阅读说明：本技术 一种检测抗胰腺癌天然抗体的组合物、试剂盒和方法 (Composition, kit and method for detecting anti-pancreatic cancer natural antibody ) 是由 韩传伟 胡颖 于 2020-06-30 设计创作，主要内容包括：本发明涉及一种用于检测抗胰腺癌天然抗体的组合物、由该组合物制成的试剂盒、使用该组合物或该试剂盒检测样品中抗胰腺癌天然抗体浓度的方法、以及该组合物或该试剂盒在样品抗胰腺癌天然抗体检测中的应用。本发明利用与目标抗胰腺癌天然抗体完全互补的3种线性抗原多肽,实现对样品中抗胰腺癌天然抗体的定性和定量检测,可用于筛查富含抗胰腺癌天然抗体的健康人血浆和体外实验研究。(The invention relates to a composition for detecting anti-pancreatic cancer natural antibodies, a kit prepared from the composition, a method for detecting the concentration of anti-pancreatic cancer natural antibodies in a sample by using the composition or the kit, and application of the composition or the kit in detection of the anti-pancreatic cancer natural antibodies in the sample. The invention realizes qualitative and quantitative detection of the anti-pancreatic cancer natural antibody in a sample by using 3 linear antigen polypeptides completely complementary with the target anti-pancreatic cancer natural antibody, and can be used for screening healthy human plasma rich in the anti-pancreatic cancer natural antibody and in-vitro experimental research.)

1. A composition for detecting natural antibodies against pancreatic cancer, comprising two or three combinations of the following three antigenic polypeptides:

H-GLSLDASMHSQLRILDSKFRRTRPLEC-OH；

H-YHHGLSALKPIRTTSKHQHPVDNAGLFSCD-OH; and

H-DYWTNTEKMEKRLHAVPAANTVKFRCPD-OH。

2. the composition of claim 1, wherein: the weight concentration ratio of the combination of the two antigen polypeptides in the composition is 1:1, and the weight concentration ratio of the three antigen polypeptides is 3:3:4 in sequence.

3. A kit comprising the composition of claim 1 or 2.

4. A kit according to claim 3 comprising a dry microtiter plate vacuum seal packaged with a non-metallic medical packaging material, said three antigenic polypeptide compositions being coated in wells of the microtiter plate.

5. The kit according to claim 4, wherein the non-metallic medical packaging material is glass or medical plastic.

6. An in vitro method for the detection of natural antibodies against pancreatic cancer of non-diagnostic interest, comprising the detection of natural antibodies against pancreatic cancer in a sample using a composition according to claim 1 or 2 or a kit according to any one of claims 3 to 5.

7. A method according to claim 6 comprising mixing the three antigenic polypeptides in proportion and then coating with a Maleimide (Maleimide) activated microtiter plate, incubating overnight at 4 ℃, washing the plate and performing a step-wise loading assay.

8. The method according to claim 7, wherein the step-wise loading analysis comprises double-well testing of a sample to be tested, simultaneously providing 2 negative control wells and 2 positive control wells; diluting plasma with an analysis solution, diluting a horseradish peroxidase-labeled goat anti-human IgG antibody, washing a plate, adding 100 mu l of a mixed solution of 3,3',5,5' -Tetramethylbenzidine (TMB) and peroxidase into each hole, keeping the plate away from light at room temperature for 20-30 minutes, adding 50 mu l of a 10-12% sulfuric acid solution into each hole to serve as a stop solution, and then detecting a light density value with a microplate reader, wherein the detection wavelength is 450nm, and the reference wavelength is 630 nm.

9. Use of the composition according to claim 1 or 2 or of the kit according to claims 3-5 for the detection of natural antibodies against pancreatic cancer in a test sample of non-diagnostic interest in vitro.

10. Use of the composition of claim 1 or 2 for the preparation of a reagent for natural antibodies against pancreatic cancer.

Technical Field

The invention belongs to the technical field of immunology, and relates to a composition, a kit and a method for detecting an anti-pancreatic cancer natural antibody.

Background

Malignant tumors are one of the major killers of human health. According to recent statistics, the number of newly-discovered cancer patients worldwide is about one thousand five million, and the number of cancer-caused deaths is over eight million each year. In china, the number of new cancers is about 300 to over ten thousand each year, the most common tumor is lung cancer, which is 80 to over ten thousand each year, and gastric cancer, which is about 40 to over ten thousand each year. In the malignancy degree, pancreatic cancer occupies the first place, the average annual survival rate is less than 25%, and the five-year survival rate is less than 5%. The main reasons for the low survival rate of tumor patients are two: firstly, the early detection is difficult, and secondly, an effective treatment means is lacked.

According to the recent research on natural antibodies, more than 50% of antibodies in human blood belong to natural antibodies, are mainly secreted by B1 lymphocytes and do not need specific antigen stimulation. Natural antibodies are involved in various physiological regulation and immune function stabilization of the body and serve as a bridge between innate immunity and specific immune systems. It is noted that some natural antibodies have the function of immune surveillance, and can clear malignant cells formed in vivo at any time and maintain homeostasis. Therefore, natural antibodies that maintain a certain level may play a role in preventing cancer. It is speculated that the risk of developing tumors in natural antibody deficient or negative patients may be significantly higher than in normal populations. The establishment of a method for detecting natural anti-cancer antibodies is helpful for researching and developing the tumor occurrence and development rules and finding a method for effectively treating tumors. Recently, a biotechnology limited company introduced a detection technology of natural anti-cancer antibodies in human plasma. According to the research, 5-10% of healthy human plasma is rich in natural anti-cancer antibodies, and the individuals have extremely strong immune monitoring function and extremely low risk of cancer in the lifetime. Laboratory studies prove that the healthy human plasma rich in natural anti-cancer antibodies can obviously inhibit the growth and proliferation of various cancer cells, including primary liver cancer, gastric cancer, nasopharyngeal cancer, lung cancer, oral cancer and the like. Clinical study of case contrast in a certain hospital shows that the plasma rich in natural anti-cancer antibodies has obvious treatment effect on diffuse liver cancer in stage B and multiple liver cancer and is also very effective on patients with postoperative recurrence of primary liver cancer. Follow-up studies lasting 3 years prove that the median survival time of the patients with the liver cancer in the B stage through the single interventional therapy is 20 months, while the median survival time of the patients is more than 30 months by the interventional therapy combined infusion of the plasma rich in natural anti-cancer antibodies. The treatment method has the greatest advantages of no drug resistance, no obvious side effect, and wide clinical value and application prospect.

Pancreatic cancer is a malignant tumor of digestive tract with high malignancy degree and difficult diagnosis and treatment, and is second to colorectal cancer at position 2 of death cause of digestive tract cancer. Pancreatic cancer occurs in approximately 75% of patients in the head of the pancreas, and the pathological diagnosis of pancreatic ductal epithelial adenocarcinoma, the exact cause of which is not yet clear, is in 90% of patients. The treatment of pancreatic cancer is comprehensive treatment mainly by operation, but the prognosis is very poor, and the survival rate 5 years after the operation is generally 10-15%. Other treatments for pancreatic cancer include radiation, chemotherapy, and interventional therapies, but are not ideal. Therefore, the development of new methods for treating pancreatic cancer is a major medical problem to be solved urgently.

Adenosine triphosphate binding cassette transporters (ABC transporters) are capable of mediating the transport of a variety of substrate molecules inside and outside the cell. ABC transporters are divided into 7 subfamilies (ABC a-G) based on sequence homology and transmembrane domain topology. To date, there are 49 members of the ABC family found in humans, and the proteins involved in drug transport have been mainly concentrated in several subfamilies, ABCB, ABCC and ABCG. Research finds that ABC transporter pump function is related to reduction of drug aggregation in tumor cells, is a main reason for drug resistance generation of tumor cells, and becomes an important defense mechanism for preventing the tumor cells from being attacked by chemotherapeutic drugs. ABCC5 belongs to one of the ABC transporter superfamily members, and this transporter is capable of binding adenosine triphosphate and using energy to drive the transport of a variety of different molecules across cell membranes. Research shows that ABCC5 is highly expressed in pancreatic cancer cells and is probably one of the reasons for insensitivity of pancreatic cancer chemotherapy. In addition, histochemical research proves that Fibroblast growth factor type 2 receptors (Fibroblast growth factor receptor 2, FGFR2) are also highly expressed in pancreatic cancer cells, have close relationship with the prognosis of patients, and can be used as important target molecules for immune targeted therapy.

Disclosure of Invention

The invention mainly provides a group of linear polypeptide antigen compositions for detecting concentrations of natural antibodies in human plasma against ABCC5 and FGFR2, a kit prepared from the compositions, and a method for detecting natural antibodies in human plasma against pancreatic cancer by using the antigen polypeptide compositions or the kit, and the application value and the prospect of the antigen polypeptide compositions are clarified. The antigenic polypeptides are derived from human ABCC5 and FGFR2 protein sequences, two of which are derived from ABCC5 transporter (SEQ ID NO:1 and SEQ ID NO:2) and one is derived from FGFR2 receptor protein (SEQ ID NO: 3). The italicized and underlined regions shown in tables 1 and 2 represent the antigenic polypeptide amino acid sequences. These epitopes are capable of specific binding to ABCC5 and FGFR2 natural antibodies in human plasma, respectively.

TABLE 1 human ABCC5 protein sequence

TABLE 2 human FGFR2 protein sequence

The term "anti-pancreatic cancer natural antibody" as defined in the present invention refers to an antibody or a mixture of antibodies naturally present in a healthy human body, and may be involved in inhibiting the development and progression of various malignant tumors including pancreatic cancer. The detection of the anti-pancreatic cancer natural antibody has a value for prompting the risk of tumor onset, and is favorable for further developing a technical means for early diagnosis and tumor treatment. In a particular embodiment of the invention, the anti-pancreatic cancer natural antibody is a natural antibody or a mixture of natural antibodies recognizing one or more of the antigenic epitope sequences of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.

The inventor analyzes the Human leukocyte antigen II (HLA-II) epitope and B cell epitope on the ABCC5 and FGFR2 protein sequences by using an immunoinformatics method and an epitope mapping technology, screens an amino acid sequence with high affinity, and further designs HLA-II restrictive epitopes and linear antigen polypeptides which can be recognized by most Human antigen presenting cells.

According to the biological characteristics of the protein epitope, the invention carries out immune informatics prediction and simulation aiming at a plurality of epitopes, and designs two linear antigen polypeptides which are completely complementary with a target antibody in human plasma in space structure and configuration respectively through analyzing various parameters related to antigenicity, and the amino acid sequences of the linear antigen polypeptides are shown in Table 3.

TABLE 3 detection of Linear antigen polypeptide sequences of ABCC5 and FGFR2 Natural antibodies in human plasma

The three antigen polypeptides are synthesized by a solid phase chemical method and used for preparing an ELISA antibody detection kit, and the concentrations of ABCC5 and FGFR2 natural antibodies in the plasma of healthy individuals are detected according to a set process specification. The antigen polypeptide can be prepared into a simple and easy-to-use convenient kit in practical application, is packaged in a vacuum sealing manner by using non-metallic materials such as glass, medical plastics and the like, and can be stored for more than 6 months at the temperature of 4 ℃. Briefly, the three antigenic polypeptides were coated on a Maleimide (Maleimide) activated 96-well microtiter plate. Drying in an oven at 40-45 deg.C (40-45 deg.C), vacuum sealing and packaging with non-metal packaging material to obtain the kit.

Thus, according to one of the present invention, there is provided a detection reagent for detecting ABCC5 and FGFR2 natural antibodies, comprising three antigenic polypeptides selected from the group consisting of:

H-GLSLDASMHSQLRILDSKFRRTRPLEC-OH(SEQ ID NO:1)；

H-YHHGLSALKPIRTTSKHQHPVDNAGLFSCD-OH (SEQ ID NO: 2); and H-DYWTNTEKMEKRLHAVPAANTVKFRCPD-OH (SEQ ID NO: 3).

In some embodiments, the detection reagent consists of the three antigenic polypeptides.

In some embodiments, the three antigenic polypeptides are high purity preparations, preferably chemically synthesized with a purity > 90%.

In some embodiments, when the detection reagent comprises three antigenic polypeptides, the three antigenic polypeptides are used in admixture. When used in combination, the three antigenic polypeptides may be mixed in a weight concentration ratio (3:3: 4). In a preferred embodiment, the three antigenic polypeptides are mixed in a (3:3:4) concentration by weight ratio.

According to another aspect of the present invention, there is provided a kit comprising the above-mentioned detection reagent composition.

In some embodiments, the kit includes a microplate, the detection reagents being coated within the wells of the microplate.

In some embodiments, in the kit, the microplate coated with the detection reagent is dried and then vacuum-sealed with a non-metallic medical packaging material. In some embodiments, the microplate is a Maleimide (Maleimide) activated 96-well microplate.

In other preferred embodiments, the non-metallic medical packaging material is glass or medical plastic.

In other embodiments, the kit further comprises a positive control and/or a negative control. In some embodiments, the positive control and/or the negative control are coated on a microtiter plate.

According to another aspect of the invention there is provided a method of detecting ABCC5 and FGFR2 natural antibodies in a sample using the above-described detection reagent or the above-described kit, preferably the method is an in vitro, non-diagnostic detection technique.

In some embodiments, the methods comprise detecting the concentration of ABCC5 and FGFR2 natural antibodies in a test sample by an antigen-antibody binding reaction using the detection reagents described above.

In a preferred embodiment, the "detecting the concentrations of the ABCC5 and FGFR2 natural antibodies in the sample to be tested by antigen-antibody binding reaction" is carried out by an enzyme-linked immunosorbent assay (ELISA) method.

In a more preferred embodiment, the enzyme-linked immunosorbent assay is a sandwich ELISA.

In some embodiments, the method is performed by: (1) activating a 96-well micro-detection plate by using Maleimide (Maleimide) coated with the detection reagent to perform step-by-step sample addition analysis, and detecting an optical density value (OD) in each well by using a microplate reader; (2) antibody levels were expressed as Positive Sample Ratios (PSR).

In a more preferred embodiment of the process according to the invention,the step (1) comprises the steps of setting a sample to be detected with double composite holes, simultaneously setting 2 negative control holes and 2 positive control holes, diluting plasma with an analysis solution, diluting a horse radish peroxidase-labeled goat anti-human IgG antibody, washing a plate, adding 100 mu l of 3,3',5,5' -Tetramethylbenzidine (TMB) and peroxidase mixed solution into each hole, keeping the room temperature away from light for 20-30 minutes, and adding 50 mu l of a 10-12% sulfuric acid solution (10-12% H) of stop buffer into each hole₂SO₄) Then, the Optical Density (OD) value was measured with a microplate reader, the detection wavelength was 450nm, and the reference wavelength was 630 nm.

In a more preferred embodiment, the sample is human plasma, more preferably single individual plasma.

In a more preferred embodiment, the individual's plasma is plasma from a single healthy individual.

In a more preferred embodiment, the specific operation steps of the step (1) are as follows:

1. before the operation, the three antigen polypeptides used were dissolved in 5.0mg/ml (mg/ml) stock solutions of 67% acetic acid, respectively, and then stored in a refrigerator at-20 degrees centigrade (within a range of ± 2 degrees centigrade).

2. When the operation is started, the antigen polypeptide is diluted into 10-30 microgram/milliliter (mu g/ml) working solution by using a coating solution, wherein the coating solution is 0.1M phosphate buffer solution containing 0.15M sodium chloride and 10mM EDTA, and the pH value is 7.0-7.4.

3. A96-well micro-detection plate (Thermoscientific, USA) activated by Maleimide (Maleimide) is coated with a working solution, after overnight incubation at 4 ℃, the plate is washed for 3 times by a washing solution, wherein the washing solution is 0.1M phosphate buffer solution containing 0.15M sodium chloride and 0.1% TWEEN-20, and the pH value is 7.0-7.4.

4. Then the sample is loaded and analyzed step by step according to the following steps:

a) the plasma sample to be tested is provided with double wells, and is further provided with 2 negative control wells (NC, a reference substance is a negative control solution without ABCC5 and FGFR2 natural antibodies, such as bovine serum albumin (provided by Sigma-Aldrich company), so that the experimental index value of the used antigen polypeptide in an ABCC5 and FGFR2 natural antibody negative reaction system can be reflected), and 2 positive control wells (PC, a reference substance is a mixture of anti-human ABCC5 and FGFR2 antibodies, so that the experimental index value of the used antigen polypeptide in an ABCC5 and FGFR2 natural antibody positive reaction system can be reflected).

b) Diluting a blood plasma sample to be detected by 1:200 by using an analysis solution, wherein the analysis solution is the same as an antigen coating solution, namely 0.1M phosphate buffer solution containing 0.15M sodium chloride and 10mM EDTA, the pH is 7.0-7.4, 100 mu l of the buffer solution is added into each hole, incubation is carried out at the room temperature of 20-25 ℃ for 1-2 hours, and then the plate is washed for 3 times.

c) And (3) diluting the horseradish peroxidase-labeled goat anti-human IgG antibody (used for verifying whether the substance to be detected in the plasma is a specific antibody) by using an analysis solution (namely 0.1M phosphate buffer solution containing 0.15M sodium chloride and 10mM EDTA, wherein the pH value is between 7.0 and 7.4), wherein the dilution ratio of the antibody is 1:20000 to 1:50000, 100 mu l of the antibody is added into each hole, and the incubation is carried out at the room temperature of 20-25 ℃ for 1-2 hours.

d) After washing the plate 3 times with a washing solution (i.e., 0.1M phosphate buffer containing 0.15M sodium chloride and 0.1% TWEEN-20, pH 7.0-7.4), 100. mu.l of a mixture of 3,3',5,5' -Tetramethylbenzidine (TMB) and peroxidase was added to each well, and the plate was protected from light at room temperature for 20-30 minutes.

e) Add 50. mu.l stop solution 12% sulfuric acid solution (12% H) per well₂SO₄) Then, the Optical Density (OD) value was measured with a microplate reader, the detection wavelength was 450nm, and the reference wavelength was 630 nm. The detection process was completed within 10 minutes after the addition of stop solution, thereby quantitatively analyzing the levels of ABCC5 and FGFR2 natural antibodies in the plasma of the individual.

In the random sampling analysis of the population, the data obtained by each individual detection can be analyzed, and the Positive Sample Ratio (PSR) is used for judging the relative level of ABCC5 and FGFR2 natural antibodies in the plasma.

In some embodiments, the Positive Sample Ratio (PSR) in step (2) above is calculated as follows:

PSR ═ OD value-OD of sample to be measured_NCValue of]/[ Positive standard OD value-OD_NCValue of]NC is a negative control for each sample.

In another aspect of the invention, there is provided the use of the above detection reagent, the above kit or the above method for detecting ABCC5 and FGFR2 natural antibodies in a sample. In some embodiments, the use is for in vitro, non-diagnostic purposes.

In another aspect of the invention, the application of the detection reagent in preparing a reagent for detecting the ABCC5 and FGFR2 natural antibodies in a sample is also provided.

In a preferred embodiment, the sample is human plasma, more preferably single individual plasma.

In a more preferred embodiment, the individual's plasma is plasma from a single healthy individual.

Based on the scheme, the invention provides the ABCC5 and FGFR2 natural antibody detection technology which is high in precision, simple and easy to operate and moderate in cost, and further provides a semi-quantitative (or relative quantitative) analysis and application scheme for the ABCC5 and FGFR2 natural antibodies on the basis, thereby laying an important foundation for developing brand-new early diagnosis tumor and early implementation treatment strategies based on healthy human plasma ABCC5 and FGFR2 natural antibodies.

The invention provides a simple and convenient human plasma ABCC5 and FGFR2 natural antibody detection means, which can be used for qualitatively or relatively quantitatively detecting the levels of ABCC5 and FGFR2 natural antibodies and assisting in quantitatively determining the levels of plasma ABCC5 and FGFR2 natural antibodies of different individuals. The natural antibody levels of the ABCC5 and the FGFR2 are reduced or negative individuals are likely to have higher pancreatic cancer onset risks, and the method has important values of predicting pancreatic cancer onset risks and early diagnosing the ABCC5 and the FGFR2 pancreatic cancers. The individuals with plasma ABCC5 and FGFR2 natural antibody negative detected by the product of the invention may have higher pancreatic cancer onset risk, can be clinically followed up and tracked, and is suitable for further developing technical means for early pancreatic cancer discovery and treatment implementation.

The ABCC5 and FGFR2 natural antibody method can be used for pancreatic cancer biological research, discussing pancreatic cancer generation mechanism, tumor cell immune escape mechanism, immune monitoring mechanism and the like.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the invention. The primary objects and other advantages of the invention may be realized and attained by the instrumentalities particularly pointed out in the specification.

Drawings

FIG. 1 is a schematic diagram showing the results of changes in cell activity.

Examples

1. Healthy human plasma ABCC5 and FGFR2 natural antibody screening

Plasma samples of healthy donors were screened for ABCC5 and FGFR2 natural antibody levels at a blood station in guangdong province. The three polypeptide antigens (see table 3) used in the experiment are synthesized by chemical solid phase synthesis, the purity is 95%, and the following operation steps are specifically carried out:

(1) prior to the procedure, each antigen was dissolved in 67% acetic acid to 5.0mg/ml stock and stored in a freezer at-20 ℃.

(2) At the beginning of the operation, the three antigens were first diluted to 20. mu.g/ml with coating solution and then mixed in weight volume at a ratio of 3:3: 4. The coating solution was 0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA, and the pH (pH) value was found to be 7.2.

(3) The mixed three antigen polypeptides were then coated on a Maleimide (Maleimide) activated 96-well assay plate (Thermo Scientific, USA), incubated overnight at 4 ℃ for 15 hours, and then the plate was washed 3 times with a wash solution of 0.1M phosphate buffer containing 0.15M sodium chloride and 0.1% TWEEN-20, which was measured to have a pH of 7.2.

(4) The fractional loading analysis was then as follows:

a) for each antigen test, duplicate wells were set, and 2 negative control wells (NC, reference bovine serum albumin (Sigma-Aldrich) and 2 positive control wells (PC, reference mixture of anti-human ABCC5 and FGFR2 antibodies, Sigma-Aldrich) were set.

b) Plasma was diluted 1:200 with the same assay as the antigen coating, 0.1M phosphate buffer containing 0.15M sodium chloride and 10mM EDTA, and the pH was 7.2, 100. mu.l was added per well, and incubated at room temperature for 1.5 hours.

c) After washing the plate 3 times with the aforementioned wash solution (i.e., 0.1M phosphate buffer containing 0.15M sodium chloride and 0.1% TWEEN-20, pH 7.2 measured), horseradish peroxidase-labeled goat anti-human IgG (supplied by Sigma-Aldrich) was diluted with the assay solution at an antibody working concentration of 1:30000, 100. mu.l per well and incubated at room temperature for 1.5 hours.

d) After washing the plate 3 times with the aforementioned wash solution (i.e., 0.1M phosphate buffer containing 0.15M sodium chloride and 0.1% TWEEN-20, pH 7.2), 100. mu.l of 3,3',5,5' -Tetramethylbenzidine (TMB) and peroxidase mixture (provided by Life Technologies) was added to each well, and incubated at room temperature in the dark for 25 minutes.

e) Add 50. mu.l stop solution 12% sulfuric acid solution (12% H) per well₂SO₄) And then, detecting the Optical Density (OD) value by using an enzyme-labeling instrument, wherein the detection wavelength is 450nm, the reference wavelength is 630nm, the detection is finished within 10 minutes after the stop solution is added, and the subsequent steps are used for carrying out quantitative comparative analysis on the ABCC5 and FGFR2 natural IgG antibodies according to the result.

3. Natural IgG antibody positive and negative plasma

When analyzing the data obtained by the detection, Positive Sample Ratio (PSR) is adopted to judge the levels of ABCC5 and FGFR2 natural IgG antibodies in the plasma, and the PSR calculation formula is as follows: PSR ═ OD value-OD of sample to be measured_NCValue of]/[ Positive standard OD value-OD_NCValue of]NC is a negative control for each sample.

The plasma of 100 healthy persons is screened, two plasma portions with the highest and lowest PSR values are selected, and are respectively mixed during filtration sterilization, so that high-antibody mixed plasma (plasma group 1) and low-antibody mixed plasma (plasma group 2) are obtained, are subpackaged and are stored in a-20 ℃ refrigerator and are unfrozen at room temperature before being used for cell culture.

4. Pancreatic cancer cell Activity assay

Pancreatic cancer cell line SW1990 cells (provided by tumor Hospital, national academy of medical sciences) were selected for activity experiments. SW1990 cells were cultured in 96-well plates in RPMI1640 medium (supplied by GIBCO) containing 10% fetal bovine serum, 2500 cells/100. mu.l per well, and 9 replicate wells were inoculated with each group of plasma-treated cells. Cells were at 37 degrees (. degree. C.) and 95% carbon dioxide (CO)₂) After 24 hours of incubation under conditions, the supernatant was removed and 100. mu.l of RPMI1640 medium containing 20% human plasma was added and incubation continued for 48 hours with only 100. mu.l of cell culture medium added to reagent blank wells. To measure the cell activity, 10. mu.l of CCK-8 cell counting solution (supplied from Sigma-Aldrich) was added, and after incubation for 2 hours, the Optical Density (OD) was measured at a wavelength of 450nm using a 96-well reading trigger, and the cell activity was calculated. Cell activity ═ plasma 1OD value-blank OD value]/[ plasma 2OD value-blank OD value]. As shown in table 4 and figure 1, healthy human plasma enriched with anti-pancreatic cancer antibodies had a significant inhibitory effect on SW1990 cell activity.

TABLE 4 Effect of healthy human plasma enriched with anti-pancreatic cancer antibodies on SW1990 cell Activity

Note: each set of 9 duplicate wells had a t-test degree of freedom of 16.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the invention. The primary objects and other advantages of the invention may be realized and attained by the structure particularly pointed out in the written description and claims hereof.

Sequence listing

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