Human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit and application thereof

Abstract

The invention relates to a human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit and application thereof. The kit comprises an elisa plate, an anti-human lactoferrin antibody, an elisa secondary antibody, a confining liquid, a color developing agent and a stop solution. A nitrocellulose membrane in Western-Blot is replaced by an elisa plate with non-specific adsorption protein, and along with the addition of a sample, an anti-human lactoferrin antibody and an enzyme-labeled secondary antibody, a lactoferrin-primary antibody-secondary antibody compound is formed. And adding a color developing agent to carry out color development reaction, wherein the absorbance value of the sample is positively correlated with the content of the lactoferrin contained in the sample. And calculating the content of human lactoferrin in the sample according to the absorbance value. The method for detecting the human lactoferrin in the fermentation liquor has the advantages of reality, economy, high efficiency and the like.

Description

Technical Field

[0001] The invention relates to the technical field of milk-based protein detection, in particular to a human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit and application thereof. Background Art

[0002] Lactoferrin is a glycoprotein with a molecular weight of around 80 kDa that is present in the exocrine fluids of most mammals. The concentration of lactoferrin in human breast milk fluctuates throughout lactation, with concentrations gradually decreasing from colostrum to mature milk. Colostrum is rich in Lf, with a content of approximately 6.7 g / L, while the concentration in mature milk is approximately 3.7 g / L. Parallel to this trend, bovine colostrum may contain up to 2 g / L of lactoferrin, which then declines to a range of 0.02 g / L-0.5 g / L in mature milk. In addition, lactoferrin is one of the components found in neutrophils, which serve as the host's main defense against invading pathogens. Therefore, during periods of disease, lactoferrin levels may also be elevated, making it a valuable biomarker for certain diseases. It is now a common ingredient in a variety of commercial products, from nutritional supplements to infant formula and even cosmetics, where it can enhance iron absorption and promote healthy development.

[0003] In the past two decades, many methods for measuring lactoferrin content have been reported. The most commonly used methods are enzyme immunoassay and reversed-phase liquid chromatography. Lactoferrin can be detected and quantified using cation exchange high-performance liquid chromatography, which is rapid, simple and reliable. ELISA uses specific monoclonal antibodies and is able to detect lactoferrin levels in milk, whey and cheese with a sensitivity of 18 ng / mL. Several other detection methods have also been reported for lactoferrin detection, such as radial immunodiffusion, capillary electrophoresis and several sensors (surface plasmon resonance sensors, fluorescent biosensors and boron affinity amplified dynamic light scattering immunosensors) (see the paper "Lactoferrin and Its Detection Methods: A Review"). These different methods reflect the continued pursuit of more effective and accurate quantification methods to meet various research and industrial needs. Nowadays, the lactoferrin detection kits sold on the market are relatively expensive, and it costs about 30 yuan to detect one sample on average. Therefore, it is very feasible to establish an efficient and inexpensive method for lactoferrin detection. Summary of the invention

[0004] To solve the above technical problems, the present invention provides a human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit and its application, which can be used for quantitative detection of human lactoferrin in fermentation broth. The present invention replaces the nitrocellulose membrane in Western-Blot with an enzyme-labeled plate with non-specific adsorption protein, and forms a lactoferrin-primary antibody-secondary antibody complex with the addition of sample, anti-human lactoferrin antibody and enzyme-labeled secondary antibody. A color developer is added to perform a color development reaction, and the sample absorbance value is positively correlated with the content of lactoferrin contained therein. The content of human lactoferrin in the sample is calculated by the absorbance value.

[0005] The present invention is achieved through the following technical solutions:

[0006] The first object of the present invention is to provide a human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit, comprising: an enzyme-labeled plate, an anti-human lactoferrin antibody, an enzyme-labeled secondary antibody, a blocking solution, a color developer and a stop solution.

[0007] In one embodiment of the present invention, the human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit further comprises a human lactoferrin standard.

[0008] In one embodiment of the present invention, the enzyme-labeled secondary antibody is a goat anti-mouse antibody labeled with horseradish peroxidase.

[0009] In one embodiment of the present invention, the anti-human lactoferrin antibody is obtained by diluting the anti-human lactoferrin antibody mother solution by 1000-6000 times; the concentration of the anti-human lactoferrin antibody mother solution is 1 mg / mL;

[0010] And / or, the enzyme-labeled secondary antibody is obtained by diluting the enzyme-labeled secondary antibody mother solution by 1000-2000 times; the concentration of the enzyme-labeled secondary antibody mother solution is 1 mg / mL.

[0011] In one embodiment of the present invention, the color developer includes a color solution A and a color solution B; the color solution A includes CH3COONa, citric acid and H2O2; the color solution B includes EDTA-Na2, citric acid, glycerol and 3,3',5,5'-tetramethylbenzidine.

[0012] In one embodiment of the present invention, the blocking solution is a TBST solution containing skimmed milk powder; the TBST solution includes NaCl, Tris and Tween-20;

[0013] The stop solution is a 1M-2M hydrochloric acid solution.

[0014] The second object of the present invention is to provide a method for detecting human lactoferrin, comprising the human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit.

[0015] The third object of the present invention is to provide the use of the human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit in detecting human lactoferrin in microbial fermentation broth (eg, Trichoderma reesei fermentation broth).

[0016] In one embodiment of the present invention, the concentration of human lactoferrin in the fermentation broth is 0.78 mg / L to 0.01 mg / L.

[0017] In one embodiment of the present invention, the method of application is:

[0018] After adding the sample to the ELISA plate for reaction, it is blocked;

[0019] Anti-human lactoferrin antibody was added for reaction, then enzyme-labeled secondary antibody was added for reaction, and finally a color developer was added for reaction. After the reaction was terminated, the resulting solution was quantitatively analyzed. The human lactoferrin content in the sample was positively correlated with the absorbance value at a wavelength of 450 nm.

[0020] The above technical solution of the present invention has the following advantages compared with the prior art:

[0021] The invention provides a human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit and its application. The human lactoferrin detection method in fermentation broth provided by the invention has the advantages of authenticity, economy, high efficiency and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0023] Figure 1 The invention discloses an establishment process of detecting human lactoferrin by Indirect ELISA (indirect competitive enzyme-linked immunosorbent assay kit); A: comparison of detection principles of Indirect ELISA and Sandwich ELISA (double antibody sandwich enzyme-linked immunosorbent assay kit) on the market; B: color change of human lactoferrin standard products with different concentrations detected by Indirect ELISA; C: preparation of standard curve of Indirect ELISA; D: selection of working concentration of primary antibody of Indirect ELISA; E: selection of working concentration of secondary antibody of Indirect ELISA;

[0024] Figure 2 The invention compares the detection results of the indirect competitive enzyme-linked immunosorbent assay kit (ELISA) of the present invention and the commercial sandwich double antibody kit for detecting the content of recombinant human lactoferrin in the fermentation broth of the engineered strain of Trichoderma reesei; wherein, 1, commercial sandwich double antibody kit; 2, Indirect ELISA. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0026] The invention provides a human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit and an economical and efficient human lactoferrin detection method.

[0027] The detection method of the present invention is to replace the nitrocellulose membrane in Western-Blot with an ELISA plate with non-specific adsorption protein, and form a lactoferrin-primary antibody-secondary antibody complex with the addition of a sample, a primary antibody (anti-human lactoferrin antibody) and a secondary antibody (horseradish peroxidase-labeled goat anti-mouse antibody). A color developer is added to perform a color development reaction, and the sample absorbance value is positively correlated with the content of lactoferrin contained therein. The content of human lactoferrin in the sample is calculated by the absorbance value.

[0028] Furthermore, the detection method is called Indirect ELISA.

[0029] Furthermore, the operation process of the economical and efficient human lactoferrin detection method is as follows:

[0030] A: Preparation of samples and standards: dilute the samples and standards to appropriate concentrations with PBS buffer.

[0031] B: Preparation of blocking solution: Dissolve skim milk powder in TBST solution to prepare a TBST solution containing 5% skim milk powder.

[0032] C: Preparation of primary and secondary antibodies: dilute the primary antibody (anti-human lactoferrin antibody) and secondary antibody (horseradish peroxidase-labeled goat anti-mouse antibody) to the corresponding multiples using TBST solution.

[0033] D: Add sample, take out the ELISA plate and add sample or standard, incubate at 37℃ for 90min, and wash four times with washing solution.

[0034] E: Blocking: add blocking solution for blocking, incubate at 37℃ for 50min, and wash four times with washing solution.

[0035] F: Add the primary antibody (anti-human lactoferrin antibody) diluted with TBST solution to the ELISA plate, incubate at 37°C for 60 minutes, and wash four times with washing solution.

[0036] G: Add the secondary antibody (horseradish peroxidase-labeled goat anti-mouse antibody) diluted with TBST solution to the ELISA plate, incubate at 37°C for 60 min, and wash four times with washing solution.

[0037] H: Add color developing reagent and incubate at 37°C for 15 min.

[0038] I: Add 2M hydrochloric acid to terminate the reaction. Measure the absorbance of the sample in the reaction well at 450nm (A). The concentration of human lactoferrin in the sample is related to A. 450nm The concentration of human lactoferrin in the sample was calculated by drawing a standard curve and using four-parameter fitting software.

[0039] The present invention also optimizes the dilution multiple of the antibody and verifies important parameters such as the specificity and accuracy of the Indirect ELISA detection method.

[0040] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0041] The reagents used in this example are prepared as follows:

[0042] TBST: 8 g NaCl, 2.4 g Tris, 1 mL Tween-20, add deionized water to make up to 1 L.

[0043] Washing solution: Add 500 μL of Tween-20 to 1L of PBS buffer.

[0044] Color developer: Solution A (13.6 g CH3COONa, 1.6 g citric acid, 0.3 mL 30% H2O2, dilute to 500 mL with deionized water), Solution B (0.2 g EDTA-Na2, 0.95 g citric acid, 20 mL glycerol, 0.15 g TMB (3,3',5,5'-tetramethylbenzidine), dilute to 500 mL with deionized water). When using, mix equal volumes of Solution A and Solution B.

[0045] Example 1 Specific process of Indirect ELISA detection

[0046] Add 100μL of gradient diluted standard (0.78mg / L~0.01mg / L) or diluted sample (concentration within the detection range) to the ELISA plate for 90min reaction. The ELISA plate can non-specifically adsorb proteins; the washed ELISA plate is blocked with 300μL of blocking solution for 50min. At this time, the blocking solution will block the unbound wells in the ELISA plate to prevent the unbound wells on the ELISA plate from binding to the antibody and causing the measured value to be high. Next, 90 μL of a 6000-fold diluted primary antibody (anti-human lactoferrin antibody, purchased from Shenzhen Youpin Biotechnology Co., Ltd., item number: YP100103, mother liquor concentration of 1 mg / mL) was added to the washed ELISA plate for 60 min, at which time the primary antibody will specifically bind to the lactoferrin molecules bound to the ELISA plate; then, 90 μL of a 2000-fold diluted secondary antibody (horseradish peroxidase HRP-labeled goat anti-mouse antibody, purchased from Shenzhen Youpin Biotechnology Co., Ltd., item number: YP8485 36-H, mother liquor concentration is 1mg / mL) is added to the washed ELISA plate and reacted for 60min. At this time, the secondary antibody labeled with horseradish peroxidase HRP will specifically bind to the primary antibody; after washing the plate, 90μL of color developer is added and reacted for 15min; if there are different concentrations of human lactoferrin in the sample in the reaction well, HRP will make the colorless TMB turn into different shades (positive correlation) of blue substances, and after adding 50μL of stop solution (2M hydrochloric acid), the reaction well will turn yellow. The final color development degree of different concentrations of human lactoferrin is different ( Figure 1 Finally, the absorbance of the reaction well sample was measured at 450 nm (A), and the concentration of human lactoferrin in the sample was related to A 450nm The concentration of human lactoferrin in the sample was calculated by drawing a standard curve and using four-parameter fitting software ( Figure 1 C in the figure). Since its detection process and materials are different from those of the commercially available Sandwich ELISA, it forms a lactoferrin-primary antibody-secondary antibody complex, so it is called Indirect ELISA ( Figure 1 A).

[0047] The average price of commercially available human lactoferrin detection kits for each sample is about 30 yuan (taking the human lactoferrin detection kit of Shenggong Bioengineering (Shanghai) Co., Ltd. as an example, article number: D711315-0096). The present invention is inspired by protein blotting experiments and enzyme-linked immunosorbent assays, and the indirect competitive enzyme-linked immunosorbent assay kit established only costs about 1-2 yuan to detect a sample on average, greatly reducing the detection cost of human lactoferrin. In this experiment, the biggest cost is the purchase of primary antibodies and ELISA plates. Wherein the ELISA plate is purchased from Thermo Fisher Scientific Inc. (article number: 442404), the cost of each ELISA plate is about 60 yuan, and the average investment of a sample on the ELISA plate is 0.625 yuan. The primary antibody is purchased from Shenzhen Youpin Biotechnology Co., Ltd., and the price of each primary antibody is 1620 yuan (100 μL, article number YP100103), and the average investment of a sample on the primary antibody is 0.243 yuan. The prices of other reagents (such as secondary antibodies, skimmed milk powder, TMB, etc.) are very cheap. In summary, it only takes a small amount of capital investment to establish an indirect competitive ELISA kit.

[0048] Example 2 Optimization of primary antibody working concentration

[0049] The working concentration of the primary antibody in implementation case 1 was optimized to avoid excessive antibody addition, increase experimental costs, and reduce waste. The primary antibody in case 1 was diluted to 1000×, 2000×, 4000×, 6000×, and 8000×, and the secondary antibody was 1000×. The experiment was carried out according to the operating procedures, and the absorbance of the reaction well samples was finally measured at 450nm.

[0050] The results show that Figure 1 D), there was no significant difference in the absorbance of the primary antibody at dilutions of 1000×, 2000×, 4000×, and 6000×. When the primary antibody was diluted to 8000×, the absorbance decreased significantly. Therefore, the primary antibody diluted 6,000 times was selected for subsequent experiments.

[0051] Example 3 Optimization of secondary antibody working concentration

[0052] The working concentration of the secondary antibody in implementation case 1 was optimized. The secondary antibody in case 1 was diluted to 1000×, 2000×, 4000×, 6000×, and 8000×, and the primary antibody was 6000×. The experiment was carried out according to the operation flow, and the absorbance of the reaction well sample was finally measured at 450nm.

[0053] The results show that Figure 1In E), there was no significant difference in the absorbance of the secondary antibody at dilutions of 1000× and 2000×. When the secondary antibody was diluted to 4000×, the absorbance decreased significantly. Therefore, the secondary antibody diluted 2,000 times was selected for subsequent experiments.

[0054] Example 4 Verification of the accuracy and specificity of the Indirect ELISA detection method

[0055] Different samples (human lactoferrin, casein, bovine serum albumin, elastin peptide, cellulase) were prepared into specified concentrations and operated according to the process of implementation case 1. The working concentrations of primary and secondary antibodies were used according to implementation cases 2 and 3.

[0056] The results show (Table 1) that the Indirect ELISA method can detect the content of human lactoferrin well. When the concentration of human lactoferrin is 0.4 and 0.2 mg / L, the recovery rate reaches 86.27% and 90.06% respectively. In addition, the Indirect ELISA was used to detect some common proteins. The results showed that the Indirect ELISA had no reaction with casein, bovine serum albumin, elastin peptide, cellulase and other substances, indicating that the Indirect ELISA method has good specificity.

[0057] Table 1 Recovery rate of Indirect ELISA

[0058]

[0059]

[0060] "-" indicates that the target product was not detected. "n" indicates the number of repetitions.

[0061] Example 5 Determination of Inter-Assay and Intra-Assay Coefficients of Variation of Indirect ELISA Assay

[0062] Human lactoferrin was prepared to a specified concentration and operated according to the process of Example 1. The working concentrations of the primary and secondary antibodies were used according to Examples 2 and 3. When the inter-assay coefficient of variation was determined, the inter-assay variation was determined for 3 replicates over 3 days. When the intra-assay coefficient of variation was determined, the intra-assay variation was determined by 4 replicates over one day.

[0063] Inter-assay precision shows the reproducibility between tests performed on different days. Intra-assay validation shows the reproducibility between wells within the assay plate. The test results show (Table 2) that the coefficient of variation (CV) is less than 10% for both inter-assay precision and intra-assay precision (Table 2). It is generally believed that the coefficient of variation needs to be less than 10%, which ensures that the results obtained are consistent over time and between kits. The results show that the Indirect ELISA has good reproducibility.

[0064] Table 2 Determination of inter-assay and intra-assay coefficients of variation

[0065]

[0066] Notes: a : Coefficient of variation CV (%) = (SD / X) * 100, where SD is the standard deviation of the sample and X is the mean of the sample. "n" represents the number of repetitions

[0067] Comparative Example 1

[0068] This comparative example provides a commercially available sandwich double antibody kit purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0069] Comparative Example 2

[0070] This comparative example provides a commercially available sandwich double antibody kit, which was purchased from Shenzhen Youpin Biotechnology Co., Ltd.

[0071] Test Case

[0072] In western blot, with the successive addition of primary and secondary antibodies, antibody conjugates are formed, and this structure can achieve the purpose of signal amplification, thereby enabling micro-detection of the target substance. Among the commercially available human lactoferrin test kits, the sensitivity of the test kits produced by different manufacturers varies. By comparing the detection limits of the test kits produced by different manufacturers with the test kit prepared by Example 1 of the present invention, it is shown that the test kit developed by the present invention has a lower detection limit and good sensitivity, and can detect low concentrations of human lactoferrin (Table 3).

[0073] Table 3 Comparison of detection limits of human lactoferrin kits

[0074]

[0075] The commercially available sandwich double antibody kit and the Indirect ELISA prepared in Example 1 of the present invention were used to detect the human lactoferrin content in the fermentation broth of the engineered strain Trichoderma reesei CJ2095 (see patent CN117487675A). The detection results of the two methods were 452.71 mg / L and 438.62 mg / L, respectively. The results showed that there was no significant difference between the results of the two detection methods ( Figure 2 ). At the same time, the detection method established in this experiment was compared with the commercially available human lactoferrin detection kit in terms of intra-plate coefficient of variation, inter-plate coefficient of variation, and cell supernatant recovery rate. From the comparison results (Table 4), it can be seen that the detection method established by the present invention has a similar effect to the commercially available human lactoferrin detection kit. In summary, it can be seen that the detection method established by the present invention has a good effect in terms of detection quality.

[0076] Table 4 Quality comparison of human lactoferrin kit

[0077]

[0078] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit, characterized in that: include: ELISA plate, anti-human lactoferrin antibody, enzyme-labeled secondary antibody, blocking solution, color developer and stop solution.

2. The human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit also includes a human lactoferrin standard product.

3. The human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The enzyme-labeled secondary antibody is a goat anti-mouse antibody labeled with horseradish peroxidase.

4. The human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The anti-human lactoferrin antibody is obtained by diluting the anti-human lactoferrin antibody mother solution by 1000-6000 times; the concentration of the anti-human lactoferrin antibody mother solution is 1 mg / mL; And / or, the enzyme-labeled secondary antibody is obtained by diluting the enzyme-labeled secondary antibody mother solution by 1000-2000 times; the concentration of the enzyme-labeled secondary antibody mother solution is 1 mg / mL.

5. The human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The color developer comprises a color solution A and a color solution B; the color solution A comprises CH3COONa, citric acid and H2O2; the color solution B comprises EDTA-Na2, citric acid, glycerol and 3,3',5,5'-tetramethylbenzidine.

6. The human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit according to claim 1, characterized in that: The blocking solution is a TBST solution containing skimmed milk powder; the TBST solution includes NaCl, Tris and Tween-20; The stop solution is a 1M-2M hydrochloric acid solution.

7. A method for detecting human lactoferrin, characterized in that: The invention comprises the human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit according to any one of claims 1 to 6.

8. Use of the human lactoferrin indirect competitive enzyme-linked immunosorbent assay kit according to any one of claims 1 to 6 in detecting human lactoferrin in microbial fermentation broth.

9. The use according to claim 1, characterized in that: The concentration of human lactoferrin in the fermentation broth is 0.78 mg / L to 0.01 mg / L.

10. The use according to claim 1, characterized in that: The method of application is: After adding the sample to the ELISA plate for reaction, it is blocked; Anti-human lactoferrin antibody was added for reaction, then enzyme-labeled secondary antibody was added for reaction, and finally a color developer was added for reaction. After the reaction was terminated, the resulting solution was quantitatively analyzed. The human lactoferrin content in the sample was positively correlated with the absorbance value at a wavelength of 450 nm.

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