A combined reagent kit for detecting prostate disease

Abstract

This invention relates to the field of in vitro diagnostics, and particularly to a combined diagnostic kit for prostate diseases. The invention provides reagents including pH, peroxidase, elastase, lecithin bodies, citric acid, zinc, and acid phosphatase under mild reaction conditions, enabling rapid differentiation of inflammatory and non-inflammatory prostatitis, benign prostatic hyperplasia, and non-prostatic genital tract infections using a single test card. Through extensive experimentation, this invention has developed a one-step reaction scheme suitable for the dry chemical determination of acid phosphatase and lecithin bodies in seminal plasma. This method eliminates the need for a stop solution, achieving a one-step reaction under mild conditions that will not cause damage to operators or fully automated instruments. The samples used are seminal plasma or semen, allowing for large sample volumes and easy acquisition, significantly improving diagnostic efficiency for doctors and patient comfort.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostics, and in particular to a combined diagnostic kit for prostate diseases. Background Technology

[0002] Chronic prostatitis (CP) is a group of diseases characterized by pain or discomfort around the pelvis and abnormal urination. It is often caused by certain pathogens or other non-infectious factors. In 1995, the National Institutes of Health (NIH) classified prostatitis into four types based on basic and clinical research at the time: Type I (acute bacterial prostatitis), Type II (chronic bacterial prostatitis), Type III (chronic pelvic pain syndrome), and Type IV (asymptomatic prostatitis).

[0003] Prostatitis is a common disease in adult men, often manifesting as urinary abnormalities such as frequent urination, urgency, painful urination, difficulty urinating, and a feeling of incomplete bladder emptying; discomfort or pain in the perineum, lower abdomen, penis, scrotum, lower back, and sacrum; and symptoms of premature ejaculation and erectile dysfunction. Common diagnostic methods for prostatitis include routine microscopic examination of prostatic massage fluid, transrectal ultrasound, and the four-glass method. Routine microscopic examination of prostatic massage fluid can determine the presence and severity of inflammation by reporting the number of white blood cells, lecithin bodies, epithelial cells, and granular cells in the prostatic fluid. Transrectal ultrasound can indicate prostatitis by detecting prostatic stones or calcifications, heterogeneous prostatic echogenicity, and dilation of the periprostatic venous plexus. The four-glass method can classify prostatitis by analyzing the number of white blood cells and bacterial culture results in initial urine, midstream urine, prostatic massage fluid, and urine after prostatic massage. Existing methods for microscopic examination of prostatic massage fluid are highly subjective, with inconsistent judgment criteria among different physicians, and prolonged observation under a microscope can easily cause visual fatigue. Since different types of prostatitis do not have specific imaging manifestations, it is impossible to use ultrasound to classify prostatitis. Although the four-cup method can classify prostatitis, it is cumbersome and time-consuming.

[0004] Benign prostatic hyperplasia (BPH), also known as benign prostatic hyperplasia, is the most common benign disease causing urinary disorders in middle-aged and elderly men. It is mainly characterized by histological hyperplasia of the prostatic stroma and glandular components, anatomical enlargement of the prostate (BPE), urodynamic bladder outlet obstruction (BOO), and clinical symptoms primarily consisting of lower urinary tract symptoms (LUTS).

[0005] Benign prostatic hyperplasia (BPH) is a common disease in elderly men, often manifesting as urinary frequency, progressive difficulty urinating, urinary retention, and hematuria. Common detection methods include ultrasound examination, residual urine volume measurement, and urodynamic testing. However, existing detection techniques for BPH are cumbersome and time-consuming, lacking a simple and reliable laboratory testing method.

[0006] Existing prostatitis diagnostic kits lack clear diagnostic rules and cannot provide clinicians with definitive diagnostic results; furthermore, the samples used in these kits are difficult to collect, resulting in small sample volumes. Meanwhile, existing seminal plasma diagnostic kits contain numerous indicators with unclear correlations to diseases, making it difficult to provide clinicians with more valuable diagnostic results; moreover, the acid phosphatase well stop solution in these kits is a strongly alkaline solution, which can easily cause damage to operators and automated machines; furthermore, the methodology for measuring lecithin bodies is not applicable to seminal plasma, resulting in poor specificity. Summary of the Invention

[0007] In view of this, the kit and application reaction conditions provided by the present invention are mild, the sample used is seminal plasma or semen, the sample volume is large and easy to obtain.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides reagents, including one or more of elastase detection reagents, peroxidase detection reagents, or lecithin body detection reagents;

[0010] The elastase detection reagent includes Meo-Suc-Ala-Ala-Pro-Val-PNA;

[0011] The peroxidase detection reagent includes TOOS, chromogen, and chromogenic solution;

[0012] The lecithin body detection reagent includes cholesterol oxidase, TOOS, peroxidase, and chromogen;

[0013] The elastase detection reagent does not include a colorimetric solution;

[0014] The lecithin body detection reagent does not include a colorimetric solution.

[0015] In some specific embodiments of the present invention, the above-mentioned reagents further include one or more of pH detection reagents, citric acid detection reagents, zinc detection reagents, and acid phosphatase detection reagents;

[0016] The pH testing reagent includes bromothymol blue sodium salt;

[0017] The citric acid detection reagent includes hydroxylamine hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and a stop solution;

[0018] The zinc detection reagent includes sodium 4-(2-pyridiniazo)-resorcinol;

[0019] The acid phosphatase detection reagent includes Guzis B, tartaric acid, 5-bromo-4-chloro-3-indole phosphate, N-acetyl-L-cysteine, imidazole, and sodium molybdate, and has a pH value of 5.0–6.0.

[0020] The high-concentration acid phosphatase detection reagent includes Guzi B, tartaric acid, 5-bromo-4-chloro-3-indole phosphate, N-acetyl-L-cysteine, imidazole, and sodium molybdate, and has a pH value of 7.0–8.0.

[0021] In some specific embodiments of the present invention, the above-mentioned reagents are:

[0022] The elastase detection reagent further includes a buffer solution; and / or

[0023] The peroxidase detection reagent further includes a buffer solution; and / or

[0024] The lecithin body detection reagent also includes a buffer solution and an enzyme protectant; and / or

[0025] The zinc detection reagent also includes a buffer solution; and / or

[0026] The 5-bromo-4-chloro-3-indole phosphate comprises disodium 5-bromo-4-chloro-3-indole phosphate; and / or

[0027] The buffer solution includes HEPES buffer, Tris-HCl buffer, or phosphate buffer; and / or

[0028] The enzyme protectant is PEG6000.

[0029] In some specific embodiments of the present invention, the above-mentioned reagents are:

[0030] The concentration of Meo-Suc-Ala-Ala-Pro-Val-PNA is 4–10 mmol / L; and / or

[0031] The concentration of TOOS in the peroxidase detection reagent is 0.002–0.01 g / L; and / or

[0032] The concentration of the chromogen in the peroxidase detection reagent is 0.009-0.02 g / L; and / or

[0033] The colorimetric solution in the peroxidase detection reagent includes urea peroxide; and / or

[0034] The concentration of the urea peroxide is 0.1–1.0 g / L; and / or

[0035] The concentration of the cholesterol oxidase is 10–30 kU / L; and / or

[0036] The concentration of the peroxidase in the lecithin body detection reagent is 1–10 kU / L; and / or

[0037] The concentration of TOOS in the lecithin body detection reagent is 1–4 g / L; and / or

[0038] The concentration of the chromogen in the lecithin body detection reagent is 0.1–1 g / L; and / or

[0039] The concentration of hydroxylamine hydrochloride is 0.05–0.08 mol / L; and / or

[0040] The concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 0.02–0.04 mol / L; and / or

[0041] The concentration of the stop solution in the citric acid detection reagent is 0.02–0.04 mol / L; and / or

[0042] The concentration of the 4-(2-pyridiniazo)-resorcinol sodium is 0.01–0.17 g / L; and / or

[0043] The concentration of the fixed purple B is 0.1–0.56 g / L; and / or

[0044] The concentration of tartaric acid is 0.01–1 mol / L; and / or

[0045] The concentration of the 5-bromo-4-chloro-3-indole phosphate is 0.1–0.9 g / L; and / or

[0046] The concentration of the N-acetyl-L-cysteine ​​is 10–40 mg / mL; and / or

[0047] The concentration of the imidazole is 0.01–0.1 mol / L; and / or

[0048] The concentration of the sodium molybdate is 0.01–0.1 mol / L; and / or

[0049] The chromogen includes 4-aminoantipyrine; and / or

[0050] The terminating solution includes ferric chloride.

[0051] In some specific embodiments of the present invention, the above-mentioned reagents are:

[0052] The concentration of the HEPES buffer solution is 0.02–2 mol / L; and / or

[0053] The concentration of the Tris-HCl buffer solution is 0.01–1 mol / L; and / or

[0054] The concentration of the phosphate buffer solution is 0.02–0.2 mol / L; and / or

[0055] The concentration of PEG6000 is 1–20 g / L.

[0056] The present invention also provides the application of the above-mentioned reagents in the preparation of a combined test kit for prostatitis using seminal plasma or semen as the test sample.

[0057] In some specific embodiments of the present invention, the prostatitis detection kit described above is used to identify one or more of inflammatory prostatitis, non-inflammatory prostatitis, non-prostatic reproductive tract infection, or benign prostatic hyperplasia.

[0058] The inflammatory prostatitis includes one or more of type I prostatitis, type II prostatitis, type IIIA prostatitis, or type IV prostatitis;

[0059] The non-inflammatory prostatitis includes type IIIB prostatitis;

[0060] The criteria for interpreting the test results of the prostatitis detection kit include (A), (B), (C), and / or (D):

[0061] (A) If (1) or (2) is satisfied, and (3) is also satisfied, then the sample to be tested is an inflammatory prostatitis sample;

[0062] (1): The result of the elastase assay was positive.

[0063] (2): The result of the peroxidase detection reagent was positive;

[0064] (3): The result of the lecithin body detection reagent was positive;

[0065] (B) If one or more of (6), (7) or (8) are satisfied, and (4) and (5) are also satisfied, then the sample to be tested is a non-inflammatory prostatitis sample;

[0066] (4): The result of the peroxidase detection reagent was negative;

[0067] (5): The result of the elastase assay was negative.

[0068] (6): The result of the acid phosphatase test was positive.

[0069] (7): The zinc test result was positive as described in the zinc test reagent.

[0070] (8): The result of the citric acid test reagent was positive;

[0071] (C) If (9) or (10) is satisfied, and (11) is also satisfied, then the sample to be tested is a non-prostate reproductive tract infection sample;

[0072] (9): The result of the peroxidase detection reagent was positive;

[0073] (10): The result of the elastase assay was positive.

[0074] (11): The result of the lecithin body detection reagent was negative;

[0075] (D) If (12) is satisfied, then the sample to be tested is a benign prostatic hyperplasia sample;

[0076] (12): The result was positive when tested with the high-concentration acid phosphatase detection reagent.

[0077] The present invention also provides a kit comprising the above-described reagents, as well as acceptable excipients or adjuvants.

[0078] The present invention also provides an apparatus comprising the above-described reagent, and acceptable components.

[0079] This invention also provides a method for classifying prostatitis, including:

[0080] (I) Take the sample to be tested and mix it with the peroxidase detection reagent, the elastase detection reagent, the lecithin body detection reagent, the citric acid detection reagent, the zinc detection reagent, the acid phosphatase detection reagent, and the high-concentration acid phosphatase detection reagent mentioned above, respectively, and obtain the typing result by judgment; or

[0081] (II) Take the sample to be tested and mix it with the peroxidase detection reagent, the elastase detection reagent, the lecithin body detection reagent, the citric acid detection reagent, the zinc detection reagent, the acid phosphatase detection reagent, and the high-concentration acid phosphatase detection reagent in the above kit, respectively, and obtain the typing result by judgment; or

[0082] (III) Take the sample to be tested and mix it with the peroxidase detection reagent, the elastase detection reagent, the lecithin body detection reagent, the citric acid detection reagent, the zinc detection reagent, the acid phosphatase detection reagent and the high-concentration acid phosphatase detection reagent in the above device respectively, and obtain the typing result by judgment;

[0083] The sample to be tested includes semen or seminal plasma;

[0084] The judgment includes (A), (B), (C) and / or (D):

[0085] (A) If (1) or (2) is satisfied, and (3) is also satisfied, then the sample to be tested is an inflammatory prostatitis sample;

[0086] (1): The result of the elastase assay was positive.

[0087] (2): The result of the peroxidase detection reagent was positive;

[0088] (3): The result of the lecithin body detection reagent was positive;

[0089] (B) If one or more of (6), (7) or (8) are satisfied, and (4) and (5) are also satisfied, then the sample to be tested is a non-inflammatory prostatitis sample;

[0090] (4): The result of the peroxidase detection reagent was negative;

[0091] (5): The result of the elastase assay was negative.

[0092] (6): The result of the acid phosphatase test was positive.

[0093] (7): The zinc test result was positive as described in the zinc test reagent.

[0094] (8): The result of the citric acid test reagent was positive;

[0095] (C) If (9) or (10) is satisfied, and (11) is also satisfied, then the sample to be tested is a non-prostate reproductive tract infection sample;

[0096] (9): The result of the peroxidase detection reagent was positive;

[0097] (10): The result of the elastase assay was positive.

[0098] (11): The result of the lecithin body detection reagent was negative;

[0099] (D) If (12) is satisfied, then the sample to be tested is a benign prostatic hyperplasia sample;

[0100] (12): The result was positive when tested with the high-concentration acid phosphatase detection reagent.

[0101] The reagent kit of the present invention has the following effects:

[0102] Currently, the diagnosis of prostatitis mainly relies on a combination of medical history taking, routine prostate fluid analysis, and transrectal ultrasound. Compared to these methods, the results of this invention are objective, repeatable, easy to interpret, and require no prior experience. Compared to existing kits or patented inventions, this invention can rapidly differentiate between inflammatory and non-inflammatory prostatitis and benign prostatic hyperplasia by measuring pH, elastase, lecithin bodies, citric acid, zinc, and acid phosphatase in seminal plasma and / or semen; it has a dry chemical assay method more suitable for lecithin bodies in seminal plasma and / or semen; and it has a simpler and safer dry chemical assay method for acid phosphatase in seminal plasma and / or semen. Specifically:

[0103] (1) This invention, through a large number of clinical trials, first proposed a diagnostic rule for differentiating and detecting different types of prostatitis and diagnosing benign prostatic hyperplasia by measuring the pH value of seminal plasma and / or semen, elastase, peroxidase, lecithin bodies, citric acid, zinc, acid phosphatase and high concentration of acid phosphatase.

[0104] (2) Through extensive experimental exploration, this invention has for the first time achieved the dry chemical method for determining lecithin bodies in seminal plasma. Specifically, cholesterol oxidase, 4-aminoantipyrine and TOOS are first added to a paper disc. Cholesterol oxidase decomposes free cholesterol to produce hydrogen peroxide. Peroxidase reacts with hydrogen peroxide to produce reactive oxygen species that react with the color source substance to produce color. The free cholesterol content is directly proportional to the color development, which is of pioneering significance for the detection of lecithin bodies in seminal plasma.

[0105] (3) Through extensive experimental exploration, this invention has for the first time achieved a one-step reaction suitable for the dry chemical method of acid phosphatase determination. Specifically, the acid phosphatase substrate is disodium 5-bromo-4-chloro-3-indole phosphate and its derivatives, and the color reagent is Solid Violet B. Acid phosphatase decomposes disodium 5-bromo-4-chloro-3-indole phosphate to produce 5-bromo-4-chloro-3-indole. 5-bromo-4-chloro-3-indole can react with Solid Violet B under near-neutral conditions to produce a colorimetric reaction. Furthermore, inhibitors such as sodium tartrate, imidazole, and sodium molybdate are added to the formula to ensure that the color of the reaction does not change within the specified time. This scheme has mild reaction conditions and will not cause damage to operators or fully automated instruments.

[0106] (4) The samples used in this experiment are seminal plasma or semen. The sample size is large and easy to obtain, which can greatly improve the doctor's diagnostic efficiency and the patient's comfort. Detailed Implementation

[0107] This invention discloses a combined diagnostic kit for prostate diseases. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0108] In clinical practice, oral antibiotics are used to treat both inflammatory prostatitis (types I, II, IIIA, and IV) and non-inflammatory prostatitis (type IIIB). However, since patients with type IIIB prostatitis do not require antibiotic treatment, indiscriminate use of antibiotics can lead to antibiotic overuse. A simple and quick method to differentiate between inflammatory and non-inflammatory prostatitis would reduce antibiotic overuse. The purpose of this invention is to enable rapid differentiation between inflammatory, non-inflammatory prostatitis, and benign prostatic hyperplasia using a single test card, assisting clinicians in prescribing medication for patients.

[0109] Extensive experiments have led to the development of a one-step reaction scheme suitable for the dry chemical determination of acid phosphatase in seminal plasma. This method eliminates the need for a stop solution, achieves a one-step reaction, and operates under mild conditions, causing no harm to operators or the fully automated instrument.

[0110] Through extensive experiments, a suitable method for the determination of lecithin bodies in seminal plasma was developed, increasing the specificity from 10.71% to 96.43%.

[0111] This invention employs eight biochemical indicators to detect and differentiate between types of prostatitis and benign prostatic hyperplasia (BPH): pH value, elastase, peroxidase, lecithin bodies, citric acid, zinc, acid phosphatase, and high-concentration acid phosphatase. The test uses a card format with eight slots (holes) containing reagent pads. Different reagent pads are coated with different reagents corresponding to pH value, elastase, peroxidase, lecithin bodies, citric acid, zinc, acid phosphatase, and high-concentration acid phosphatase, respectively.

[0112] This invention uses eight biochemical indicators to detect prostatitis and benign prostatic hyperplasia (BPH): pH value, elastase, peroxidase, lecithin bodies, citric acid, zinc, acid phosphatase, and high-concentration acid phosphatase. The test uses a card with eight slots (holes) containing test strips. The test reagents used are made of materials such as cotton pulp paper, filter paper, glass fiber, or plastic.

[0113] This invention provides a kit for diagnosing inflammatory and non-inflammatory prostatitis and benign prostatic hyperplasia using semen or seminal plasma. Its feature is that it can quickly differentiate and diagnose inflammatory and non-inflammatory prostatitis and benign prostatic hyperplasia through the combined interpretation of multiple indicators.

[0114] This invention also provides a kit for diagnosing inflammatory and non-inflammatory prostatitis and benign prostatic hyperplasia using semen or seminal plasma. The kit is characterized by using seminal plasma and semen as samples, which have the advantages of large sample volume and easy access compared to prostatic fluid, which is commonly used in clinical practice. This can greatly improve the diagnostic efficiency of doctors and the comfort of patients.

[0115] This invention also provides a dry chemical method suitable for the detection of lecithin bodies in seminal plasma and / or semen, specifically by adding components such as cholesterol oxidase, peroxidase, and 4-aminoantipyrine to a paper substrate. Cholesterol oxidase decomposes free cholesterol to produce hydrogen peroxide, and peroxidase decomposes hydrogen peroxide to produce reactive oxygen species that react with the chromogenic substance to produce color.

[0116] This invention also provides a one-step reaction scheme suitable for the dry chemical determination of acid phosphatase. Specifically, the acid phosphatase substrate is disodium 5-bromo-4-chloro-3-indole phosphate and its derivatives, and the chromogenic agent is Solid Violet B. Acid phosphatase decomposes disodium 5-bromo-4-chloro-3-indole phosphate to produce 5-bromo-4-chloro-3-indole. 5-bromo-4-chloro-3-indole can react with Solid Violet B under near-neutral conditions to produce a colorimetric reaction. Furthermore, the formulation includes inhibitors such as sodium tartrate, imidazole, and sodium molybdate to ensure that the color does not change within a specified time. This scheme allows the substrate decomposition reaction and the colorimetric reaction to occur under mild conditions, achieving the goal of a one-step reaction and mild reaction conditions.

[0117] Specifically:

[0118] (1) Different reagent pads are coated with different reagents, corresponding to pH value, elastase, peroxidase, lecithin bodies, citric acid, zinc, acid phosphatase and high concentration of acid phosphatase, respectively. The specific formulas are as follows:

[0119] pH value: The solid reagent for detecting pH value is prepared by dissolving 10-50 g / L of sodium bromothymol blue and purified water. 10 μL of the solution is coated onto a carrier with a diameter of 6 mm and then dried under vacuum.

[0120] Elastase: The solid reagent for detecting elastase consists of HEPES buffer and Meo-Suc-Ala-Ala-Pro-Val-PNA. The concentration of the HEPES buffer is 0.02–2 mol / L, and the pH is 7.0–9.0. The concentration of the Meo-Suc-Ala-Ala-Pro-Val-PNA is 4 mmol / L–10 mmol / L. A 10 μL solution is prepared, coated onto a 6 mm diameter support, and then freeze-dried under vacuum.

[0121] Peroxidase: The reagent for detecting peroxidase consists of Tris-HCl buffer, TOOS, and chromogen. The concentration of Tris-HCl buffer is 0.05-1 mol / L, pH 7.2, the concentration of TOOS is 0.002-0.01 g / L, and the concentration of 4-aminoantipyrine is 0.009-0.02 g / L. A 10 μL solution is prepared and coated onto a 6 mm diameter carrier, then freeze-dried under vacuum. The chromogenic solution is 0.1 g / L-1.0 g / L urea peroxide solution.

[0122] Lecithin bodies: The solid reagent for detecting lecithin bodies consists of Tris-HCl buffer, cholesterol oxidase, TOOS, peroxidase, chromogen, and PEG6000. The concentration of Tris-HCl buffer is 0.05–1 mol / L, pH is 7.0–8.0, the concentration of cholesterol oxidase is 10–30 kU / L, the concentration of TOOS is 1%–4%, the concentration of 4-aminoantipyrine is 0.1%–0.4%, the concentration of peroxidase is 1–10 kU / L, and the concentration of PEG6000 is 0.1%–2%. A 10 μL solution is prepared, coated onto a 6 mm diameter carrier, and then freeze-dried under vacuum.

[0123] Citric acid: The solid reagent for detecting citric acid consists of hydroxylamine hydrochloride, (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), and ferric chloride. The concentration of hydroxylamine hydrochloride is 0.05–0.08 mol / L, and the concentration of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) is 0.02–0.04 mol / L. A solution is prepared with water, and 10 μL is coated onto a carrier with a diameter of 6 mm and dried under vacuum. The stop solution is a 0.02–0.04 mol / L ferric chloride solution.

[0124] Zinc: The solid reagent for zinc detection consists of phosphate buffer and sodium 4-(2-pyridiniazo)-resorcinol substrate. The concentration of phosphate buffer is 0.02-0.2 mol / L, pH is 5.0-7.0, and the concentration of sodium 4-(2-pyridiniazo)-resorcinol is 0.01-0.17 g / L. 10 μL of the prepared solution is coated onto a 6 mm diameter support and dried under vacuum.

[0125] Acid phosphatase: The solid reagent for detecting acid phosphatase consists of Solid Violet B, tartaric acid, disodium 5-bromo-4-chloro-3-indole phosphate and its derivatives, N-acetyl-L-cysteine, imidazole, and sodium molybdate. The concentrations of Solid Violet B are 0.1–0.56 g / L, tartaric acid is 0.01–1 mol / L, 5-bromo-4-chloro-3-indole phosphate is 0.1–0.9 g / L, N-acetyl-L-cysteine ​​is 10–40 mg / mL, imidazole is 0.01–0.1 mol / L, and sodium molybdate is 0.01–0.1 mol / L. The pH of the solution is adjusted to 5.0–6.0 with water and sodium hydroxide. 10 μL of the prepared solution is coated onto a 6 mm diameter support and dried under vacuum.

[0126] High-concentration acid phosphatase: The solid reagent for detecting high-concentration acid phosphatase consists of Solid Violet B, tartaric acid, disodium 5-bromo-4-chloro-3-indole phosphate and its derivatives, N-acetyl-L-cysteine, imidazole, and sodium molybdate. The concentrations are as follows: Solid Violet B: 0.1–0.56 g / L; tartaric acid: 0.01–1 mol / L; 5-bromo-4-chloro-3-indole phosphate: 0.1–0.9 g / L; N-acetyl-L-cysteine: 10–40 mg / mL; imidazole: 0.01–0.1 mol / L; sodium molybdate: 0.01–0.1 mol / L. The pH of the solution is adjusted to 7.0–8.0 with sodium hydroxide. A 10 μL solution is prepared and coated onto a 6 mm diameter support, then vacuum dried.

[0127] (2) The method of using the kit of the present invention is as follows: Dilute the sample to be tested (semen or seminal plasma) with physiological saline by 10 times, and add 30 μL of each to the pH value, elastase, lecithin body, citric acid, zinc and acid phosphatase test strips respectively. Add color development solution or stop solution as needed, and read the results according to the displayed color, as shown in Table 1.

[0128] Table 1

[0129]

[0130]

[0131] (3) Kit Application Rules

[0132] Type I, Type II, Type IIIA, and Type IV prostatitis (inflammatory prostatitis) are diagnosed based on the following criteria: either peroxidase or elastase is positive, and lecithin bodies are also positive.

[0133] Type IIIB prostatitis (non-inflammatory prostatitis) is diagnosed as follows: peroxidase and elastase are negative, while at least one of the following three indicators—acid phosphatase, zinc, and citric acid—is positive.

[0134] Non-prostatic genital tract infection, positive for peroxidase or elastase, negative for lecithin bodies.

[0135] Benign prostatic hyperplasia: High concentrations of acid phosphatase appear red in the pores.

[0136] (4) The principle of this invention is as follows: pH value is an acid-base indicator, and the pH value changes according to the color change; elastase hydrolyzes the specific substrate and turns yellow; lecithin bodies decompose to produce hydrogen peroxide through enzymatic reaction, and peroxidase turns red through the Trider reaction; zinc turns red by binding with the specific substrate; citric acid turns red by converting to hydroxamic acid and combining to form ferric hydroxamic acid; acid phosphatase turns reddish-brown by decomposing the specific substrate and undergoing a diazotization reaction.

[0137] The term "non-prostatic reproductive tract infection" as used in this article refers to infections of other reproductive tract sites besides the prostate, such as urethral infection, seminal vesicle infection, and epididymal infection.

[0138] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0139] The present invention will be further illustrated below with reference to the embodiments:

[0140] Example 1

[0141] Through extensive experiments, a suitable method for determining lecithin bodies in seminal plasma was developed. The specific experimental methods and steps are as follows:

[0142] The reagent kit of the present invention:

[0143] Lecithin body solid reagent: The solid reagent for detecting lecithin bodies consists of Tris-HCl buffer, cholesterol oxidase, peroxidase, TOOS, 4-aminoantipyrine, and PEG6000. The concentration of Tris-HCl buffer is 0.2 mol / L, pH is 7.2, the concentration of cholesterol oxidase is 10 kU / L, the concentration of TOOS is 1%, the concentration of 4-aminoantipyrine is 0.1%, the concentration of peroxidase is 1 kU / L, and the concentration of PEG6000 is 0.1%. The solution is prepared, coated onto a carrier, and then vacuum dried.

[0144] The available reagent kit contains: 0.1 g / L potassium chloride, 0.1 g / L boric acid, 0.2 g / L 5-(2-carboxyphenyl)-1-(2-hydroxy-5-sulfophenyl)-3-phenylmethyl monosodium salt; 0.2 g / L zinc chloride, adjusted to pH 7.0 with 6M sodium hydroxide and then brought to a final volume of 1 L. Take 10 μL and coat it onto a quantitative filter paper disc.

[0145] Reference reagent kit: Shanghai Hengyuan Technology Co., Ltd.; HB1002-Hu

[0146] The results are shown in Tables 2-4. The specificity increased from 10.71% in the original method to 96.43%.

[0147] Table 2

[0148]

[0149]

[0150] Table 3

[0151]

[0152] Table 4

[0153]

[0154] Example 2

[0155] pH value: The solid reagent for detecting pH value is prepared by coating a carrier with a solution of 10 g / L sodium bromothymol blue and purified water, and then vacuum drying.

[0156] Elastase: The solid reagent for detecting elastase consists of HEPES buffer and Meo-Suc-Ala-Ala-Pro-Val-PNA. The HEPES buffer has a concentration of 0.02 mol / L and a pH of 7.0, and the Meo-Suc-Ala-Ala-Pro-Val-PNA has a concentration of 4 mmol / L. The solution is coated onto a carrier and then freeze-dried under vacuum.

[0157] Peroxidase: The reagent for detecting peroxidase consists of Tris-HCl buffer, TOOS, and chromogen. The concentration of Tris-HCl buffer is 0.05 mol / L, pH 7.2, the concentration of TOOS is 0.002 g / L, and the concentration of 4-aminoantipyrine is 0.009 g / L. The concentration of urea peroxide chromogenic solution is 0.1 g / L.

[0158] Lecithin bodies: The solid reagent for detecting lecithin bodies consists of Tris-HCl buffer, cholesterol oxidase, TOOS, peroxidase, chromogen, and PEG6000. The concentration of Tris-HCl buffer is 0.05 mol / L, pH is 7.0, the concentration of cholesterol oxidase is 10 kU / L, the concentration of TOOS is 10 g / L, the concentration of 4-aminoantipyrine is 1 g / L, the concentration of peroxidase is 1 kU / L, and the concentration of PEG6000 is 1 g / L. The solution is prepared, coated onto a carrier, and then freeze-dried under vacuum.

[0159] Citric acid: The solid reagent for detecting citric acid consists of hydroxylamine hydrochloride, (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), and ferric chloride. The concentration of hydroxylamine hydrochloride is 0.05 mol / L, and the concentration of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) is 0.02 mol / L. The solution is prepared, coated onto a carrier, and dried under vacuum. The stop solution is a 0.02 mol / L ferric chloride solution.

[0160] Zinc: The solid reagent for zinc detection consists of phosphate buffer and sodium 4-(2-pyridiniazo)-resorcinol substrate. The concentration of phosphate buffer is 0.02 mol / L and the pH is 5.0. The concentration of sodium 4-(2-pyridiniazo)-resorcinol is 0.01 g / L. The solution is prepared, coated onto a carrier, and dried under vacuum.

[0161] Acid phosphatase: The solid reagent for detecting acid phosphatase consists of Solid Violet B, tartaric acid, disodium 5-bromo-4-chloro-3-indole phosphate and its derivatives, N-acetyl-L-cysteine, imidazole, and sodium molybdate. The concentrations are: Solid Violet B 0.1 g / L, tartaric acid 0.01 mol / L, 5-bromo-4-chloro-3-indole phosphate 0.1 g / L, N-acetyl-L-cysteine ​​10 mg / mL, imidazole 0.01 mol / L, and sodium molybdate 0.01 mol / L. The pH of the solution is adjusted to 5.0 with sodium hydroxide. The prepared solution is coated onto a carrier and then vacuum dried.

[0162] High-concentration acid phosphatase: The solid reagent for detecting high-concentration acid phosphatase consists of Solid Violet B, tartaric acid, disodium 5-bromo-4-chloro-3-indole phosphate and its derivatives, N-acetyl-L-cysteine, imidazole, and sodium molybdate. The concentrations are: Solid Violet B 0.1 g / L, tartaric acid 0.01 mol / L, 5-bromo-4-chloro-3-indole phosphate 0.1 g / L, N-acetyl-L-cysteine ​​10 mg / mL, imidazole 0.01 mol / L, and sodium molybdate 0.01 mol / L. The pH of the solution is adjusted to 7.0 with sodium hydroxide. The prepared solution is coated onto a carrier and then vacuum dried.

[0163] Example 3

[0164] pH value: The solid reagent for detecting pH value is prepared by coating a carrier with a solution of 20.0 g / L sodium bromothymol blue and purified water, and then vacuum drying.

[0165] Elastase: The solid reagent for detecting elastase consists of HEPES buffer and Meo-Suc-Ala-Ala-Pro-Val-PNA. The HEPES buffer has a concentration of 1.0 mol / L and a pH of 8.0, and the Meo-Suc-Ala-Ala-Pro-Val-PNA has a concentration of 6 mmol / L. The solution is coated onto a carrier and then vacuum dried.

[0166] Peroxidase: The reagent for detecting peroxidase consists of Tris-HCl buffer, TOOS, and chromogen. The concentration of Tris-HCl buffer is 0.2 mol / L, pH 7.2, the concentration of TOOS is 0.008 g / L, and the concentration of 4-aminoantipyrine is 0.01 g / L. The concentration of urea peroxide chromogenic solution is 0.5 g / L.

[0167] Lecithin bodies: The solid reagent for detecting lecithin bodies consists of Tris-HCl buffer, cholesterol oxidase, TOOS, peroxidase, chromogen, and PEG6000. The concentration of Tris-HCl buffer is 0.2 mol / L, pH is 7.5, the concentration of cholesterol oxidase is 20 kU / L, the concentration of TOOS is 20 g / L, the concentration of 4-aminoantipyrine is 2 g / L, the concentration of peroxidase is 5 kU / L, and the concentration of PEG6000 is 10 g / L. The solution is prepared, coated onto a carrier, and then vacuum dried.

[0168] Citric acid: The solid reagent for detecting citric acid consists of hydroxylamine hydrochloride, (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), and ferric chloride. The concentration of hydroxylamine hydrochloride is 0.07 mol / L, and the concentration of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) is 0.03 mol / L. The solution is prepared, coated onto a carrier, and dried under vacuum. The stop solution is a 0.03 mol / L ferric chloride solution.

[0169] Zinc: The solid reagent for zinc detection consists of phosphate buffer and sodium 4-(2-pyridiniazo)-resorcinol substrate. The concentration of phosphate buffer is 0.1 mol / L and the pH is 6.0. The concentration of sodium 4-(2-pyridiniazo)-resorcinol is 0.05 g / L. The solution is prepared, coated onto a carrier, and dried under vacuum.

[0170] Acid phosphatase: The solid reagent for detecting acid phosphatase consists of Solid Violet B, tartaric acid, disodium 5-bromo-4-chloro-3-indole phosphate and its derivatives, N-acetyl-L-cysteine, imidazole, and sodium molybdate. The concentrations are: Solid Violet B 0.3 g / L, tartaric acid 0.1 mol / L, 5-bromo-4-chloro-3-indole phosphate 0.4 g / L, N-acetyl-L-cysteine ​​25 mg / mL, imidazole 0.05 mol / L, and sodium molybdate 0.05 mol / L. The pH of the solution is adjusted to 5.5 with sodium hydroxide. The prepared solution is coated onto a carrier and then vacuum dried.

[0171] High-concentration acid phosphatase: The solid reagent for detecting high-concentration acid phosphatase consists of Solid Violet B, tartaric acid, disodium 5-bromo-4-chloro-3-indole phosphate and its derivatives, N-acetyl-L-cysteine, imidazole, and sodium molybdate. The concentrations are: Solid Violet B 0.3 g / L, tartaric acid 0.1 mol / L, 5-bromo-4-chloro-3-indole phosphate 0.4 g / L, N-acetyl-L-cysteine ​​25 mg / mL, imidazole 0.05 mol / L, and sodium molybdate 0.05 mol / L. The pH of the solution is adjusted to 7.5 with sodium hydroxide. The prepared solution is coated onto a carrier and then vacuum dried.

[0172] Example 4

[0173] pH value: The solid reagent for detecting pH value is prepared by coating a carrier with a solution of 50 g / L sodium bromothymol blue and purified water, and then vacuum drying.

[0174] Elastase: The solid reagent for detecting elastase consists of HEPES buffer and Meo-Suc-Ala-Ala-Pro-Val-PNA. The HEPES buffer has a concentration of 2.0 mol / L and a pH of 9.0, and the Meo-Suc-Ala-Ala-Pro-Val-PNA has a concentration of 10 mmol / L. The solution is coated onto a carrier and then freeze-dried under vacuum.

[0175] Peroxidase: The reagent for detecting peroxidase consists of Tris-HCl buffer, TOOS, and chromogen. The concentration of Tris-HCl buffer is 1 mol / L, pH 7.2, the concentration of TOOS is 0.01 g / L, and the concentration of 4-aminoantipyrine is 0.02 g / L. The concentration of urea peroxide chromogenic solution is 1.0 g / L.

[0176] Lecithin bodies: The solid reagent for detecting lecithin bodies consists of Tris-HCl buffer, cholesterol oxidase, TOOS, peroxidase, chromogen, and PEG6000. The concentration of Tris-HCl buffer is 1 mol / L, pH is 8.0, the concentration of cholesterol oxidase is 30 kU / L, the concentration of TOOS is 40 g / L, the concentration of 4-aminoantipyrine is 4 g / L, the concentration of peroxidase is 10 kU / L, and the concentration of PEG6000 is 20 g / L. The solution is prepared, coated onto a carrier, and then freeze-dried under vacuum.

[0177] Citric acid: The solid reagent for detecting citric acid consists of hydroxylamine hydrochloride, (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), and ferric chloride. The concentration of hydroxylamine hydrochloride is 0.08 mol / L, and the concentration of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) is 0.04 mol / L. The solution is prepared, coated onto a carrier, and dried under vacuum. The stop solution is a 0.04 mol / L ferric chloride solution.

[0178] Zinc: The solid reagent for zinc detection consists of phosphate buffer and the substrate sodium 4-(2-pyridiniazo)-resorcinol. The concentration of the phosphate buffer is 0.2 mol / L and the pH is 7.0. The concentration of sodium 4-(2-pyridiniazo)-resorcinol is 0.17 g / L. The solution is prepared, coated onto the carrier, and dried under vacuum.

[0179] Acid phosphatase: The solid reagent for detecting acid phosphatase consists of Solid Violet B, tartaric acid, disodium 5-bromo-4-chloro-3-indole phosphate and its derivatives, N-acetyl-L-cysteine, imidazole, and sodium molybdate. The concentrations are: Solid Violet B 0.56 g / L, tartaric acid 1 mol / L, 5-bromo-4-chloro-3-indole phosphate 0.9 g / L, N-acetyl-L-cysteine ​​40 mg / mL, imidazole 0.1 mol / L, and sodium molybdate 0.1 mol / L. The pH of the solution is adjusted to 6.0 with sodium hydroxide. The prepared solution is coated onto a carrier and then vacuum dried.

[0180] High-concentration acid phosphatase: The solid reagent for detecting high-concentration acid phosphatase consists of Solid Violet B, tartaric acid, disodium 5-bromo-4-chloro-3-indole phosphate and its derivatives, N-acetyl-L-cysteine, imidazole, and sodium molybdate. The concentrations are: Solid Violet B 0.56 g / L, tartaric acid 1 mol / L, 5-bromo-4-chloro-3-indole phosphate 0.9 g / L, N-acetyl-L-cysteine ​​40 mg / mL, imidazole 0.1 mol / L, and sodium molybdate 0.1 mol / L. The pH of the solution is adjusted to 8.0 with sodium hydroxide. The prepared solution is coated onto a carrier and then vacuum dried.

[0181] Example 5: Reagent Kit Application Rules

[0182] Samples: A total of 607 semen samples were collected, including 21 samples from patients with type I prostatitis, 76 samples from patients with type II prostatitis, 124 samples from patients with type IIIA prostatitis, 188 samples from patients with type IIIB prostatitis, 12 samples from patients with type IV prostatitis, 22 samples from patients with BPH, 16 samples from patients with other reproductive tract infections, and 148 samples from healthy volunteers. The samples were randomly shuffled. Samples 1-300 were used to summarize the diagnostic rules, while samples 301-607 were used to verify the diagnostic rules.

[0183] Experimental methods: The assay was performed using the kit described in Example 2. Specific raw data are shown in Table 5, and the relevant performance data of the kit are shown in Table 6. The following diagnostic rules were derived from the data analysis:

[0184] Type I, Type II, Type IIIA, and Type IV prostatitis (inflammatory prostatitis) are diagnosed based on the following criteria: either peroxidase or elastase is positive, and lecithin bodies are also positive.

[0185] Type IIIB prostatitis (non-inflammatory prostatitis) is diagnosed as follows: peroxidase and elastase are negative, while at least one of the following three indicators—acid phosphatase, zinc, and citric acid—is positive.

[0186] For non-prostatic genital tract infections, the diagnostic criteria are positive for peroxidase or elastase and negative for lecithin bodies.

[0187] Benign prostatic hyperplasia: High concentration acid phosphatase pores positive.

[0188] Table 5

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204] Table 6

[0205] Types of diseases Sensitivity Specificity Inflammatory prostatitis 92.13％ / Non-inflammatory prostatitis 88.61％ / BHP 85.71％ / Non-CP reproductive tract infection 91.67％ / Normal control / 96.34％

[0206] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A combined diagnostic reagent for prostatitis, characterized in that, This includes elastase detection reagents, peroxidase detection reagents, and lecithin body detection reagents; The elastase detection reagent includes Meo-Suc-Ala-Ala-Pro-Val-PNA; The peroxidase detection reagent includes TOOS, chromogen, and chromogenic solution; The lecithin body detection reagent includes cholesterol oxidase, TOOS, peroxidase, and chromogen; The elastase detection reagent does not include a colorimetric solution; The lecithin body detection reagent does not include a colorimetric solution.

2. The prostatitis detection reagent as described in claim 1, characterized in that, It also includes one or more of the following: pH test reagent, citric acid test reagent, zinc test reagent, acid phosphatase test reagent, or high-concentration acid phosphatase test reagent; The pH testing reagent includes bromothymol blue sodium salt; The citric acid detection reagent includes hydroxylamine hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and a stop solution; The zinc detection reagent includes sodium 4-(2-pyridiniazo)-resorcinol; The acid phosphatase detection reagent includes Guzis B, tartaric acid, 5-bromo-4-chloro-3-indole phosphate, N-acetyl-L-cysteine, imidazole, and sodium molybdate, and has a pH value of 5.0~6.

0. The high-concentration acid phosphatase detection reagent includes Guzi B, tartaric acid, 5-bromo-4-chloro-3-indole phosphate, N-acetyl-L-cysteine, imidazole, and sodium molybdate, and has a pH value of 7.0~8.

0.

3. The prostatitis detection reagent as described in claim 2, characterized in that, include: The elastase detection reagent further includes a buffer solution; and / or The peroxidase detection reagent further includes a buffer solution; and / or The lecithin body detection reagent also includes a buffer solution and an enzyme protectant; and / or The zinc detection reagent also includes a buffer solution; and / or The 5-bromo-4-chloro-3-indole phosphate includes disodium 5-bromo-4-chloro-3-indole phosphate.

4. The prostatitis detection reagent as described in claim 3, characterized in that, include: The buffer solution includes HEPES buffer, Tris-HCl buffer, or phosphate buffer; and / or The enzyme protectant includes PEG6000.

5. The prostatitis detection reagent as described in claim 4, characterized in that, include: The concentration of Meo-Suc-Ala-Ala-Pro-Val-PNA is 4~10 mmol / L; and / or The concentration of TOOS in the peroxidase detection reagent is 0.002~0.01 g / L; and / or The concentration of the chromogen in the peroxidase detection reagent is 0.009-0.02 g / L; and / or The concentration of the chromogenic solution in the peroxidase detection reagent is 0.1~1.0 g / L; and / or The concentration of the cholesterol oxidase is 10~30 kU / L; and / or The concentration of the peroxidase in the lecithin body detection reagent is 1~10 kU / L; and / or The concentration of TOOS in the lecithin body detection reagent is 1~4 g / L; and / or The concentration of the chromogen in the lecithin body detection reagent is 0.1~1 g / L; and / or The concentration of hydroxylamine hydrochloride is 0.05~0.08 mol / L; and / or The concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 0.02~0.04 mol / L; and / or The concentration of the stop solution in the citric acid detection reagent is 0.02~0.04 mol / L; and / or The concentration of the 4-(2-pyridiniazo)-resorcinol sodium is 0.01~0.17 g / L; and / or The concentration of the fixed purple B is 0.1~0.56 g / L; and / or The concentration of tartaric acid is 0.01~1 mol / L; and / or The concentration of the 5-bromo-4-chloro-3-indole phosphate is 0.1~0.9 g / L; and / or The concentration of the N-acetyl-L-cysteine ​​is 10~40 mg / mL; and / or The concentration of the imidazole is 0.01~0.1 mol / L; and / or The concentration of the sodium molybdate is 0.01~0.1 mol / L; and / or The chromogen includes 4-aminoantipyrine; and / or The colorimetric solution includes urea peroxide; and / or The terminating solution includes ferric chloride.

6. The prostatitis detection reagent as described in claim 5, characterized in that, include: The concentration of the HEPES buffer is 0.02~2 mol / L; and / or The concentration of the Tris-HCl buffer solution is 0.01~1 mol / L; and / or The concentration of the phosphate buffer solution is 0.02~0.2 mol / L; and / or The concentration of PEG6000 is 1~20 g / L.

7. The use of the prostatitis detection reagent as described in any one of claims 1 to 6 in the preparation of a prostatitis detection kit using seminal plasma or semen as the test sample.

8. The application as described in claim 7, characterized in that, The prostatitis detection kit is used to differentiate between one or more of the following: inflammatory prostatitis, non-inflammatory prostatitis, non-prostatic genital tract infection, or benign prostatic hyperplasia. The inflammatory prostatitis includes one or more of type I prostatitis, type II prostatitis, type IIIA prostatitis, or type IV prostatitis; The non-inflammatory prostatitis includes type IIIB prostatitis; The criteria for interpreting the test results of the prostatitis detection kit include (A), (B), (C), and / or (D): (A) If (1) or (2) is satisfied, and (3) is also satisfied, then the sample to be tested is an inflammatory prostatitis sample; (1): The result of the elastase assay was positive. (2): The result of the peroxidase detection reagent was positive; (3): The result of the lecithin body detection reagent was positive; (B) If one or more of (6), (7) or (8) are satisfied, and (4) and (5) are also satisfied, then the sample to be tested is a non-inflammatory prostatitis sample; (4): The result of the peroxidase detection reagent was negative; (5): The result of the elastase assay was negative. (6): The result of the acid phosphatase test was positive. (7): The zinc test result was positive as described in the zinc test reagent. (8): The result of the citric acid test reagent was positive; (C) If (9) or (10) is satisfied, and (11) is also satisfied, then the sample to be tested is a non-prostate reproductive tract infection sample; (9): The result of the peroxidase detection reagent was positive; (10): The result of the elastase assay was positive. (11): The result of the lecithin body detection reagent was negative; (D) If (12) is satisfied, then the sample to be tested is a benign prostatic hyperplasia sample; (12): The result was positive when tested with the high-concentration acid phosphatase detection reagent.

9. A reagent kit, characterized in that, It includes the prostatitis diagnostic reagent as described in any one of claims 1 to 6, and acceptable excipients or adjuvants.

Images