as follows:

chromatographic conditions are as follows: using C18 chromatographic column or C8 chromatographic column; the column temperature is 30 ℃; the mobile phase A is an aqueous solution containing an ion exchanger; mobile phase B has two forms: 1) a mixed solution of an ion exchanger and an organic solvent; or 2) a mixed solution of an ion exchanger, an organic solvent and water; gradient is 0-8min, 5% B-40% B; 8-8.1min, 40% B-100% B; 8.1-10min, 100% B; 10-10.1min, 100% B-5% B; 10.1-15min, 5% B; flow rate: 0.2-0.5 mL/min;

mass spectrum conditions: an ion source: ESI + mode; mass spectrometry: a triple quadrupole mass spectrometer; flow rate of the atomizer: 3L/min;

flow rate of heater: 10L/min; interface temperature: 200 ℃; DL temperature: 235 ℃; heating module temperature: 400 ℃; flow rate of drying gas: 10L/min; interface voltage: 3 kV.

Preferably, the conversion method is: volume V of the sample taken₁The volume of the liquid to be measured is V after constant volume₂Obtaining the content C of the component to be detected in the liquid to be detected according to the standard working curve_MeasuringIf the content C of the component to be detected in the sample to be detected is equal to C_Measuring×V₂/V₁。

More preferably, the sample pretreatment method comprises the following specific steps: adding 100 μ L pertussis toxin product or diphtheria-pertussis vaccine into 100 μ L protein denaturant solution, placing in a column incubator, and incubating at 60-80 deg.C for 15 min; adding 10 mu L of disulfide bond breaking reagent solution and 270 mu L of ammonium bicarbonate solution, and reacting for 60 minutes at 60-80 ℃; cooling to room temperature, adding 10 mu L iodoacetamide solution, and reacting at room temperature in a dark place; adding 5 mu L of trypsin solution, reacting overnight at 37 ℃, and adding 5 mu L of acid solution; centrifuging at 14000r/min for 15 minutes to obtain supernatant, namely enzymolysis liquid.

Further preferably, the trypsin solution in the sample pretreatment method is a solution obtained by dissolving 20. mu.g of trypsin in 1mL of 100mmol/L ammonium bicarbonate solution, i.e., the concentration is 20. mu.g/mL.

Further preferably, the ion exchanger in chromatographic conditions is a mass-compatible acid or salt, or a mixture of acid and salt; the salt is ammonium formate or ammonium acetate, and the acid is formic acid, acetic acid or trifluoroacetic acid.

In the mobile phase A, when the ion exchanger selects salt, the concentration of the ion exchanger aqueous solution is 0-20 mmol/L;

when the ion exchanger selects acid, the volume ratio of the acid to water is 0-10: 100-1000;

in the mobile phase B, when the ion exchanger is acid, the volume ratio of the ion exchanger to the organic solvent to the water is 1-2: 200-1000: 0-1000;

when the ion exchanger is a salt, the salt is dissolved in water to form a salt water solution, and the ratio of the organic solvent: the volume ratio of the saline solution is 100-60: 0 to 40, wherein the concentration of the saline solution is 0 to 20 mmol/L.

Further preferably, the organic solvent in the mobile phase B comprises methanol, acetonitrile and ethanol.

Further preferably, the protein denaturant solution in the sample pretreatment method is urea, sodium dodecyl sulfate, sodium octane sulfonate and RapidGest^SFOne of solutions, the concentration of the protein denaturant is 10mmol/L-10 mol/L; the acid solution is one of aqueous solutions of formic acid, acetic acid and trifluoroacetic acid; the volume ratio of formic acid, acetic acid, trifluoroacetic acid to water is 100-1: 0-100; the disulfide bond breaking reagent solution is dithiothreitol and tri (2-carboxyethyl) phosphine (also called TCEP), and the concentration of the disulfide bond breaking reagent solution is 10mmol/L～2mol/L。

Further preferably, the concentration of the ammonium bicarbonate solution in the sample pretreatment method is 10-500 mmol/L; more preferably 50 to 200 mmol/L.

Further preferably, the concentration of the iodoacetamide solution in the sample pretreatment method is 10mmol/L to 2mol/L, and further preferably 1 mol/L.

Preferably, the standard working curve is drawn by the following method:

(1) preparing a standard stock solution: dissolving standard pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin solid with ammonium bicarbonate water solution to obtain standard stock solutions with concentrations of 60, 153, 125, 143 and 100 μ g/mL;

(2) preparation of standard solutions: respectively taking each protein standard substance stock solution into a special centrifugal tube for low adsorption proteomics, uniformly mixing, carrying out enzymolysis according to an enzymolysis step, and after enzymolysis, fixing the volume of each enzymolysis solution to 1mL by using water to respectively obtain standard solutions with 6 concentrations;

(3) drawing a standard working curve: analyzing the standard solution by high performance liquid chromatography tandem mass spectrometry to obtain a chromatogram of the standard substance, and obtaining a standard working curve according to the relation between the standard solution concentration and the corresponding chromatographic peak area;

the chromatographic peak area is the sum of the chromatographic peak areas of at least three characteristic peptide sections.

The mass detector detection mode is multi-ion selective monitoring (MRM), and the mass detection parameters are shown in tables 1 and 2.

TABLE 1 Pertussis Toxin (PT) subunit S1-S5 Mass Spectrometry detection parameters

Is a quantitative ion

TABLE 2 PRN, FAH, FIM and ACT Mass Spectrometry detection parameters

Is a quantitative ion

The invention adopts a high performance liquid chromatography tandem mass spectrometry method to establish a high-flux, high-selectivity and high-sensitivity quantitative method for pertussis toxin products and pertussis vaccine active proteins, namely pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin. The method has the following advantages: 1) the method uses the high performance liquid chromatography tandem mass spectrometry for simultaneous quantification of pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin in pertussis toxin products and pertussis vaccines for the first time, solves the problems of poor repeatability, poor selectivity and the like of the quality control method of the original pertussis toxin products and pertussis vaccines, and makes up the defects that the ELISA method cannot be used for comparing the protein contents of vaccines of different manufacturers and the defects that each protein needs to be prepared and detected independently in the ELISA method; 2) compared with other reported high performance liquid chromatography tandem mass spectrometry quantitative proteins, the matrix of an application object (pertussis toxin products and diphtheria-pertussis-tetanus vaccine) is more complex, the method finally screens characteristic peptide segments which can be used for qualitative and quantitative analysis of various active vaccine proteins from the complex vaccine matrix for the first time through a series of researches such as matrix screening, repeatability inspection, linear inspection and the like, and other reported protein peptide segments of the peptide segments are different and cannot be obtained through a protein search library or simply obtained through the amino acid sequences of the vaccine proteins. The work curve obtained by the peak area of the obtained peptide segment and the protein concentration can realize the simultaneous quantification of 5 subunits of pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin in pertussis toxin products and pertussis vaccines of different manufacturers and batches.

The invention has the beneficial effects that:

1. the invention has the characteristic of multiple purposes by one method. The traditional method for quantifying the vaccine protein, such as an ELISA method, cannot be used for comparing the content of the active protein in vaccine products of different manufacturers. The method can realize the content determination of 5 subunits of pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin in various products (pertussis toxin products and pertussis vaccines), can be used for comparing the content of active protein in pertussis toxin products and pertussis vaccines of different batches of the same manufacturer and evaluating the repeatability of the product batches, can also be used for quantitative and qualitative analysis of the active protein in the pertussis toxin products and the pertussis vaccines of different manufacturers, and can provide reference for the establishment of related product quality control standards.

2. High selectivity and resistance to matrix interference. Through a series of experiments, including enzymolysis repeatability inspection, linear inspection, matrix screening and other experiments, 5 subunits (S1, S2, S3, S4 and S5) of pertussis toxin, filamentous hemagglutinin, adhesin, pilin and adenylate cyclase toxin are screened out; the obtained characteristic peptide fragments are different from other reported protein peptide fragments, and the peptide fragments can not be obtained by searching the existing protein library. Because the active protein can be enzymolyzed to obtain a plurality of different peptide fragments, the peptide fragments obtained by the method can not be deduced from the amino acid sequence of the active protein, and the peptide fragments are obtained by searching and verifying through a large number of experiments. The characteristic peptide segment obtained by optimization has high selectivity and matrix interference resistance, and can be used for quantitative and qualitative research of active protein in pertussis toxin products and pertussis vaccines.

3. And (3) the speed is high. In a traditional vaccine protein quantification method, such as an ELISA method, antibodies need to be prepared for different proteins, and one antibody can only be used for measuring one protein, so that the flux is low. The method can realize simultaneous content determination of 5 subunits (S1-S5) of Pertussis Toxin (PT) and other four active proteins (FAH, PRN, FIM and ACT) by one-time enzymolysis, one-time sample injection, liquid phase separation and MS detection within 15 minutes;

4. the quantitative determination has qualitative function at the same time. The method can be used for content determination of 5 proteins and qualitative analysis of four proteins, and can realize integrity identification of four protein peptide fragments. Specifically, the peptide fragments of the corresponding proteins in the protein standard and the sample are compared, and the integrity of the peptide fragments of the related proteins in the vaccine is qualitatively analyzed.

5. The invention adopts 2 stationary phases and a plurality of mobile phases to realize the simultaneous quantitative and qualitative analysis of 5 target proteins and subunits, and provides better retention and separation selectivity for different samples, thereby realizing the pretreatment of one sample and the simultaneous quantification of 5 different active proteins in pertussis toxin products and pertussis vaccines.

Drawings

FIG. 1a matrix screening of Pertussis Toxin (PT) characteristic peptide fragments; FIG. 1b filamentous hemagglutinin (FAH) characteristic peptide fragment matrix screening; FIG. 1c matrix screening of adhesin (PRN) signature peptide fragments; FIG. 1d pilin (FIM) characteristic peptide fragment matrix screening; FIG. 1e Adenylate Cyclase Toxin (ACT) characteristic peptide fragment matrix screening;

FIG. 2a is a partial enlarged view of a Pertussis Toxin (PT) characteristic peptide fragment spectrum of an active protein standard solution; FIG. 2b is a graph of a characteristic peptide fragment of filamentous hemagglutinin (FAH) in an active protein standard solution; FIG. 2c is a spectrum of a peptide fragment characteristic of adhesin (PRN) in a standard solution of activated protein; FIG. 2d is a graph of pilin (FIM) characteristic peptide fragment of active protein standard solution; FIG. 2e is a graph of the characteristic peptide fragment of Adenylate Cyclase Toxin (ACT) in the active protein standard solution;

FIG. 3a European Union pertussis toxin standard characterization; FIG. 3b is a graph comparing the peptides of Chinese Pertussis Toxin (PT) and European Union Pertussis Toxin (PT);

FIG. 4a WHO first generation Pertussis Toxin (PT) standard characterization; FIG. 4b is a graph comparing the peptides of Chinese Pertussis Toxin (PT) and WHO first generation Pertussis Toxin (PT);

FIG. 5a WHO second generation Pertussis Toxin (PT) standard characterization; FIG. 5b is a graph comparing the peptides of Chinese Pertussis Toxin (PT) and WHO second generation Pertussis Toxin (PT);

figure 6a british Pertussis Toxin (PT) standard characterisation; FIG. 6b is a comparison of the peptides of Chinese Pertussis Toxin (PT) and British Pertussis Toxin (PT);

FIG. 7a determination of Pertussis Toxin (PT) content in the first batch of pertussis toxin product from manufacturer 1; FIG. 7b determination of Pertussis Toxin (PT) content in the second batch of pertussis toxin product from manufacturer 1; FIG. 7c determination of Pertussis Toxin (PT) content in the third batch of pertussis toxin product from manufacturer 1;

FIG. 8a determination of Pertussis Toxin (PT) content in the first pertussis toxin product from manufacturer 2; FIG. 8b determination of Pertussis Toxin (PT) content in the second batch of pertussis toxin product from manufacturer 2; FIG. 8c determination of Pertussis Toxin (PT) content in the third batch of pertussis toxin product from manufacturer 2;

FIG. 9 determination of Pertussis Toxin (PT) content in the pertussis toxin product from manufacturer 3;

FIG. 10 determination of Pertussis Toxin (PT) content in the pertussis toxin product of manufacturer 4;

FIG. 11 determination of Pertussis Toxin (PT) content in pertussis toxin product from manufacturer 5;

FIG. 12a determination of Pertussis Toxin (PT) content in 6 diphtheria-pertussis-tetanus vaccine from manufacturer 6; FIG. 12b determination of filamentous hemagglutinin (FAH) content in the 6 diphtheria-pertussis-tetanus vaccine of manufacturer; FIG. 12c determination of adhesin (PRN) content in the 6 diphtheria-pertussis-tetanus vaccine from manufacturer 6; FIG. 12d determination of pilin (FIM) content in 6 diphtheria-pertussis-tetanus vaccine of manufacturer; FIG. 12e manufacturer 6 Adenylate Cyclase Toxin (ACT) assay;

FIG. 13a determination of Pertussis Toxin (PT) content in 7 diphtheria-pertussis-tetanus vaccine of manufacturer; FIG. 13b determination of filamentous hemagglutinin (FAH) content in 7 Baikal vaccine from manufacturer; FIG. 13c determination of adhesin (PRN) content in 7 Baikubush vaccine from manufacturer; FIG. 13d determination of pilin (FIM) content in 7 diphtheria-pertussis-tetanus vaccine of manufacturer; FIG. 13e manufacturer 7 Adenylate Cyclase Toxin (ACT) assay;

FIG. 14a determination of Pertussis Toxin (PT) content in 8 diphtheria-pertussis-tetanus vaccine of manufacturer; FIG. 14b determination of filamentous hemagglutinin (FAH) content in 8 diphtheria-pertussis-tetanus vaccine of manufacturer; FIG. 14c determination of adhesin (PRN) content in 8 diphtheria-pertussis-tetanus vaccine from manufacturer 8; FIG. 14d determination of pilin (FIM) content in 8 diphtheria-pertussis-tetanus vaccine of manufacturer; FIG. 14e manufacturer 8 Adenylate Cyclase Toxin (ACT) assay;

FIG. 15a determination of Pertussis Toxin (PT) content in 9 diphtheria-pertussis-tetanus vaccine of manufacturer; FIG. 15b determination of filamentous hemagglutinin (FAH) content in 9 diphtheria-pertussis-tetanus vaccine of manufacturer; FIG. 15c determination of adhesin (PRN) content in 9 diphtheria-pertussis-tetanus vaccine; FIG. 15d determination of pilin (FIM) content in 9 diphtheria-pertussis-tetanus vaccine of manufacturer; FIG. 15e manufacturer 9 Adenylate Cyclase Toxin (ACT) assay;

FIG. 16a determination of Pertussis Toxin (PT) content in10 diphtheria-pertussis-tetanus vaccine from manufacturer; FIG. 16b determination of filamentous hemagglutinin (FAH) content in10 Baikukou vaccine from manufacturer; FIG. 16c determination of adhesin (PRN) content in10 diphtheria-pertussis-tetanus vaccine from manufacturer; FIG. 16d determination of pilin (FIM) content in10 diphtheria-pertussis-tetanus vaccine of manufacturer; FIG. 16e manufacturer 10 Adenylate Cyclase Toxin (ACT) assay;

FIG. 17a determination of Pertussis Toxin (PT) content in 11 diphtheria-pertussis-tetanus vaccine from manufacturer; FIG. 17b determination of filamentous hemagglutinin (FAH) content in 11 diphtheria-pertussis-tetanus vaccine of manufacturer; FIG. 17c determination of adhesin (PRN) content in 11 diphtheria-pertussis-tetanus vaccine; FIG. 17d determination of pilin (FIM) content in 11 diphtheria-pertussis-tetanus vaccine manufactured by manufacturer; FIG. 17e manufacturer 11 Adenylate Cyclase Toxin (ACT) content assay.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and examples, which are provided for the purpose of illustration only and are not intended to limit the scope of the invention.