A method for extracting and quantitatively detecting ApoJ on a tear test strip, and a kit and application thereof

Abstract

The present invention discloses a method for extracting and quantitatively detecting ApoJ on a tear test strip, as well as a kit and application thereof. Sodium N-lauroylsarcosine is used to extract ApoJ on the tear test strip, and a method for quantitatively detecting the content of ApoJ in tears by ELISA is established. The concentration of the ApoJ protein solution has a linear relationship with the OD value at a wavelength of 450 nm, and the regression linear equation is y = 1.7182x, where the correlation coefficient R<supgt;2< / supgt> = 0.9936. Through the method of the present invention, the content of ApoJ in the tears of dry eye patients can be quickly obtained, so as to judge the fundus barrier condition of dry eye patients, providing a rapid and convenient means for subsequent diagnosis and treatment, and having the value of application and popularization.

Description

Technical Field

[0001] The present invention relates to the technical field of detection, and particularly relates to a method for extracting and quantitatively detecting ApoJ on a tear test paper, a kit thereof, and an application. Background Art

[0002] Dry eye is an ocular surface disease caused by various disorders of different functions and ocular surface protection mechanisms. It is one of the ocular surface diseases with the highest incidence in ophthalmology. The onset of dry eye is caused by multiple factors, and the absence of ocular surface tissues and tear film can all cause dry eye. Current research shows that nerve function, sex hormones, and inflammatory factors play important roles in regulating tear secretion. Dry eye is a disease caused by any reason resulting in abnormalities in the quality, quantity, and kinetics of tears, thereby leading to unstable tear film and / or abnormalities of the ocular surface, accompanied by ocular discomfort symptoms. There are many causes of dry eye. Mild dry eye patients caused by environmental factors and personal habits (such as long-term use of computers and long-term exposure to air-conditioned environments) only show mild symptoms without obvious damage to the ocular surface. Timely improvement of these influencing factors can make the ocular discomfort disappear. However, for dry eye caused by local or systemic definite causes (such as ocular surface chemical and thermal burns, Sjogren's syndrome, allergies, blepharitis, etc.), its pathogenesis is very complex. The ocular surface (cornea, conjunctiva, accessory lacrimal glands, and meibomian glands), the main lacrimal gland, and their neural connections form an integrated functional unit due to their close anatomical and functional connections, jointly playing a regulatory role in tear secretion and tear film formation, maintaining the health of the ocular surface. Damage to any link can lead to the destruction of the integrity and function of the tear film, thereby presenting dry eye discomfort symptoms. The continuous abnormality of the tear film can damage the normal repair or defense mechanism of the ocular surface, leading to a chronic inflammatory state of the ocular surface and lacrimal gland. Although the initial causes of dry eye are different, once it enters the progressive stage, inflammation becomes the most critical factor in the pathogenesis of dry eye, and apoptosis, neural regulation, and sex hormones also participate in the pathogenesis of dry eye together. Therefore, different types of dry eye show similar pathophysiological changes.

[0003] The Schirmer I test is one of the main means for screening dry eye clinically. It has obvious precise graduations, is easy to observe, is an objective examination, is simple and easy to operate, and has a relatively high accuracy. The specific operation method is to use a test paper of 5 mm × 35 mm, bend one end by 5 mm, place it in the inner 1 / 3 of the lower eyelid conjunctival sac, and let the rest hang on the skin surface. Gently close the eyes, and after 5 minutes, measure the length of the test paper wetted by tears. The results of the Schirmer I test are as follows: the results of normal people are 10 - 15 mm / 5 min, <10 mm is low secretion, and <5 mm is dry eye.

[0004] Apolipoprotein J (ApoJ, also known as clusterin) is a multifunctional glycoprotein that is widely present in various tissues and body fluids of the human body. ApoJ is a heterodimeric protein with a relative molecular mass of 75-80 kDa and can bind to human plasma high-density lipoprotein (HDL) and very high-density lipoprotein (VHDL). The function of ApoJ is complex and it participates in various in vivo physiological processes including the regulation of complement function, inhibition of apoptosis and inflammation, and participation in lipid transport. In 1996, ApoJ was proved to be the most highly expressed transcript in the human cornea, and the protein product was localized to the apical layer of the corneal and conjunctival mucosal epithelium. ApoJ is also present in human tears. ApoJ is prominent at the liquid-tissue interface and its expression is significantly reduced in severe dry eye disease. In a preclinical stress mouse model mimicking human dry eye disease, when the eyes are under dry stress, the expression levels of ApoJ mRNA and protein in the ocular surface epithelial cells are both reduced by about 30%. Local application of ApoJ to the mouse eyes can provide complete protection, manifested as ApoJ being able to seal the eye surface to prevent the penetration of fluorescein dye. ApoJ selectively binds to the ocular surface under dry stress in vivo and in vitro and binds to the key barrier component LGALS3 (galectin-3), further inhibiting the proteolysis of ocular surface structural proteins (such as LGALS3 and OCLN). By localizing in this way, ApoJ not only physically seals the ocular surface barrier, but also protects the barrier cells and prevents further damage to the barrier structure. ApoJ has the ability to protect ocular surface proteins from enzymatic degradation, possibly by interacting with MMP9, thereby inhibiting the protease activity of MMP9. In various inflammatory conditions in humans and mice, depletion of CLU in the ocular surface epithelial cells can be seen, leading to squamous metaplasia and keratinized epithelium. This indicates that ApoJ may play a special role in maintaining mucosal epithelial differentiation.

[0005] The tear secretion test can only preliminarily judge the tear secretion situation by the length of tear infiltration within a certain time, but it cannot know whether the fundus barrier of the tested person is normal. Summary of the Invention

[0006] In view of this, the present invention aims to propose a method for extracting and quantitatively detecting ApoJ on a tear test strip, its kit and application, so as to further analyze the expression of ApoJ in dry eye patients by using the tear test strip used in the tear secretion test, and provide a basis for the diagnosis and subsequent treatment of dry eye.

[0007] To achieve the above object, the technical solution of the present invention is realized as follows:

[0008] On the one hand, the present invention proposes a method for extracting and quantitatively detecting ApoJ on a tear test strip, including the following steps:

[0009] S1. Preparation of standard product: Prepare a protein solution from natural or recombinant ApoJ protein.

[0010] Preparation of control product: Place the tear test strip into the protein solution, then take it out and air-dry.

[0011] Preparation of test product: Air-dry the tear test strip from the tear secretion experiment.

[0012] S2. Preparation of protein extraction solution: Prepare a protein extraction solution from sodium lauroyl sarcosinate.

[0013] S3. Protein extraction: Add the protein extraction solution to the tear test strips in step S1 respectively. After mixing, let it stand overnight at 4°C. After centrifugation, obtain the supernatant, and then the extracted protein solution can be obtained.

[0014] S4. ApoJ quantitative detection method: Use the ELISA method to detect the content of ApoJ in the standard product and obtain the regression linear equation; Take the protein solutions extracted from the control product and the test product, detect the absorbance values through the ELISA method, and compare with the regression linear equation to obtain the ApoJ concentrations of the control product and the test product.

[0015] Further, the specific operation steps for obtaining the regression linear equation by the ELISA method in step S4 are as follows:

[0016] S41. Dilute the protein solution of the standard product to different concentrations with the diluent.

[0017] S42. Add 100 μL of the sample to each well in the coated plate, gently shake to mix evenly, cover with the plate sticker and react at 37°C for 2 hours.

[0018] S43. Discard the liquid and centrifuge dry; Add 100 μL of the antibody working solution labeled with biotin antibody to each well, cover with a new plate sticker and react at 37°C for 1 hour.

[0019] S44. Discard the liquid in the wells and centrifuge dry, wash the plate 3 times, soak for 2 minutes each time, 200 μL per well, and centrifuge dry.

[0020] S45. Add 100 μL of the avidin working solution labeled with horseradish peroxidase to each well, cover with a new plate sticker and react at 37°C for 1 hour.

[0021] S46. Discard the liquid in the wells and centrifuge dry, wash the plate 5 times, soak for 2 minutes each time, 200 μL per well, and centrifuge dry.

[0022] S47. Sequentially add 90 μL of the TMB substrate chromogenic solution to each well and develop color at 37°C in the dark.

[0023] S48. Sequentially add 50 μL of the TMB stop solution to each well to terminate the reaction.

[0024] S49. Measure the OD value of each well at a wavelength of 450 nm with an enzyme-labeled instrument within 5 minutes after the reaction termination, and establish a regression linear equation.

[0025] Further, in the preparation of the control in step S1, the natural or recombinant ApoJ protein is formulated into a protein solution with a concentration of 50 μg / mL.

[0026] Further, in step S2, sodium lauroyl sarcosinate is formulated into a 1% protein extraction solution.

[0027] Further, in step S3, centrifuge at 12000 - 15000 rpm for 3 minutes to obtain the supernatant.

[0028] Further, in step S4, taking the concentration of the protein solution of the standard product as the abscissa and the OD value as the ordinate, plot a standard curve and calculate the regression linear equation.

[0029] Further, in step S4, the regression linear equation is y = 1.7182x, where the correlation coefficient R 2 = 0.9936.

[0030] Further, in step S41, the dilution solution is a PBST solution containing 1% BSA.

[0031] Further, in step S43, the antibody working solution is a PBST solution containing 1% BSA with ApoJ antibody.

[0032] Further, in steps S44 and S46, the washing solution used for washing the plate is a PBST solution.

[0033] Compared with the prior art, the method for ApoJ extraction and quantitative detection on a tear test strip of the present invention has the following advantages: using sodium lauroyl sarcosinate to strip and collect ApoJ protein from tears, and then quantitatively detecting the content of ApoJ on the tear test strip by an ELISA detection method, so as to judge the fundus barrier condition of dry eye patients, providing a rapid and convenient means for subsequent diagnosis and treatment, and the ELISA detection method has high detection sensitivity.

[0034] On the other hand, the present invention provides a kit for ApoJ extraction and quantitative detection on a tear test strip, and the kit is detected by the method for ApoJ extraction and quantitative detection on a tear test strip as described above.

[0035] On the other hand, the present invention proposes an application of a kit for ApoJ extraction and quantitative detection on a tear test strip in dry eye.

[0036] The advantages of the kit and its application and the above method for ApoJ extraction and quantitative detection on the tear test strip over the prior art are the same, and will not be elaborated here. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0038] Figure 1 is the standard curve of the present invention; DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention will be further described below in conjunction with specific embodiments. It should be noted first that the data in the following experimental examples were obtained by the inventors through a large number of experiments. Due to space limitations, only a part of them are shown in the specification, and those of ordinary skill in the art can understand and implement the present invention based on this data. These embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these changes or modifications also fall within the scope protected by this application.

[0040] All the reagents used in the method provided by the present invention can be purchased from the market. The ApoJ used as a standard can be natural or recombinant.

[0041] Example 1 A method for ApoJ extraction and quantitative detection on a tear test strip

[0042] Specifically, it includes the following steps:

[0043] S1. Sample preparation

[0044] S11. Preparation of standard:

[0045] Prepare a protein solution from natural or recombinant ApoJ protein,

[0046] S12. Confirmation of the saturated volume of the tear test strip:

[0047] First, cut the unused tear test strip into 3 mm / segments, and respectively suck 1 - 5 μL of PBS solution, and observe the volume that can make the tear test strip fully absorb the PBS solution. The result is that using 2 μL of PBS solution can completely infiltrate the test strip and there is basically no liquid residue;

[0048] S13. Preparation of control:

[0049] Prepare a protein solution of 50 μg / mL with natural or recombinant ApoJ protein using PBS solution. Moisten unused tear test strips with the protein solution, then place the test strips in an EP tube and air-dry them to simulate the tear test strips being infiltrated by tears.

[0050] S14. Preparation of test samples:

[0051] Place the tear test strips from the patient's tear secretion experiment in an EP tube and air-dry them.

[0052] S2. Preparation of protein extraction solution:

[0053] Take 1 g of sodium lauroyl sarcosinate (SKL) and dissolve it in 100 mL of PBS solution to prepare a PBS solution containing 1% SKL.

[0054] S3. Protein extraction:

[0055] Take out the air-dried tear test strips from step S1. The tear test strips are the tear test strips of the control and the test samples. Observe the wetting and staining of the tears. Take 3 mm from the middle part of the wetted area as needed and put it into an EP tube. Add 200 μL of protein extraction solution, pipette several times with a pipette gun or mix well with a shaker, and place it at 4 °C overnight. Finally, centrifuge at 13000 rpm for 3 min and take the supernatant to obtain the extracted protein solution.

[0056] S4. ApoJ quantitative detection method:

[0057] Use the ELISA method to detect the content of standard ApoJ and obtain the regression linear equation; take the protein solutions extracted from the control and test samples, detect them by the ELISA method, obtain the absorbance values, and compare them with the regression linear equation to obtain the ApoJ concentrations of the control and test samples.

[0058] Among them, the specific operation steps for obtaining the regression linear equation by the ELISA method are as follows:

[0059] S41. Dilute the protein solution of the standard product to different concentrations with a diluent; the diluent is a PBST solution containing 1% BSA;

[0060] S42. Add 100 μL of the sample to each well in the coated plate, gently shake to mix evenly, cover with a plate sticker and react at 37 °C for 2 hours;

[0061] S43. Discard the liquid and centrifuge dry; add 100 μL of the biotin antibody-labeled antibody working solution to each well, cover with a new plate sticker and react at 37 °C for 1 hour; the antibody working solution is a PBST solution containing 1% BSA with ApoJ antibody;

[0062] S44. Discard the liquid in the wells, centrifuge to dry, wash the plate 3 times, soak for 2 min each time, 200 μL per well, centrifuge to dry;

[0063] S45. Add 100 μL of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate sticker, and react at 37 °C for 1 hour;

[0064] S46. Discard the liquid in the wells, centrifuge to dry, wash the plate 5 times, soak for 2 min each time, 200 μL per well, centrifuge to dry; The washing solution used for washing the plate is PBST solution.

[0065] S47. Sequentially add 90 μL of TMB substrate chromogenic solution to each well, and develop color in the dark at 37 °C;

[0066] S48. Sequentially add 50 μL of TMB stop solution to each well to terminate the reaction;

[0067] S49. Measure the OD value of each well with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm within 5 min after terminating the reaction; Use the concentration of the protein solution of the standard product as the abscissa and the OD value as the ordinate to draw a standard curve.

[0068] The concentration of the ApoJ protein solution has a linear relationship with the OD value at a wavelength of 450 nm. The specific standard curve is as Figure 1 shown. The regression linear equation is y = 1.7182x, where the correlation coefficient R 2 = 0.9936.

[0069] The specific experimental data are shown in Table 1:

[0070] Table 1

[0071] Concentration ng / mL 0 0.03125 0.0625 0.125 0.25 0.5 1 OD450nm 0.115 0.174 0.24 0.367 0.585 1.022 1.777

[0072] Detect the OD value of the control or the sample to be tested, and find out the corresponding concentration from the regression linear equation, and then multiply by the dilution factor to obtain the actual concentration of ApoJ in the control or the sample to be tested.

[0073] Furthermore, compare the concentration of ApoJ in the control product calculated by the regression linear method obtained by the ELISA detection method of the present invention with the actual concentration of ApoJ in the control product. The results of the two are consistent, indicating that the detection method of the present invention is accurate and reliable.

[0074] The expression of ApoJ significantly decreases in severe dry eye disease. Using ApoJ as a drug can not only resist the drying stress that causes dry eye disease but also prevent the downstream activation of the pathological pathway of dry eye disease by MMP9. If the expression level of ApoJ in the tears of dry eye patients can be quickly detected, it can be determined whether the dry eye patients have damaged fundus barriers caused by low ApoJ content in the tears, providing a basis for later diagnosis and treatment. If the expression level of ApoJ in the tears of some dry eye patients is relatively low, ApoJ can be used as a drug for the patients. Therefore, using the detection method of the present invention, first, sodium lauroyl sarcosinate is used to strip and elute the ApoJ protein on the tear test paper. The sodium lauroyl sarcosinate solution can also help the ApoJ protein maintain a certain monomer structure, inhibit protein aggregation, and facilitate the measurement of the expression level of ApoJ. Then, the expression level of ApoJ on the tear test paper is accurately detected by the ELISA method. By comparing the expression level of ApoJ on the tear test paper in the tear secretion experiment of dry eye patients with that in the tear secretion experiment of normal people, when the expression level of ApoJ in dry eye patients is lower than that in normal people, it indicates that the fundus barriers of dry eye patients are damaged and corresponding treatment is needed in a timely manner.

[0075] Example 2 Assembly of ELISA Kit

[0076] The specific composition of the ELISA kit is as follows: (1) One blank enzyme-labeled plate; (2) One ApoJ standard; (3) Sodium lauroyl sarcosinate; (4) Biotinylated antibody working solution (the antibody working solution is a PBST solution containing 1% BSA of ApoJ antibody), horseradish peroxidase-labeled avidin working solution; (5) PBS buffer, washing solution (PBST solution), substrate chromogenic solution (TMB system), and termination solution (TMB system).

[0077] Example 3 Detection of Clinical Samples

[0078] Four dry eye patients were randomly selected from a hospital for tear secretion experiments. After the obtained tear test paper was air-dried, 3 mm of it was cut and placed into an EP tube. Sodium lauroyl sarcosinate was prepared into a 1% protein extraction solution, and 200 μL of the protein extraction solution was added to the air-dried tear test paper. It was pipetted several times with a pipette or mixed evenly with a shaker and placed at 4°C overnight. Finally, it was centrifuged at 13000 rpm for 3 min to take the supernatant, and the extracted protein solution was obtained. The ELISA detection method was used for determination, and the OD value at a wavelength of 450 nm was recorded. The content of ApoJ in the tear sample was calculated using the standard curve. The specific results are shown in Table 2.

[0079] Table 2

[0080] Number Right eye mg / L Left eye mg / L 1 16.36 10.08 2 19.80 15.78 3 26.75 11.03 4 29.43 35.86

[0081] As can be seen from Table 2, among dry eye patients, the content of ApoJ in tears is also different. For example, the content of ApoJ in the left and right eyes of Patient No. 4 is 35.86 mg / L and 29.43 mg / L respectively, while the content of ApoJ in the left and right eyes of Patient No. 1 is 10.08 mg / L and 16.36 mg / L respectively. The content of ApoJ in the tears of Patient No. 1 decreased significantly, and the fundus barrier was damaged.

[0082] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for extracting and quantitatively detecting ApoJ on a tear test strip, characterized in that, It includes the following steps: S1. Preparation of standard product: Prepare a protein solution from natural or recombinant ApoJ protein. Preparation of control product: Immerse the tear test strip in the protein solution, then take it out and air-dry it. Preparation of test product: Air-dry the tear test strip from the tear secretion experiment. S2. Preparation of protein extraction solution: Prepare a protein extraction solution from sodium lauroyl sarcosine. S3. Protein extraction: Add the protein extraction solution to the tear test strips in step S1 respectively. After mixing evenly, place them at 4°C overnight, centrifuge, and obtain the supernatant to get the extracted protein solution. S4. ApoJ quantitative detection method: Use the ELISA method to detect the standard product and obtain a regression linear equation. Take the protein solutions extracted from the test product and the control product, detect them by the ELISA method to obtain absorbance values, compare with the regression linear equation, and obtain the ApoJ concentrations of the test product and the control product.

2. The method for ApoJ extraction and quantitative detection on a tear test strip according to claim 1, wherein The specific operation steps for obtaining the regression linear equation by the ELISA method in step S4 are as follows: S41. Dilute the protein solution of the standard product to different concentrations with a diluent. S42. Add 100 μL of the sample to each well of the coated plate, gently shake to mix evenly, cover with a plate sticker, and react at 37°C for 2 hours. S43. Discard the liquid and centrifuge dry; add 100 μL of the antibody working solution labeled with biotin antibody to each well, cover with a new plate sticker, and react at 37°C for 1 hour. S44. Discard the liquid in the wells and centrifuge dry; wash the plate 3 times, soak for 2 minutes each time, 200 μL per well, and centrifuge dry. S45. Add 100 μL of the avidin working solution labeled with horseradish peroxidase to each well, cover with a new plate sticker, and react at 37°C for 1 hour. S46. Discard the liquid in the wells and centrifuge dry; wash the plate 5 times, soak for 2 minutes each time, 200 μL per well, and centrifuge dry. S47. Sequentially add 90 μL of the TMB substrate chromogenic solution to each well and develop color at 37°C in the dark. S48. Sequentially add 50 μL of the TMB stop solution to each well to terminate the reaction. S49. Measure the OD values of each well with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm within 5 minutes after terminating the reaction, and make a regression linear equation.

3. A method for extracting and quantitatively detecting ApoJ on a tear test strip according to claim 1, characterized in that, In the preparation of the control product in step S1, prepare a protein solution of 50 μg / mL from natural or recombinant ApoJ protein.

4. A method for extracting and quantitatively detecting ApoJ on a tear test strip according to claim 1, characterized in that, In step S2, prepare a 1% protein extraction solution from sodium lauroyl sarcosine.

5. A method for extracting and quantitatively detecting ApoJ on a tear test strip according to claim 1, characterized in that, In step S3, centrifuge at 12000 - 15000 rpm for 3 minutes to obtain the supernatant.

6. A method for ApoJ extraction and quantitative detection on a tear test strip according to claim 1, characterized in that, In step S4, use the concentration of the protein solution of the standard product as the abscissa and the OD value as the ordinate to draw a standard curve and calculate the regression linear equation.

7. A method for extracting and quantitatively detecting ApoJ on a tear test strip according to claim 1, characterized in that, In the step S4, the regression linear equation is y = 1.7182x, where the correlation coefficient R 2 = 0.9936.

8. A method for ApoJ extraction and quantitative detection on a tear test strip according to claim 2, characterized in that, In step S43, the antibody working solution is a PBST solution containing 1% BSA with ApoJ antibody.

9. A kit for the extraction and quantitative detection of ApoJ on a tear test strip, characterized in that, The kit is detected by a method for ApoJ extraction and quantitative detection on a tear test strip as described in any one of claims 1 - 8.

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