CN115541568A - ApoJ extraction and quantitative detection method on tear test paper, kit and application thereof - Google Patents
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Abstract
The invention discloses an ApoJ extraction and quantitative detection method on tear test paper, a kit and application thereof. Extracting ApoJ on tear test paper by using sodium dodecyl sarcosinate, and establishing a method for quantitatively detecting the content of ApoJ in tears by using ELISA. The concentration of the ApoJ protein solution is linear with the OD at 450nm wavelength with a regression linear equation of y =1.7182x, with the correlation coefficient R 2 =0.9936. The method can quickly obtain the content of ApoJ in tears of dry eye patients, thereby judging the condition of fundus barriers of the dry eye patients, providing a quick and convenient means for subsequent diagnosis and treatment, and having application and popularization values.
Description
Technical Field
The invention relates to the technical field of detection, in particular to an ApoJ extraction and quantitative detection method on tear test paper, a kit and application thereof.
Background
Dry eye is an ocular surface disease caused by various disorders of functions and ocular surface protection mechanisms, and is one of the ocular surface diseases with the highest incidence in ophthalmology, the incidence of dry eye is caused by various factors, and dry eye is caused by the deficiency of ocular surface tissues and tear film. Studies have now shown that nerve functions, sex hormones, inflammatory factors and the like play an important role in regulating tear secretion. Dry eye is a disease in which the tear film is unstable and/or the surface of the eye is abnormal due to abnormalities in the quality and quantity and kinetics of tears caused by any cause, with the attendant symptoms of ocular discomfort. The xerophthalmia is caused by a plurality of etiological factors, and mild xerophthalmia patients (such as a computer used for a long time and an air-conditioning environment used for a long time) caused by environmental factors, personal habits and the like only have mild symptoms without obvious ocular surface damage, and the discomfort of eyes can disappear by timely improving the influencing factors; however, the pathogenesis of dry eye disease (such as chemical and thermal burns on the ocular surface, sj69ren syndrome, allergy, blepharitis, etc.) caused by local or systemic specific etiology is very complicated. The ocular surface (cornea, conjunctiva, accessory lacrimal gland and meibomian gland), the main lacrimal gland and the nerve connection among the main lacrimal gland and the accessory lacrimal gland form a whole functional unit due to close anatomical and functional connection, the regulation and control functions on tear secretion and tear film formation are exerted together, the ocular surface health is maintained, the damage of any link can cause the damage of the tear film integrity and function, thus the uncomfortable symptom of xerophthalmia is caused, the continuous abnormality of the tear film can damage the normal repair or defense mechanism of the ocular surface, and the ocular surface and the tear gland are in a chronic inflammation state. Although the initial etiology of dry eye varies, once it has progressed, inflammation becomes the most critical factor in the pathogenesis of dry eye, and apoptosis, neuromodulation, sex hormones, etc. also participate together in the pathogenesis of dry eye, and thus different types of dry eye exhibit similar pathophysiological changes.
The tear secretion test (Schirmeri) is one of the main clinical means for screening xerophthalmia, has obvious precise scales, is easy to observe, is an objective examination, is simple and easy to operate, and has high accuracy. The specific operation method is that a piece of test paper with the size of 5mm multiplied by 35mm is used, one end of the test paper is bent by 5mm, the test paper is placed in a 1/3 conjunctival sac at the inner side of the lower eyelid, the other part of the test paper is hung on the surface of the skin, the eyes are slightly closed, and the length of the test paper which is wetted by the tear water is measured after 5 minutes. The results of the tear secretion experiments were: normal persons had results of 10-15mm/5min, low secretion < 10mm and dry eye < 5 mm.
Apolipoprotein J (ApoJ), a versatile glycoprotein, is widely found in various tissues and fluids of the human body. Apolipoprotein J is a heterodimeric protein with a relative molecular mass of 75-80kDa and is capable of binding to human plasma High Density Lipoprotein (HDL) and Very High Density Lipoprotein (VHDL). Apolipoprotein J is functionally complex and involved in a variety of physiological processes in vivo including regulation of complement function, inhibition of apoptosis and inflammation, and in lipid transport. ApoJ was shown to be the most highly expressed transcript in human cornea in 1996, with protein products localized to the apical layer of 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. In a preclinical stress mouse model that mimics human dry eye disease, the expression levels of ApoJ mRNA and protein in ocular surface epithelial cells are both reduced by about 30% when the eye is subjected to drying pressure. Complete protection was provided by topical application of ApoJ to the mouse eye, as demonstrated by the ability of ApoJ to seal the surface of the eye from penetration by fluorescein dye. ApoJ selectively binds to the ocular surface under the effect of dry stress in vivo and in vitro and binds to LGALS3 (galectin-3), a key barrier component, further inhibiting proteolysis of ocular surface structural proteins (e.g., LGALS3 and OCLN). Positioned in this way, apoJ not only physically seals the ocular surface barrier, but also protects the barrier cells, preventing further damage to the barrier structure. ApoJ has the ability to protect eye surface proteins from enzymatic degradation, possibly through interaction with MMP9, thereby inhibiting the protease activity of MMP 9. Under various inflammatory conditions in humans and mice, depletion of CLU from ocular surface epithelial cells can be seen, leading to squamous metaplasia and keratinized epithelium. This suggests that ApoJ may have a particular role in maintaining mucosal epithelial differentiation.
The lacrimal secretion test can only preliminarily judge the lacrimal secretion condition through the length of the lacrimal infiltration in a certain time, but cannot know whether the ocular fundus barrier of the tested person is normal.
Disclosure of Invention
In view of the above, the present invention is directed to a method for extracting and quantitatively detecting ApoJ on tear test paper, and a kit and an application thereof, so as to further analyze the ApoJ expression of a patient with dry eye syndrome by using tear test paper used in a tear secretion experiment, and provide a basis for the diagnosis and subsequent treatment of dry eye syndrome.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
on one hand, the invention provides a method for extracting and quantitatively detecting ApoJ on tear test paper, which comprises the following steps:
s1, preparing a standard substance: preparing natural or recombinant ApoJ protein into protein solution,
preparation of control: putting the tear test paper into a protein solution, taking out and drying in the shade;
preparing a to-be-detected product: drying tear test paper for tear secretion test in the shade;
s2, preparing a protein extracting solution: preparing protein extracting solution from sodium dodecyl sarcosinate;
s3, protein extraction: adding protein extracting solutions into the tear test paper obtained in the step S1 respectively, uniformly mixing, standing overnight at 4 ℃, centrifuging to obtain supernate, and thus obtaining an extracted protein solution;
s4, an ApoJ quantitative detection method: detecting the content of ApoJ in the standard substance by using an ELISA method, and obtaining a regression linear equation; and (3) taking the protein solutions extracted from the reference substance and the to-be-detected substance, detecting by an ELISA method to obtain an absorbance value, and comparing the absorbance value with the regression linear equation to obtain the ApoJ concentrations of the reference substance and the to-be-detected substance.
Further, the specific operation steps of obtaining the regression linear equation by the ELISA method in step S4 are as follows:
s41, diluting the protein solution of the standard substance to different concentrations by using a diluent;
s42, adding 100 mu L of sample into each hole in the coated plate, slightly shaking and uniformly mixing, covering the plate, and reacting for 2 hours at 37 ℃;
s43, discarding the liquid, and spin-drying; adding 100 mu L of biotin antibody-labeled antibody working solution into each hole, covering a new plate, and reacting at 37 ℃ for 1 hour;
s44, discarding liquid in the holes, spin-drying, washing the plate for 3 times, soaking for 2min each time, and spin-drying at a rate of 200 mu L per hole;
s45, adding 100 mu L of horseradish peroxidase labeled avidin working solution into each hole, covering a new plate, and reacting for 1 hour at 37 ℃;
s46, discarding liquid in the holes, spin-drying, washing the plate for 5 times, soaking for 2min each time, and spin-drying at a rate of 200 mu L per hole;
s47, sequentially adding 90 mu L of TMB substrate color development solution into each hole, and developing at 37 ℃ in a dark place;
s48, adding 50 mu L of TMB stop solution into each hole in sequence, and stopping reaction;
s49, measuring the OD value of each hole by using an enzyme-labeling instrument within 5min after the reaction is stopped at the wavelength of 450nm, and making a regression linear equation.
Further, in the step S1 of preparing the reference substance, the natural or recombinant ApoJ protein is prepared as a protein solution of 50 μ g/mL.
Further, in the step S2, sarcosyl is prepared into a 1% protein extract.
Further, in the step S3, the supernatant is obtained by centrifuging at 12000-15000rpm for 3 min.
Further, in the step S4, a standard curve is drawn with the concentration of the protein solution of the standard as an abscissa and the OD value as an ordinate, and a regression linear equation is calculated.
Further, in step S4, the regression linear equation is y =1.7182x, where the correlation coefficient R is 2 ＝0.9936。
Further, in step S41, the dilution is 1% BSA in PBST solution.
Further, in step S43, the antibody working solution was a 1% BSA in PBST solution containing ApoJ antibody.
Further, the washing solution used for washing the plate in steps S44 and S46 is a PBST solution.
Compared with the prior art, the method for extracting and quantitatively detecting ApoJ on tear test paper has the following advantages: the ApoJ protein is stripped and collected from tears by utilizing the sodium dodecyl sarcosine, and then the content of the ApoJ on tear test paper is quantitatively detected by an ELISA detection method, so that the condition of the fundus barrier of a xerophthalmia patient is judged, a rapid and convenient means is provided for subsequent diagnosis and treatment, and the detection sensitivity of the ELISA detection method is high.
On the other hand, the invention provides a kit for extracting and quantitatively detecting ApoJ on tear test paper, and the kit is used for detecting ApoJ on tear test paper by using the method for extracting and quantitatively detecting ApoJ on tear test paper.
On the other hand, the invention provides application of the kit for ApoJ extraction and quantitative detection on the tear test paper in xerophthalmia.
The advantages of the kit and the application thereof are the same as the advantages of the ApoJ extraction and quantitative detection method on the tear test paper compared with the prior art, and the details are not repeated.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation of the invention. In the drawings:
FIG. 1 is a standard curve of the present invention;
Detailed Description
The invention will be further illustrated with reference to specific embodiments. It should be noted at first that the data in the experimental examples described below are obtained by the inventors through a large number of experiments, limited to the space, only a part of which is shown in the specification, and those skilled in the art can understand and implement the present invention under the data. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Furthermore, it should be understood that various changes or modifications to the invention may be made by those skilled in the art after reading the disclosure of the present invention, and these changes or modifications also fall within the scope of the protection of the present application.
The reagents used in the methods provided by the present invention are commercially available. Wherein the ApoJ serving as a standard product can be natural or recombinant.
Example 1 ApoJ extraction and quantitative detection method on tear test paper
The method specifically comprises the following steps:
s1. Sample preparation
S11, preparing a standard product:
preparing natural or recombinant ApoJ protein into protein solution,
s12, confirming the saturated volume of the tear test paper:
firstly, cutting unused tear test paper into 3 mm/section, respectively sucking 1-5 microliter volume of PBS solution, observing the volume capable of making tear test paper fully suck dry PBS solution, and obtaining the result: the test paper can be completely soaked by PBS solution with the volume of 2 mu L and basically has no liquid residue;
s13, preparation of a reference substance:
preparing natural or recombinant ApoJ protein into a protein solution of 50 mu g/mL by using a PBS solution, infiltrating unused tear test paper by using the protein solution, then putting the test paper into an EP tube, drying in the shade, and simulating the tear test paper to be infiltrated by tears;
s14, preparation of a to-be-detected product:
put the tear test paper of the patient's tear secretion experiment into the EP tube and dry in the shade.
S2, preparing a protein extracting solution:
1g of Sarcosyl (SKL) was dissolved in 100mL of PBS solution to prepare a PBS solution containing 1% of SKL.
S3, protein extraction:
taking out the dried tear test paper in the step S1, wherein the tear test paper is the tear test paper of a reference product and the tear test paper of a product to be detected, observing the condition that tears are soaked and dyed, taking 3mm of the tear test paper in the middle part of the soaked interval as required, putting the tear test paper into an EP (EP) tube, adding 200 mu L of protein extracting solution, blowing and beating the tear test paper for a plurality of times by using a pipette or uniformly mixing the tear test paper with an oscillator, and standing the tear test paper at 4 ℃ overnight. Finally, centrifuging at 13000rpm for 3min and taking the supernatant to obtain the extracted protein solution.
S4, an ApoJ quantitative detection method:
detecting the content of a standard product ApoJ by using an ELISA method, and obtaining a regression linear equation; and (3) taking the protein solutions extracted from the reference substance and the to-be-detected substance, detecting by an ELISA method to obtain an absorbance value, and comparing the absorbance value with the regression linear equation to obtain the ApoJ concentrations of the reference substance and the to-be-detected substance.
The specific operation steps for obtaining the regression linear equation by the ELISA method are as follows:
s41, diluting the protein solution of the standard product to different concentrations by using a diluent; the diluent is a 1% bsa in PBST;
s42, adding 100 mu L of sample into each hole in the coated plate, slightly shaking and uniformly mixing, covering the plate, and reacting for 2 hours at 37 ℃;
s43, discarding the liquid, and spin-drying; adding 100 mu L of biotin antibody labeled antibody working solution into each hole, covering a new plate, and reacting for 1 hour at 37 ℃; the antibody working fluid is a PBST solution of 1% bsa containing ApoJ antibody;
s44, discarding liquid in the holes, spin-drying, washing the plate for 3 times, soaking for 2min each time, and spin-drying at a rate of 200 mu L per hole;
s45, adding 100 mu L of horse radish peroxidase labeled avidin working solution into each hole, covering a new plate, and reacting for 1 hour at 37 ℃;
s46, discarding liquid in the holes, spin-drying, washing the plate for 5 times, soaking for 2min each time, and spin-drying at a rate of 200 mu L per hole; the washing solution used for washing the plate is PBST solution.
S47, sequentially adding 90 mu L of TMB substrate color development solution into each hole, and developing at 37 ℃ in a dark place;
s48, sequentially adding 50 mu L of TMB stop solution into each hole, and stopping the reaction;
s49, measuring the OD value of each hole by using an enzyme-labeling instrument within 5min after the reaction is terminated and at the wavelength of 450 nm; and (4) drawing a standard curve by taking the concentration of the protein solution of the standard substance as an abscissa and the OD value as an ordinate.
The concentration of ApoJ protein solution is linear with OD at 450nm, and the standard curve is shown in FIG. 1. The regression linear equation is y =1.7182x with a correlation coefficient R 2 ＝0.9936。
Specific experimental data are shown in table 1:
TABLE 1
| Concentration ng/ |
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 |
And detecting the OD value of the reference substance or the to-be-detected substance, finding out the corresponding concentration by the regression linear equation, and multiplying the concentration by the dilution factor to obtain the actual concentration of the ApoJ in the reference substance or the to-be-detected substance.
Furthermore, the concentration of the control ApoJ calculated by the regression linear method obtained by the ELISA detection method is compared with the actual concentration of the control ApoJ, and the results of the comparison and the actual concentration of the control ApoJ are consistent, so that the detection method is accurate and reliable.
ApoJ expression is markedly reduced in severe dry eye, and the use of ApoJ as a drug can both counteract the drying pressure that causes dry eye and prevent downstream MMP9 from activating the dry eye pathological pathway. If the expression quantity of ApoJ in tears of xerophthalmia patients can be quickly detected, whether the xerophthalmia patients belong to the damaged ocular fundus barrier caused by low content of ApoJ in tears can be judged, and basis is provided for later diagnosis and treatment. If the amount of ApoJ expressed in the tear fluid of a patient with dry eye is low, apoJ may be administered to the patient as a drug. Therefore, by adopting the detection method, firstly, the ApoJ protein on the tear test paper is stripped and eluted by utilizing the sodium dodecyl sarcosinate, and the sodium dodecyl sarcosinate solution can help the ApoJ protein to maintain a certain monomer structure, inhibit the aggregation of the protein and facilitate the measurement of the expression quantity of the ApoJ; and then accurately detecting the expression amount of ApoJ on the tear test paper by an ELISA method. By comparing the ApoJ expression amount on the tear test paper for the dry eye patient tear secretion test with the ApoJ expression amount on the tear test paper for the normal human tear secretion test, when the ApoJ expression amount of the dry eye patient is lower than that of a normal human, the condition that the ocular fundus barrier of the dry eye patient is damaged is indicated, and corresponding treatment needs to be performed in time.
Example 2 Assembly of ELISA kits
The ELISA kit comprises the following specific components: (1) one blank enzyme label plate; (2) one part of ApoJ standard substance; (3) sarcosyl; (4) A biotin antibody-labeled antibody working solution (the antibody working solution is a 1% BSA (bovine serum albumin) PBST solution containing ApoJ antibody), and a horseradish peroxidase-labeled avidin working solution; (5) PBS buffer solution, washing solution (PBST solution), substrate developing solution (TMB system), and stop solution (TMB system).
EXAMPLE 3 detection of clinical samples
Randomly selecting 4 dry eye patients from a hospital to perform a tear secretion experiment, drying the obtained tear test paper in the shade, shearing 3mm of the tear test paper, and placing the tear test paper into an EP (EP) tube; preparing 1% protein extract from sarcosyl, adding 200 μ L protein extract into dried tear test paper, blowing with pipette several times or mixing with oscillator, and standing at 4 deg.C overnight. And finally centrifuging at 13000rpm for 3min, taking the supernatant to obtain an extracted protein solution, measuring by adopting an ELISA detection method, recording the OD value of 450nm wavelength, and calculating the content of ApoJ in the tear sample by using a standard curve, wherein the specific result is shown in Table 2.
TABLE 2
| Number of | mg/L for right eye | Left eye mg/ |
| 1 | 16.36 | 10.08 |
| 2 | 19.80 | 15.78 |
| 3 | 26.75 | 11.03 |
| 4 | 29.43 | 35.86 |
As can be seen from Table 2, the content of ApoJ in tears was also different in the case of dry eye patients. For example, patient No. 4 had ApoJ content in the left and right eyes of 35.86mg/L and 29.43mg/L, respectively, while patient No. 1 had ApoJ content in the left and right eyes of 10.08mg/L and 16.36mg/L, respectively. In patient No. 1, the content of ApoJ in tear is reduced, and the ocular fundus barrier is damaged.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A method for extracting and quantitatively detecting ApoJ on tear test paper is characterized by comprising the following steps:
s1, preparing a standard substance: preparing natural or recombinant ApoJ protein into protein solution,
preparation of control: putting the tear test paper into a protein solution, taking out and drying in the shade;
preparation of a sample to be tested: drying tear test paper of a tear secretion experiment in the shade;
s2, preparing a protein extracting solution: preparing the sodium dodecyl sarcosinate into protein extracting solution;
s3, protein extraction: adding protein extracting solutions into the tear test paper obtained in the step S1 respectively, uniformly mixing, standing overnight at 4 ℃, centrifuging to obtain supernate, and thus obtaining an extracted protein solution;
s4, an ApoJ quantitative detection method: detecting the standard substance by using an ELISA method, and obtaining a regression linear equation; and (3) taking protein solutions extracted from the to-be-detected product and the reference product, detecting by an ELISA method to obtain an absorbance value, and comparing the absorbance value with the regression linear equation to obtain the ApoJ concentrations of the to-be-detected product and the reference product.
2. The method for extracting and quantitatively detecting ApoJ on tear test paper according to claim 1, wherein the step S4 of obtaining the regression linear equation by the ELISA method comprises the specific steps of:
s41, diluting the protein solution of the standard product to different concentrations by using a diluent;
s42, adding 100 mu L of sample into each hole in the coated plate, slightly shaking and uniformly mixing, covering the plate, and reacting for 2 hours at 37 ℃;
s43, discarding the liquid, and spin-drying; adding 100 mu L of antibody working solution marked by biotin antibody into each hole, covering a new plate and sticking the mixture for reaction for 1 hour at 37 ℃;
s44, discarding liquid in the holes, spin-drying, washing the plate for 3 times, soaking for 2min each time, and spin-drying at a rate of 200 mu L per hole;
s45, adding 100 mu L of horseradish peroxidase labeled avidin working solution into each hole, covering a new plate, and reacting for 1 hour at 37 ℃;
s46, discarding liquid in the holes, spin-drying, washing the plate for 5 times, soaking for 2min each time, and spin-drying at a rate of 200 mu L per hole;
s47, sequentially adding 90 mu L of TMB substrate color development solution into each hole, and performing light-shading color development at 37 ℃;
s48, adding 50 mu L of TMB stop solution into each hole in sequence, and stopping reaction;
s49, measuring the OD value of each hole by using an enzyme-labeling instrument within 5min after the reaction is stopped at the wavelength of 450nm, and making a regression linear equation.
3. The method of claim 1, wherein the step S1 of preparing the control comprises preparing a 50 μ g/mL protein solution of natural or recombinant ApoJ protein.
4. The method of claim 1, wherein in step S2, sarcosyl is formulated as a 1% protein extract.
5. The method for ApoJ extraction and quantitative determination on tear strip of claim 1, wherein in step S3, the supernatant is obtained by centrifugation at 12000-15000rpm for 3 min.
6. The method of claim 1, wherein in step S4, the concentration of the protein solution of the standard is used as an abscissa and the OD value is used as an ordinate to draw a standard curve and calculate a regression linear equation.
7. The method for extracting and quantitatively detecting ApoJ on tear paper according to claim 1, wherein in step S4, the regression linear equation is y =1.7182x, wherein the correlation coefficient R is 2 ＝0.9936。
8. The method of claim 2, wherein in step S43, the antibody working solution is a 1% BSA in PBST solution containing ApoJ antibody.
9. A kit for ApoJ extraction and quantitative detection on tear strip, wherein the kit is for detection by an ApoJ extraction and quantitative detection method on tear strip according to any one of claims 1 to 8.
10. An application of a kit for ApoJ extraction and quantitative detection on tear test paper in xerophthalmia.
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| CN117268877B (en) * | 2023-11-21 | 2024-02-20 | 军科正源（北京）药物研究有限责任公司 | Method for detecting nerve growth factor in human tear and method for treating tear |
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