Detection kit for serum IgG characteristic N-carbohydrate chain combination and use thereof

Abstract

The invention discloses a serum IgG characteristic N-carbohydrate chain combination detection kit and a use method thereof, and relates to the technical field of biology. The kit comprises an IgG protein purification reagent, an N-glycan release reagent, a chemical derivatization reagent and an N-glycan purification reagent. The use method comprises the following steps: purifying IgG protein, releasing and purifying N-glycans, carrying out formylation treatment to obtain chemically derived IgG N-glycans, and purifying the chemically derived IgG N-glycans by using a solid-phase extraction method based on microcrystalline cellulose to obtain the N-glycans with five different structural characteristics, namely H4N4F1, H4N5F1, H5N5F1S1, H5N4F1S1 and H5N5F1S2. On the basis of a high-throughput mass spectrometry means, the accuracy of the five kinds of characteristic N-sugar chains explored by the invention on early prediction of gastric cancer is far higher than that of clinical common biomarkers.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a detection kit for serum IgG characteristic N-sugar chain combination and use thereof. Background Art

[0002] Gastric cancer is the fifth most diagnosed malignant tumor in the world. The early stage of gastric cancer is mainly caused by Helicobacter pylori infection of the gastric mucosa, which then leads to the gradual progression from atrophic gastritis to intestinal metaplasia. Therefore, early prediction of tumors is of great significance. Early detection and screening of tumors can significantly improve the survival rate while reducing the pain and economic burden of patients. However, most patients are not diagnosed until the late stage of the tumor due to unclear or delayed clinical symptoms, or the lack of effective early diagnostic markers. Commonly used markers for gastric cancer are CA72-4, PG1 or PG2, but they lack sensitivity and specificity. Therefore, it is particularly important to discover new biomarkers with higher diagnostic or predictive accuracy for early screening of gastric cancer.

[0003] Sugar chains are involved in the occurrence or development of almost all major diseases, and many glycosylated proteins have been used for early diagnosis of tumors, such as AFP-L3 for early detection of liver cancer. Glycoscience will play a vital role in future precision medicine. More and more evidence shows that the development of gastric cancer is accompanied by changes in glycosylation. Characteristic N-glycans derived from serum IgG are potential markers for the diagnosis of gastric cancer, such as N-glycans modified by sialic acid glycosylation or galactosyl glycosylation in IgG. However, there have been no reports on the use of characteristic N-glycopeptides in specific serum IgG to predict gastric precancerous lesions.

[0004] Therefore, technicians in this field are committed to developing a serum IgG characteristic N-glycan combination detection kit based on parsing the highly differentially varied IgG N-glycans in gastric precancerous lesions, and using high-throughput mass spectrometry to quickly parse the highly differentially varied IgG N-glycans in gastric precancerous lesions. Summary of the invention

[0005] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to develop a serum IgG characteristic N-glycan combination detection kit based on parsing the IgG N-glycans with highly differential changes in gastric precancerous lesions, and to use high-throughput mass spectrometry to quickly parsing the IgG N-glycans with highly differential changes in gastric precancerous lesions.

[0006] To achieve the above-mentioned object, the present invention provides a serum IgG characteristic N-glycan combination detection kit, comprising an IgG protein purification reagent, an N-glycan releasing reagent, a chemical derivatization reagent, and an N-glycan purification reagent.

[0007] Furthermore, the IgG protein purification reagents are binding buffer, washing buffer and elution buffer; the binding buffer and washing buffer are both 0.1M phosphate buffer containing 0.15M sodium chloride and 0.02% sodium azide, and the elution buffer is 0.1M acetic acid solution.

[0008] Furthermore, the pH of the binding buffer, the washing buffer and the elution buffer is 2.5.

[0009] Furthermore, the N-glycan releasing reagent is PNGaseF buffer, denaturing buffer, NP-40 and N-glycosidase and 5% acetonitrile solution containing 0.01% trifluoroacetic acid.

[0010] Furthermore, the chemical derivatization reagents are methylamine hydrochloride, 0.5 M methylmorpholine DMSO solution, 50 mM PyAOP DMSO solution and a mixture, and the composition of the mixture is: n-butanol: ethanol: water in a ratio of 4:1:1.

[0011] Furthermore, the N-glycan purification reagent is a balanced solution, and the composition of the balanced solution is: n-butanol: ethanol: water in a ratio of 4:4:1.

[0012] The present invention also discloses a method for using a serum IgG characteristic N-sugar chain combination detection kit, comprising the following steps:

[0013] Step 1, purifying the IgG protein to obtain a dried IgG protein sample;

[0014] Step 2, releasing and purifying N-glycans, extracting the dried IgG protein sample obtained in step 1 using a solid phase extraction method based on porous graphitized carbon to obtain purified IgG N-glycans;

[0015] Step 3, treating the purified IgG N-glycan obtained in step 2 by formiamidation to obtain chemically derivatized IgG N-glycan;

[0016] Step 4: Purify the chemically derivatized IgG N-glycan obtained in step 3 by a microcrystalline cellulose-based solid phase extraction method to obtain purified N-glycans. The purified N-glycans are N-glycans with five different structural characteristics: H4N4F1, H4N5F1, H5N5F1S1, H5N4F1S1 and H5N5F1S2.

[0017] Furthermore, step 1 also includes using an affinity chromatography column and a medium- and high-pressure protein purifier to purify the IgG protein: the IgG protein purification reagent is 5uL of serum diluted to 1mL with a binding buffer, and the sample is filtered with a disposable filter head with a pore size of 0.22um, and then the sample is injected into a 1mL injection valve using a syringe that comes with the purifier.

[0018] Furthermore, step 2 also includes adding 94uL of ultrapure water to the dried IgG protein sample, and then adding 3.6uL of 10-fold diluted PNGaseF buffer and 2.4uL of denaturation buffer; after vortex mixing for 10s, centrifuging at a medium speed and boiling for 10min to obtain a sample solution; after the sample solution is cooled to room temperature, 12uL of 10% NP-40 is added; after reacting for 5min, 5units of N-glycosidase is added, and then placed in a 37° water bath overnight to obtain a sample mixture; 500uL of 5% acetonitrile solution containing 0.01% trifluoroacetic acid is added to the sample mixed solution.

[0019] Furthermore, step 4 also includes subjecting the purified N-glycans to MALDI-MS detection to obtain the relative abundance of N-glycans with five different structural characteristics, namely H4N4F1, H4N5F1, H5N5F1S1, H5N4F1S1 and H5N5F1S2.

[0020] In preferred embodiment 1 of the present invention, the experimental process for evaluating the predictive effect of serum characteristic N-glycans on gastric cancer lesions is described in detail;

[0021] In another preferred embodiment 2 of the present invention, the method for using the serum IgG characteristic N-sugar chain combination detection kit for IgG N-sugar chains is described in detail.

[0022] The beneficial technical effects of the present invention are as follows:

[0023] The present invention is based on high-throughput mass spectrometry to analyze the serum IgG N-glycome spectrum of normal people vs benign gastric disease groups vs gastric cancer patients. Statistical analysis screens out IgG N-sugar chains whose expression levels change significantly during the evolution from normal to gastric cancer, and performs receiver operating characteristic curve (ROC) evaluation on them, comparing commonly used clinical markers to discover more effective new methods for predicting gastric cancer. The prediction accuracy of the five characteristic N-sugar chains discovered is much higher than that of commonly used clinical biomarkers. The results show that the prediction ability of N-glycans for patients with benign gastric tumors is over 80%, which is much higher than the commonly used marker CA72-4.

[0024] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a distribution diagram of five characteristic N-glycans in a preferred embodiment 1 of the present invention in the early stage (Normal) vs benign gastric tumor (Benign) vs gastric cancer (Cancer) population;

[0026] Figure 2 This is a diagram of experimental results of evaluating the predictive effect of serum characteristic N-glycans on gastric cancer lesions according to a preferred embodiment 1 of the present invention. DETAILED DESCRIPTION

[0027] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0028] Example 1 Experiment to evaluate the predictive effect of serum characteristic N-glycans on gastric cancer lesions

[0029] After matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) detection and statistical analysis, the experimental results showed that the five characteristic N-glycans derived from serum IgG changed significantly in healthy people (Normal) vs benign gastric people (Benign) vs gastric cancer (Cancer), such as Figure 1 As shown in Figure 2, the predictive effect of serum characteristic N-glycans on gastric cancer lesions was evaluated. Figure 2 As shown in the figure, part A is the five characteristic N-glycan structures, which are mainly fucosylated, galactosylated, N-acetylglucosamined and sialylated. Using binary logistic regression, the expression levels of these five N-glycans were combined according to probability to obtain a new variable N-glycans. With the help of MedCalc software, ROC analysis was used to evaluate the predictive ability of N-glycans for patients with benign gastric tumors. Figure 2 As shown in Part B, the results show that the accuracy is over 80%, which is much higher than the commonly used marker CA72-4. Therefore, the present invention provides that the five characteristic N-glycans in serum IgG can be used to predict gastric cancer.

[0030] The present invention is composed of 5 N-glycans with different structural characteristics derived from human serum glycoproteins, including H4N4F1, H4N5F1, H5N5F1S1, H5N4F1S1 and H5N5F1S2. The abundance of these N-glycans in peripheral blood IgG is relatively high, and can be effectively detected using MALDI-MS. For the collected peripheral blood samples, these 5 characteristic N-glycans can be obtained through four major steps: serum collection, IgG protein purification, N-glycan release, chemical derivatization, and N-glycan purification. Further, its relative abundance can be detected by MALDI-MS. Finally, its predictive ability for gastric cancer is evaluated by statistical analysis. The results of comparative studies show that the combination of the above 5 characteristic N-glycans in human serum is 30% more accurate than the commonly used gastric cancer marker CA72-4 in clinical practice, and the comparative results are shown in Table 1. Since this indicator can accurately predict the population of early gastric cancer, it has a predictive effect on gastric precancerous lesions.

[0031] Table 1 Statistical analysis to evaluate its predictive ability for gastric cancer

[0032] index AUC Standard error 95% CI Sensitivity Specificity N-glycans 0.804 0.050 0.704-0.882 76.00％ 73.33％ CA72-4 0.502 0.078 0.391-0.612 72.00％ 10.00％

[0033] Example 2 Serum IgG Characteristics of IgG N-Glycans Use of N-Glycan Combination Detection Kit

[0034] The steps for using the serum IgG characteristic N-glycan combination detection kit for IgG N-glycans are as follows:

[0035] 1) IgG purification

[0036] The IgG protein in human serum was purified by affinity chromatography column using medium and high pressure protein purification instrument.

[0037] The purification detector is an ultraviolet detector, and the absorption wavelength is set to 280nm. 5uL of serum is diluted to 1mL with binding buffer, and the sample is filtered with a disposable filter head with a pore size of 0.22um, and then the sample is injected into the 1mL injection valve using a syringe equipped with the purifier. According to the instructions for use of the chromatographic column, the binding buffer and the washing buffer are selected as 0.1M phosphate buffer (pH 2.5) containing 0.15M sodium chloride and 0.02% sodium azide, and 0.1M acetic acid solution (pH2.5) is selected as the elution buffer. Before running the purification method, the flow rate of binding and impurity removal is set to 0.5mL / min, running for 10min, and the elution flow rate is 1mL / min, and the running time is 25min. The eluate is collected by the automatic sample collector (BioFrac Fraction Collector) of the purifier. In order to stabilize the collected eluate, a certain amount of neutralization buffer (1M Tris-Hcl) is added to it so that its final pH is around 7.0. To further verify the collected proteins, the eluate was tested by SDS-PAGE. In addition, we further tested the protein concentration of all samples by BCA protein concentration determination kit. Finally, the collected protein eluate was dialyzed, desalted, freeze-dried and concentrated, and stored in a -20℃ medical refrigerator for future use.

[0038] 2) Release and purification of N-glycans

[0039] First, add 94uL of ultrapure water to the dried IgG protein sample, then add 3.6uL of 10×PNGaseF buffer and 2.4uL of denaturation buffer. After vortex mixing for 10s, centrifuge at medium speed and boil for 10min. After the sample cools to room temperature, add 12uL of 10% NP-40. After reacting for 5min, add 5units of N-glycosidase and place in a 37° water bath overnight. Then, add 500uL of 5% acetonitrile solution containing 0.01% trifluoroacetic acid to the sample mixed solution. N-glycans in the mixed solution are extracted using a solid phase extraction method based on porous graphitized carbon (PGC).

[0040] The steps to use PDC are as follows:

[0041] ① Add 500ul of 5% ACN (acetonitrile) 0.1% TFA mixed solution to each tube of sample → then centrifuge at 12000rpm for 2 minutes;

[0042] ② Wash the column with 100% ACN for 3 times, 1000ul (1ml) each time;

[0043] ③ Wash the column and activate it with a mixed solution of 80% ACN (acetonitrile) and 0.1% TFA, 3 times, 1000ul (1ml) each time;

[0044] ④ Balance, balance with 5% ACN (acetonitrile) 0.1% TFA mixed solution, 3 times, and reserve 100-200ul of 5% ACN (acetonitrile) 0.1% TFA mixed solution for the last time;

[0045] ⑤ Add the sample and let it settle naturally for about half an hour (be careful not to suck up the sediment at the bottom of the tube when adding the sample);

[0046] ⑥ Remove impurities and rinse with a balanced solution of 5% ACN (acetonitrile) and 0.1% TFA for 3 times;

[0047] ⑦ Remove the remaining liquid by pressurizing (note that when pressurizing, insert the tube first and then tighten it. When the pressure is reached, loosen it first, then remove the tube and shut down the machine). Mark the EP tube first, corresponding to the order of sample addition, and place it under the filtered PGC column. At the same time, wipe the needle to prevent impurities. Then elute with 1000ul 40% ACN (acetonitrile) 0.1% TFA mixed solution;

[0048] ⑧Put into rotary evaporator.

[0049] 3) Formamidation and reduction of N-glycan carboxyl functional groups

[0050] The IgG N-glycans purified by PGC were further treated by formiamidation. Briefly, 25uL of DMSO solution containing 1M forammonia hydrochloride and 0.5M methylmorpholine was first added to the sample; then 25uL of DMSO solution containing 50mM PyAOP was added. After the sample was reacted at room temperature in the dark for 30min, 500uL of a solution of n-butanol: ethanol: water in a ratio of 4:1:1 was added.

[0051] 4) N-glycan purification

[0052] Finally, the derivatized IgG N-glycans were purified using a microcrystalline cellulose (MCC)-based solid phase extraction method.

[0053] The steps to use MCC are as follows:

[0054] ① Wash the column (white column) with 1000ul of pure water, 3 times;

[0055] ② Use balance solution (n-butanol: ethanol: water = 4:4:1) to balance for 3 times, 1000ul each time, and leave a liquid surface of about 1cm for the last balance;

[0056] ③ Add 500ul of balancing solution into the sample tube and mix well;

[0057] ④ Add the sample into MCC and let it settle naturally without pressurizing;

[0058] ⑤ Remove impurities and add balancing solution, 1000ul each time, 3 times.

[0059] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A serum IgG characteristic N-glycan combination detection kit, characterized in that: Including IgG protein purification reagents, N-glycan release reagents, chemical derivatization reagents, and N-glycan purification reagents.

2. The kit according to claim 1, characterized in that The IgG protein purification reagents include binding buffer, washing buffer and elution buffer; the binding buffer and washing buffer are both 0.1M phosphate buffer containing 0.15M sodium chloride and 0.02% sodium azide, and the elution buffer is 0.1M acetic acid solution.

3. The kit according to claim 1, characterized in that The pH of the binding buffer, washing buffer and elution buffer was 2.

5.

4. The kit according to claim 1, characterized in that The N-glycan releasing reagent is PNGaseF buffer, denaturing buffer, NP-40 and N-glycosidase and 5% acetonitrile solution containing 0.01% trifluoroacetic acid.

5. The kit according to claim 1, characterized in that The chemical derivatization reagents are methylamine hydrochloride, 0.5M methylmorpholine DMSO solution, 50mM PyAOP DMSO solution and a mixed solution, wherein the mixed solution is composed of n-butanol: ethanol: water in a ratio of 4:1:

1.

6. The kit according to claim 1, characterized in that The N-glycan purification reagent is a balanced solution, and the composition of the balanced solution is: n-butanol: ethanol: water in a ratio of 4:4:

1.

7. A method for using the serum IgG characteristic N-sugar chain combination detection kit according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: Step 1, purifying the IgG protein to obtain a dried IgG protein sample; Step 2, releasing and purifying N-glycans, extracting the dried IgG protein sample obtained in step 1 using a solid phase extraction method based on porous graphitized carbon to obtain purified IgG N-glycans; Step 3, treating the purified IgG N-glycan obtained in step 2 by formiamidation to obtain chemically derivatized IgG N-glycan; Step 4: Purify the chemically derivatized IgG N-glycan obtained in step 3 by a microcrystalline cellulose-based solid phase extraction method to obtain purified N-glycans. The purified N-glycans are N-glycans with five different structural characteristics: H4N4F1, H4N5F1, H5N5F1S1, H5N4F1S1 and H5N5F1S2.

8. The method according to claim 7, characterized in that The step 1 also includes purifying the IgG protein using an affinity chromatography column and a medium- and high-pressure protein purifier: 5uL of serum is diluted to 1mL with a binding buffer, and filtered with a disposable filter head with a pore size of 0.22um to obtain a sample, and then the sample is injected into a 1mL injection valve using a syringe that comes with the purifier.

9. The method according to claim 7, characterized in that The step 2 also includes adding 94uL of ultrapure water to the dried IgG protein sample, and then adding 3.6uL of 10-fold diluted PNGaseF buffer and 2.4uL of denaturation buffer; after vortex mixing for 10s, centrifuging at a medium speed and boiling for 10min to obtain a sample solution; after the sample solution is cooled to room temperature, 12uL of 10% NP-40 is added; after reacting for 5min, 5units of N-glycosidase is added, and then placed in a 37° water bath overnight to obtain a sample mixed solution; 500uL of a 5% acetonitrile solution containing 0.01% trifluoroacetic acid is added to the sample mixed solution.

10. The method according to claim 7, characterized in that The step 4 also includes subjecting the purified N-glycans to MALDI-MS detection to obtain the relative abundance of N-glycans with five different structural characteristics: H4N4F1, H4N5F1, H5N5F1S1, H5N4F1S1 and H5N5F1S2.

Images