Multicolor fluorescent diagnostic reagent for detecting PAP and preparation method thereof

Abstract

This invention relates to the field of immunoassay technology, specifically to a multicolor fluorescent diagnostic reagent for detecting PAP. The multicolor fluorescent diagnostic reagent includes Hurst dye, wheat germ lectin, fluorescein isothiocyanate, a counterstaining agent, an organic solubilizer, and a humectant. This invention incorporates multiple fluorescent dyes, enabling rapid and effective detection of glycoprotein signals in PAP patient specimens. Simultaneously, it clearly displays cell morphology in the specimens, allowing for timely exclusion of bacteria, fungi, and abnormal cells, thus helping doctors quickly and accurately determine the type of PAP in patients. Compared to the traditional PAS staining method, the multicolor fluorescent diagnostic reagent provided by this invention uses a one-step staining method, which is simple to operate, has a short detection time, and allows for rapid observation of test results, facilitating timely diagnosis and treatment for patients.

Description

Technical Field

[0001] This invention relates to the field of medical diagnostic reagent technology, specifically to a multicolor fluorescent diagnostic reagent for detecting PAP and its preparation method. Background Technology

[0002] Pulmonary alveolar proteinosis (PAP), also known as Rosen-Castleman-Liebow syndrome, is a rare, diffuse lung disease characterized by the deposition of large amounts of phospholipoprotein-like substances in the alveoli. It commonly affects young and middle-aged adults, with men being approximately three times more likely to be affected than women. The etiology is unknown, but it may be related to immune dysfunction (such as thymic atrophy, immunodeficiency, and lymphopenia). Dust, especially silica dust, can induce PAP in animals, suggesting a nonspecific reaction to certain irritants that leads to the breakdown of alveolar macrophages and the production of PAS-positive proteins.

[0003] The annual incidence and prevalence of PAP per million people are 0.36–0.49 cases and 3.7–6.2 cases, respectively. Because PAP is rare, has an insidious onset, and its clinical manifestations are nonspecific and highly individualized, patients may be asymptomatic or have only a mild cough, while severe cases can lead to respiratory failure and even death. Currently, clinicians have insufficient understanding of the diagnosis and treatment of this disease, which easily leads to misdiagnosis.

[0004] The primary diagnostic method for PAP is currently PAS staining. However, PAS staining is cumbersome, time-consuming, and prone to false positives. Furthermore, it requires additional staining with alcine blue and hematoxylin and eosin (HE) to rule out interference from other pathogens such as fungi. In summary, the current clinical method of using PAS staining to detect glycogen in PAP patients is prone to misdiagnosis and missed diagnosis, is cumbersome, and time-consuming, hindering early and rapid screening of clinical specimens and failing to meet the need for simple, rapid, and efficient clinical testing. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problems of high misdiagnosis and missed diagnosis rate, cumbersome operation and long detection cycle of PAP in the prior art, so as to provide a multicolor fluorescent diagnostic reagent for detecting PAP and its preparation method.

[0006] In one aspect, the present invention provides a multicolor fluorescent diagnostic reagent for detecting PAP, the multicolor fluorescent diagnostic reagent comprising Hurst dye, wheat germ lectin, fluorescein isothiocyanate, counterstaining agent, organic co-solvent, and humectant.

[0007] Furthermore, each 100 mL of multicolor fluorescent diagnostic reagent contains:

[0008] Hearst dye 1-2 mg;

[0009] Wheat germ lectin 0.1-1 mg;

[0010] Fluorescein isothiocyanate 0.1-0.5 mg;

[0011] Counterstain agent 0.1-0.5 mg;

[0012] 2-10 mL of organic co-solvent;

[0013] 10-20 mL of humectant;

[0014] The remainder is water.

[0015] Preferably, each 100 mL of multicolor fluorescent diagnostic reagent contains:

[0016] Hearst dye 1.5 mg;

[0017] Wheat germ lectin 0.6 mg;

[0018] Fluorescein isothiocyanate 0.4 mg;

[0019] Counterstain 0.3 mg;

[0020] 5 mL of organic co-solvent;

[0021] 12mL of humectant;

[0022] The remainder is water.

[0023] Preferably, the Hoechst dye is one or more of Hoechst 33258 and Hoechst 33342.

[0024] Preferably, the wheat germ lectin is AF488-labeled wheat germ lectin.

[0025] Preferably, the dyeing agent is an azo dye.

[0026] Preferably, the counterstain is Evans blue.

[0027] Preferably, the organic co-solvent is glycerol.

[0028] Preferably, the humectant is dimethyl sulfoxide.

[0029] Another aspect of the present invention provides a method for preparing a multicolor fluorescent diagnostic reagent for detecting PAP, comprising the following steps: fully mixing an organic co-solvent, a humectant, and a portion of water to obtain a saturated organic solvent; weighing the required amount of Hearst dye and adding it to the saturated organic solvent, stirring and dissolving it thoroughly; then weighing the required amount of wheat germ lectin into the saturated organic solvent, stirring and dissolving it thoroughly to obtain an organic solvent containing fluorescent dye; fully mixing the organic solvent containing fluorescent dye with the remaining water; sequentially adding a counterstaining agent and fluorescein isothiocyanate, and mixing thoroughly to obtain the final product.

[0030] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0031] 1. This invention incorporates multiple fluorescent dyes, enabling rapid and effective detection of glycoprotein signals in PAP patient specimens. It also clearly displays cell morphology, allowing for the timely exclusion of bacteria, fungi, and abnormal cells, thus helping doctors quickly and accurately determine the type of PAP. Compared to traditional PAS staining, the multicolor fluorescent diagnostic reagent provided by this invention uses a one-step staining method, which is simple to operate, has a short detection time, and allows for rapid observation of test results without delaying the patient's condition and treatment.

[0032] 2. Compared with the traditional PAS staining method, the multicolor fluorescent diagnostic reagent provided by this invention has a better staining effect, increases color contrast, makes differences significant, and is easy to observe; it has a higher accuracy and positive rate for detecting PAP patient samples, effectively reducing the rate of missed diagnosis and misdiagnosis. Attached Figure Description

[0033] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 This is a microscopic image of a PAP patient specimen stained with conventional PAS staining in a comparative study.

[0035] Figure 2 This is a microscopic image of a PAP patient specimen stained with multicolor fluorescent diagnostic reagent according to Example 3 of the present invention. Detailed Implementation

[0036] Example 1

[0037] This embodiment provides a multicolor fluorescent diagnostic reagent for detecting PAP. Each 100mL of the reagent contains the following components: 2mg of Hearst dye, 1mg of AF488-labeled wheat germ agglutinin, 0.5mg of fluorescein isothiocyanate, 0.5mg of counterstaining agent Evans blue, 10mL of organic solubilizer glycerol, 20mL of humectant dimethyl sulfoxide, and 70mL of pure water.

[0038] The preparation method of this multicolor fluorescent diagnostic reagent is as follows: Measure 10 mL of organic co-solvent glycerol, 20 mL of humectant dimethyl sulfoxide, and 40 mL of pure water. Mix the organic co-solvent glycerol, humectant dimethyl sulfoxide, and pure water thoroughly to obtain a saturated organic solvent. Weigh 2 mg of Hearst dye into the saturated organic solvent and stir thoroughly to dissolve. Then weigh 1 mg of AF488-labeled wheat germ agglutinin into the saturated organic solvent and stir thoroughly to dissolve. Mix the saturated organic solvent containing Hearst dye and AF488-labeled wheat germ agglutinin thoroughly with the remaining water. Finally, add 0.5 mg of counterstaining agent Evans blue and 0.5 mg of fluorescein isothiocyanate in sequence and mix thoroughly to obtain the reagent.

[0039] Example 2

[0040] This embodiment provides a multicolor fluorescent diagnostic reagent for detecting PAP. Each 100mL of the reagent contains the following components: 1mg of Hearst dye, 0.1mg of AF488-labeled wheat germ agglutinin, 0.1mg of fluorescein isothiocyanate, 0.1mg of counterstaining agent Evans blue, 2mL of organic solubilizer glycerol, 10mL of humectant dimethyl sulfoxide, and 88mL of pure water.

[0041] The preparation method of this multicolor fluorescent diagnostic reagent is as follows: Measure 2 mL of organic co-solvent glycerol, 10 mL of humectant dimethyl sulfoxide, and 30 mL of pure water. Mix the organic co-solvent glycerol, humectant dimethyl sulfoxide, and pure water thoroughly to obtain a saturated organic solvent. Weigh 0.1 mg of Hearst dye into the saturated organic solvent and stir thoroughly to dissolve. Then weigh 0.1 mg of AF488-labeled wheat germ agglutinin into the saturated organic solvent and stir thoroughly to dissolve. Mix the saturated organic solvent containing Hearst dye and AF488-labeled wheat germ agglutinin thoroughly with the remaining water. Finally, add 0.1 mg of counterstaining agent Evans blue and 0.1 mg of fluorescein isothiocyanate in sequence and mix thoroughly to obtain the reagent.

[0042] Example 3

[0043] This embodiment provides a multicolor fluorescent diagnostic reagent for detecting PAP. Each 100mL of the reagent contains the following components: 1.5mg of Hearst dye, 0.6mg of AF488-labeled wheat germ agglutinin, 0.4mg of fluorescein isothiocyanate, 0.3mg of counterstaining agent Evans blue, 5mL of organic solubilizer glycerol, 12mL of humectant dimethyl sulfoxide, and 83mL of pure water.

[0044] The preparation method of this multicolor fluorescent diagnostic reagent is as follows: Measure 5 mL of organic co-solvent glycerol, 12 mL of humectant dimethyl sulfoxide, and 30 mL of pure water. Mix the organic co-solvent glycerol, humectant dimethyl sulfoxide, and pure water thoroughly to obtain a saturated organic solvent. Weigh 1.5 mg of Hearst dye into the saturated organic solvent and stir thoroughly to dissolve. Then weigh 0.6 mg of AF488-labeled wheat germ agglutinin into the saturated organic solvent and stir thoroughly to dissolve. Mix the saturated organic solvent containing Hearst dye and AF488-labeled wheat germ agglutinin thoroughly with the remaining water. Finally, add 0.3 mg of counterstaining agent Evans blue and 0.4 mg of fluorescein isothiocyanate in sequence and mix thoroughly to obtain the reagent.

[0045] Clinical trial analysis

[0046] I. Positive Rate and Accuracy Analysis

[0047] Case specimen selection: Bronchoalveolar lavage fluid specimens from 42 suspected PAP patients were selected as clinical trial case specimens for clinical trial research. Two sets of specimens were prepared for each case, for a total of 84 case specimens.

[0048] Case specimen processing: Bronchoalveolar lavage fluid specimens were stained using the traditional PAS staining method and the multicolor fluorescent diagnostic reagent provided in Example 3 of this invention. 42 case specimens were stained using the PAS staining method and the multicolor fluorescent diagnostic reagent, respectively, and were designated as the PAS staining group and the multicolor fluorescent diagnostic reagent staining group.

[0049] The specific staining process is as follows: Select 1 mL of the case specimen, centrifuge at 3000g for 30 min, discard most of the supernatant, leave a small amount of liquid to resuspend the precipitate, smear it on 2 glass slides, and stain it with PAS staining method and the multicolor fluorescent reagent provided in Example 3 of this invention (only 1 drop needs to be added, staining for 2 minutes is sufficient).

[0050] Microscopic examination of case specimens: Case specimens from the PAS staining group and the multicolor fluorescent diagnostic reagent staining group were examined under a microscope. The number of positive cases, positive rate and accuracy of the staining results of the two staining methods were compared to evaluate the clinical detection effect of the fluorescent staining method.

[0051] The data of case specimens from the traditional PAS staining group and the multicolor fluorescent staining reagent group were counted using SPSS 22.0 statistical software. The data are expressed as number of cases (n) and rate (%). The chi-square test was used to compare the groups. If P < 0.05, the difference was considered to be statistically significant.

[0052] The formula for calculating the positive rate is as follows:

[0053] Positive rate = Number of positive cases / Total number of cases;

[0054] The accuracy calculation formula is as follows:

[0055] Accuracy = (True positives + True negatives) / (Total positives + Total negatives).

[0056] The calculation results are shown in Table 1:

[0057] Table 1 Comparison of staining effects between PAS staining and multicolor fluorescent diagnostic reagents

[0058]

[0059] Based on clinical characteristics, imaging examinations, and pathological biopsies, a total of 37 out of 42 suspected PAP patients were diagnosed with PAP, and 5 were not PAP patients. Table 1 shows that the positive rate and accuracy of the staining group using the fluorescent staining diagnostic reagent provided in Example 3 of this invention were higher, and the difference between the two staining methods was statistically significant.

[0060] II. Analysis of Staining Effect

[0061] A bronchoalveolar lavage fluid specimen from a randomly selected PAP patient was used as the case specimen, forming two groups of case specimens. The bronchoalveolar lavage fluid specimens were stained using the traditional PAS staining method and the multicolor fluorescent diagnostic reagent prepared in Example 3 of this invention, respectively. The staining steps were the same as in the positive rate and accuracy analysis experiment. The microscopic examination results are as follows: Figure 1 and Figure 2 As shown; where, Figure 1 This is a microscopic image obtained using the traditional PAS staining method. Figure 2 This is a microscopic image of staining with the multicolor fluorescent diagnostic reagent prepared in Example 3 of this invention. (Comparison) Figure 1 and Figure 2 It can be seen that the multicolor fluorescent diagnostic reagent prepared in Example 3 of the present invention has a better staining effect. A large number of glycoprotein fragments can be seen in an irregular emerald green shape without oil immersion, which contrasts clearly with the light black background, increases the color contrast, makes the difference significant, and is easy to observe. At the same time, the detection efficiency is higher when observed with a fluorescence microscope.

[0062] In the formulation of this invention, Hearst dye stains the cell nuclei in the case specimen, producing blue fluorescence imaging under the UV band of a fluorescence microscope; AF488-labeled wheat germ lectin dye stains glycoproteins and cell membranes in the case specimen, producing green fluorescence imaging under the B band of a fluorescence microscope. The complete cell morphology can be scanned by switching bands, and after cell fusion, it appears as a blue cell nucleus and a green cell membrane, which can detect the presence of tumor cells; fluorescein isothiocyanate makes the green staining effect of glycoproteins brighter and more lasting; Evans blue is a counterstaining agent, which increases the color contrast after counterstaining the case specimen, facilitating clear imaging of bacteria; dimethyl sulfoxide is an organic co-solvent, which makes the powder dissolve better and maintains the stability of the solution; glycerol is a humectant, which can increase the overall humidification and refractive index of the multicolor fluorescent diagnostic reagent, improve the contrast, and thus make the staining effect of the multicolor fluorescent diagnostic reagent clearer. By switching the light source wavelength of a fluorescence microscope, a single case specimen can be tested for glycoproteins and cell morphology simultaneously, allowing for timely exclusion of abnormal cells, fungi, and bacteria, which helps in the rapid diagnosis of PAP disease.

[0063] In summary, the multicolor fluorescent diagnostic reagent provided by this invention allows for the clinical diagnosis of PAP patient specimens with only one drop of staining solution, requiring only two minutes of staining. Combined with a fluorescence microscope for microscopic examination, the detection time is short, the efficiency is higher, and the operation is simple, facilitating the rapid diagnosis of PAP patients in clinical practice.

[0064] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A multicolor fluorescent diagnostic reagent for detecting PAP, characterized in that, The multicolor fluorescent diagnostic reagent comprises Hurst dye, AF488-labeled wheat germ agglutinin, fluorescein isothiocyanate, Evans blue counterstain, glycerol, and dimethyl sulfoxide; each 100 mL of the multicolor fluorescent diagnostic reagent contains: Hearst dye 1-2 mg; AF488-labeled wheat germ lectin 0.1-1 mg; Fluorescein isothiocyanate 0.1-0.5 mg; Counterstain with Evans blue 0.1-0.5 mg; 2-10 mL of glycerol; 10-20 mL of dimethyl sulfoxide; The remainder is water.

2. The multicolor fluorescent diagnostic reagent for detecting PAP as described in claim 1, characterized in that, Each 100mL of multicolor fluorescent diagnostic reagent contains: Hearst dye 1.5 mg; 0.6 mg of AF488-labeled wheat germ lectin; Fluorescein isothiocyanate 0.4 mg; Evans blue, 0.3 mg, was used as a counterstain. 5 mL of glycerol; 12 mL of dimethyl sulfoxide; The remainder is water.

3. The multicolor fluorescent diagnostic reagent for detecting PAP as described in claim 1 or 2, characterized in that, The Hoechst dye is one or more of Hoechst 33258 and Hoechst 33342.

4. A method for preparing a multicolor fluorescent diagnostic reagent for detecting PAP as described in any one of claims 1-3, characterized in that, The process includes the following steps: Saturated organic solvent is obtained by thoroughly mixing glycerol, dimethyl sulfoxide, and a portion of water; the required amount of Hearst dye is weighed and added to the saturated organic solvent, and after thorough stirring to dissolve, the required amount of wheat germ lectin dye is weighed and added to the saturated organic solvent, and after thorough stirring to dissolve, an organic solvent containing fluorescent dye is obtained; the organic solvent containing fluorescent dye is thoroughly mixed with the remaining water, and Evans blue and fluorescein isothiocyanate are added sequentially and thoroughly mixed to obtain the final product.

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