CN117782765A

CN117782765A - Diluent for fecal occult blood detection kit and preparation method and application thereof

Info

Publication number: CN117782765A
Application number: CN202311857720.5A
Authority: CN
Inventors: 郭亚; 林燕清; 陆婷婷
Original assignee: ZHUHAI KEYU BIOLOGICAL ENGINEERING CO LTD
Current assignee: ZHUHAI KEYU BIOLOGICAL ENGINEERING CO LTD
Priority date: 2023-12-29
Filing date: 2023-12-29
Publication date: 2024-03-29

Abstract

The application relates to the field of diluents, in particular to a diluent for a fecal occult blood detection kit, a preparation method and application thereof. The diluent for the fecal occult blood detection kit comprises the following components: 0.05-0.3g/L chelating agent, 0.05-5g/L serum albumin, 0-0.5mL/L preservative, 0.01-0.03g/L sodium cholate, 0.02-0.05 g/L3-sulfopropyl tetradecyl dimethyl betaine, 0.005-0.02g/L sodium laureth sulfate; the buffer was made up for the remainder and maintained at a pH of 7.0-7.8. The method and the device can improve the detection accuracy of fecal occult blood and expand the application range of the diluent.

Description

Diluent for fecal occult blood detection kit and preparation method and application thereof

Technical Field

The application relates to the field of diluents, in particular to a diluent for a fecal occult blood detection kit, a preparation method and application thereof.

Background

The normal phenomenon is that about 0.5-1.5mL of blood is lost every day in the process of updating the gastrointestinal mucosa epithelium of the gastrointestinal tract of a resident person. However, when the daily bleeding amount is as small as about 5mL, the blood in the stool is not observed by naked eyes, and the blood can be detected only by a chemical test, the blood is called as the occult fecal bleeding (FOB), which is called as occult fecal bleeding for short. Often this small insignificant amount of gastrointestinal bleeding is normally colored and asymptomatic, but it is indeed an important manifestation of organic lesions of the digestive tract.

There are many rapid detection kits on the market, and more commonly, a colloidal gold method kit is used. The immune colloidal gold technology is used for detecting hemoglobin in human feces by a double antibody sandwich method. When the sample contains human hemoglobin, the human hemoglobin reacts with the marked Hb2 monoclonal antibody to form a complex, the complex moves forward along the nitrocellulose membrane to a detection area under the action of chromatography and is captured by Hb1 monoclonal monomer on the nitrocellulose membrane, and a red reaction line appears or does not appear in the detection area to obtain a detection result.

After sampling, the sample needs to be diluted with a diluent and then hemoglobin in the red blood cells is released. In practical research and development, it is found that some positive results cannot be accurately detected due to incomplete release of hemoglobin or low content of original hemoglobin, so that detection accuracy is affected. Therefore, there is still a need for improvement.

Disclosure of Invention

In order to improve the detection accuracy of fecal occult blood, the application provides a diluent for a fecal occult blood detection kit, and a preparation method and application thereof.

In a first aspect, the present application provides a diluent for a fecal occult blood detection kit, which adopts the following technical scheme: the diluent for the fecal occult blood detection kit comprises the following components: 0.05-0.3g/L chelating agent, 0.05-5g/L serum albumin, 0-0.5mL/L preservative, 0.01-0.03g/L sodium cholate, 0.02-0.05 g/L3-sulfopropyl tetradecyl dimethyl betaine, 0.005-0.02g/L sodium laureth sulfate; the buffer was made up for the remainder and maintained at a pH of 7.0-7.8.

When a sample is detected by a colloidal gold method, the red blood cells in the sample need to be destroyed by a surfactant, so that hemoglobin is released. However, when the hemoglobin content is too low, no positive result may be detected, and there is a limitation in sensitivity and specificity. In this case, if the machine microscopic examination (stool analyzer) can be performed, the accuracy of the judgment can be improved by combining the number and the form of the erythrocytes.

However, if the detection is required, the red blood cells cannot be damaged seriously, and the shape of the red blood cells needs to be maintained to a certain extent, so that the accurate judgment effect can be achieved.

Therefore, the development of the diluent which can be simultaneously matched with the detection of the kit and the detection of the on-machine is initiated and the compatibility of the diluent is improved.

By adopting the technical scheme, under the joint coordination of sodium cholate, 3-sulfopropyl tetradecyl dimethyl betaine and sodium lauryl polyoxyethylene ether sulfate, the method is milder, can not completely destroy red blood cells, has lower damage degree, but can release hemoglobin, and can be detected by a kit.

And the usage amount of sodium cholate, 3-sulfopropyl tetradecyl dimethyl betaine and sodium lauryl polyoxyethylene ether sulfate is further limited, so that the kit can more sensitively capture a small amount of hemoglobin, and the detection accuracy of the colloidal gold method kit can be effectively improved.

Preferably, the diluent comprises the following components: 0.1-0.2g/L chelating agent, 0.05-3g/L serum albumin, 0-0.5mL/L preservative, 0.01-0.02g/L sodium cholate, 0.025-0.035 g/L3-sulfopropyl tetradecyl dimethyl betaine, 0.005-0.01g/L sodium lauryl polyoxyethylene ether sulfate.

Preferably, the mass ratio of the sodium cholate to the sodium lauryl polyoxyethylene ether sulfate is 1: (2-3): (0.5-1.0) based on the mass of sodium cholate.

By adopting the technical scheme, the contents of all components in the diluent are further limited, particularly the dosage of sodium cholate, 3-sulfopropyl tetradecyl dimethyl betaine and sodium lauryl polyoxyethylene ether sulfate is further limited, and the method has more stable and proper treatment effect on erythrocytes with different contents in a sample, is favorable for keeping the rough form of the erythrocytes and releasing inherent hemoglobin, so that the method can be simultaneously applied to detection of a kit and detection of an on-machine.

Preferably, the buffer solution is at least one of phosphate buffer solution, borate buffer solution and citrate buffer solution.

Preferably, the chelating agent is at least one or more of EDTA-dipotassium or EDTA-disodium.

Preferably, the serum albumin is at least one or more of bovine serum albumin, horse serum albumin and human serum albumin.

Preferably, the serum albumin is bovine serum albumin.

By adopting the technical scheme, a proper amount of bovine serum albumin is added into the system, and the combination of the antibody and the nonspecific protein can be placed, so that the detection accuracy is effectively improved.

In a second aspect, the present application provides a method for preparing a diluent for a fecal occult blood detection kit, which adopts the following technical scheme:

the preparation method of the diluent for the fecal occult blood detection kit comprises the following steps: mixing and stirring the components uniformly to obtain the finished product.

The diluent provided by the application is quite simple in preparation method, can be prepared by uniformly mixing various components without too many harsh conditions, and is suitable for popularization and use in industry.

In a third aspect, the present application provides an application of a diluent for a basic fecal occult blood detection kit in fecal analysis instrument detection.

As described above, the diluent provided by the application not only can be used in the detection of the kit, but also can be used in the detection of the on-machine analysis, so that the universality and the application range of the diluent are obviously improved, and the detection steps can be effectively simplified.

In summary, the present application has the following beneficial effects:

1. the diluent provided by the application can be used as the diluent of the kit of the colloidal gold method, and also can be used as the diluent in a follow-up fecal analyzer, and the diluent can better keep the morphology of red blood cells for on-line observation, and can release hemoglobin for rapid detection of the kit, so that the diluent has wider applicability.

2. The diluent of the kit can enable the kit to capture a small amount of hemoglobin more sensitively by further selecting specific sodium cholate, 3-sulfopropyl tetradecyl dimethyl betaine and sodium lauryl polyoxyethylene ether sulfate and limiting the contents of the three in a system under the cooperation of the three, and can effectively improve the detection accuracy of the colloidal gold method kit.

Drawings

FIG. 1 is a graph of the test results of the dilutions of example 1 of the present application in test 1.

FIG. 2 is a graph of the test results of the dilutions of example 4 of the present application in test 1.

FIG. 3 is a graph of the test results of the dilutions of comparative example 4 of the present application in test 1.

Fig. 4 is a graph of the test results of the dilutions of comparative example 5 of the present application in test 1.

Fig. 5 is a view of the dilution of example 1 of the present application for 60 minutes in test 2.

Fig. 6 is a view of the dilution of example 4 of the present application for 60 minutes in test 2.

Fig. 7 is a view of the dilution of comparative example 1 of the present application for 60 minutes in test 2.

Fig. 8 is a view of the dilution of comparative example 5 of the present application for 60 minutes in test 2.

Fig. 9 is a view of the dilution of comparative example 6 of the present application for 60 minutes in test 2.

Detailed Description

The present application is described in further detail below with reference to the drawings and examples.

The raw materials used in the following examples and comparative examples are all commercially available products.

The diluent for the fecal occult blood detection kit comprises the following components:

(1) Chelating agent:

the chelating agent may be one or more of EDTA-dipotassium, EDTA-disodium, etc.

The chelating agent may be used in the diluent in an amount of any of 0.05g/L, 0.08g/L, 0.10g/L, 0.12g/L, 0.15g/L, 0.18g/L, 0.20g/L, 0.22g/L, 0.25g/L, 0.28g/L, 0.3g/L, etc.

(2) Serum albumin:

the serum albumin can be one of bovine serum albumin, horse serum albumin and human serum albumin.

The amount of serum albumin used in the diluent may be any of 0.05g/L, 0.08g/L, 0.12g/L, 0.35g/L, 0.68g/L, 0.92g/L, 1.25g/L, 2.35g/L, 3.00g/L, 4.32g/L, 5.0g/L, etc.

(3) Preservative:

the preservative is mainly used for preservation and can be Proclin300.

The amount of the preservative to be used in the diluent may be any of 0, 0.01mL/L, 0.05mL/L, 0.1mL/L, 0.2mL/L, 0.3mL/L, 0.4mL/L, 0.5mL/L, etc.

(4) Sodium cholate:

the amount of sodium cholate used in the diluent may be any of 0.01g/L, 0.015g/L, 0.02g/L, 0.025g/L, 0.03g/L, and the like.

(5) 3-sulfopropyl tetradecyldimethyl betaine: the amount of 3-sulfopropyl tetradecyldimethyl betaine may be any of 0.02g/L, 0.025g/L, 0.03g/L, 0.035g/L, 0.04g/L, 0.045g/L, 0.05g/L, etc.

(6) Sodium lauryl polyoxyethylene ether sulfate:

the amount of sodium laureth sulfate used in the diluent may be any of 0.005g/L, 0.008g/L, 0.01g/L, 0.015g/L, 0.02g/L, etc.

(7) Buffer solution:

the buffer may be one or more of phosphate buffer, borate buffer, citrate buffer.

(8) The mass ratio of the sodium cholate to the 3-sulfopropyl tetradecyl dimethyl betaine to the sodium lauryl polyoxyethylene ether sulfate can be 1:2:0.5, which may be 1:2.5:0.8, which may be 1:3:1, which may be 1:2:1, etc.

Examples

Example 1

The diluent for the fecal occult blood detection kit comprises the following components in 1L: 0.15g chelating agent, 1g serum albumin, 0.1mL preservative, 0.015g sodium cholate, 0.032g 3-sulfopropyl tetradecyldimethyl betaine, 0.009g sodium laureth sulfate, the buffer supplementing the balance to 1L.

The pH of the dilution was 7.4.

The chelating agent is EDTA-disodium, the serum albumin is bovine serum albumin, the preservative is Proclin300, and the buffer solution is phosphate buffer solution (0.1M, 0.85% NaCl).

The specific amounts of the components are shown in Table 1.

The application also provides a preparation method of the diluent for the fecal occult blood detection kit, which comprises the following steps: adding chelating agent, serum albumin, preservative, sodium cholate, 3-sulfopropyl tetradecyl dimethyl betaine and sodium lauryl polyoxyethylene ether sulfate into buffer solution, and stirring uniformly to obtain diluent.

Example 2

The dilution for the fecal occult blood detection kit is different from example 1 in that the mass ratio of sodium cholate, 3-sulfopropyl tetradecyl dimethyl betaine and sodium lauryl polyoxyethylene ether sulfate is 1:2:0.5. namely, 0.015g of sodium cholate, 0.030g of 3-sulfopropyl tetradecyl dimethyl betaine and 0.0075g of sodium lauryl polyoxyethylene ether sulfate.

The specific amounts of the components are shown in Table 1.

Example 3

The dilution for the fecal occult blood detection kit is different from example 1 in that the mass ratio of sodium cholate, 3-sulfopropyl tetradecyl dimethyl betaine and sodium lauryl polyoxyethylene ether sulfate is 1:3:1. namely, 0.015g of sodium cholate, 0.045g of 3-sulfopropyl tetradecyl dimethyl betaine and 0.015g of sodium lauryl polyoxyethylene ether sulfate.

The specific amounts of the components are shown in Table 1.

Examples 4 to 5

The dilution for fecal occult blood test kit is different from example 1 in the amounts of the components, and is specifically shown in Table 1.

TABLE 1

Comparative example

Comparative example 1

The diluent for the fecal occult blood test kit is different from example 1 in that sodium cholate is replaced with tween 80, namely 0, 0.015g of sodium cholate.

Comparative example 2

A diluent for a fecal occult blood test kit is different from example 1 in that 3-sulfopropyl tetradecyl dimethyl betaine is replaced by cocamidopropyl betaine, namely, 0g of 3-sulfopropyl tetradecyl dimethyl betaine and 0.032g of cocamidopropyl betaine.

Comparative example 3

The dilution for fecal occult blood test kit is different from example 1 in that sodium laureth sulfate is replaced with sodium laurylsulfate, namely, 0.009g of sodium laureth sulfate.

Comparative example 4

The dilution for the fecal occult blood test kit is different from example 1 in that sodium cholate, 3-sulfopropyl tetradecyl dimethyl betaine, and sodium laureth sulfate are omitted, and the buffer is used to supplement the amount.

Comparative example 5

A diluent for a fecal occult blood test kit is different from example 1 in that sodium cholate is 0.005g, 3-sulfopropyl tetradecyldimethyl betaine is 0.01g, and sodium laureth sulfate is 0.001g.

Comparative example 6

A diluent for a fecal occult blood test kit is different from example 1 in that sodium cholate is 0.15g, 3-sulfopropyl tetradecyl dimethyl betaine is 0.32g, and sodium laureth sulfate is 0.09g.

Application example 1

The application of the diluent for the fecal occult blood detection kit in the detection of fecal analysis instruments.

Sampling a flat spoon (about 0.3-0.5 g) at 6 different positions of the fecal sample by using a sampling spoon, inserting the sampling spoon with the fecal sample into a sample collecting cup, placing the sample collecting cup into an instrument sample rack, placing the sample collecting cup into a sample feeding tray of a fecal analyzer, setting parameters of the fecal analyzer, and starting detection operation.

The fecal analyzer automatically performs the operation of adding 5-10ml of diluent and stirring the mixed sample.

In this application example, the diluent of example 1 was used as the diluent.

Performance test

1. And (3) rapid detection of the kit: human hemoglobin was prepared at 1mg/ml, and then diluted with the dilutions of examples 1-5, comparative examples 1-6 to give 6. Mu.g/ml of the sample to be tested.

3 drops (about 120 mu L) of the sample to be tested are dropped into the test card loading well of the FOB detection kit (colloidal gold method).

Wait 3-5 minutes, interpret the results, see if the detection zone has a red reaction line, and record the results in table 2.

2. And (3) detecting: 10mL of the dilutions of examples 1-5 and comparative examples 1-6 were placed in a sharp-bottomed tube, and 10. Mu.L of fresh blood was measured with a pipette and added and shaken well. Observations were made immediately with a fecal analyzer (in-house microscope) and pictures were kept. After 30 minutes, the test was carried out on-machine in the same manner and the pictures were retained. After 60 minutes, the test was carried out on-machine in the same manner and the pictures were kept. And comparing the observation results of 30 minutes and 60 minutes by taking the test picture just on the machine as a control basis. The difference in morphology between the red blood cells immediately after the completion of the measurement was determined, and the results are shown in Table 2.

If the cell morphology is not greatly different and has no swelling, cracking and shrinkage, the representing effect is good; if the difference in cell morphology is large, the effect is not good.

3. And (3) detecting the accuracy of the kit:

human hemoglobin was formulated at 1mg/ml and then diluted with the dilutions of examples 1-5, comparative examples 1-6 to give 200ng/ml, 400ng/ml, 600ng/ml, 800ng/ml, 1000ng/ml, 1200ng/ml, 1500ng/ml, 2. Mu.g/ml, 4. Mu.g/ml, 6. Mu.g/ml, 8. Mu.g/ml of the test sample.

Then, 3 drops (about 120. Mu.L) of the sample to be tested were dropped into the test card well of the FOB test kit (colloidal gold method). Wait 3-5 minutes, interpret the results, see if the detection zone has a red reaction line. The sample to be tested with the lowest concentration, at which a positive result was detected, was the sensitivity of the kit, and the results are recorded in table 2.

The lower the concentration of human hemoglobin corresponding to the sensitivity of the kit, the better the effect of the representative diluent.

TABLE 2

As can be seen from the test results of examples 1 to 5 in Table 2, the dilutions of examples 1 to 5 were used in the rapid test of the kit to show a positive result; in the on-machine detection, the morphology of the red blood cells can be well maintained, and the red blood cells can be observed. Comparative example 4 was based on example 1, omitting sodium cholate, 3-sulfopropyl tetradecyl dimethyl betaine, and sodium laureth sulfate. From the results of the test, the dilution of comparative example 4 can well maintain the state of red blood cells in the on-machine test, but is not compatible with the rapid test of the kit. The dilution provided by the application is characterized in that specific sodium cholate, 3-sulfopropyl tetradecyl dimethyl betaine and sodium lauryl polyoxyethylene ether sulfate are selected and used in combination, the dosage of the three components in a system is further limited, and the prepared dilution can be simultaneously suitable for detection of a kit (colloidal gold method) and microscopic examination on a machine.

Comparative examples 1 to 3 are dilutions prepared by optionally replacing one of sodium cholate, 3-sulfopropyl tetradecyl dimethyl betaine, and sodium laureth sulfate based on example 1. Although the dilutions of comparative examples 1-3 can be used in rapid detection of the kit, the morphology of the red blood cells is severely destroyed during the on-machine detection, and the condition can not be judged by microscopic examination and combination.

Comparative examples 5 and 6 are based on example 1, and the dilution obtained by changing the specific amounts of sodium cholate, 3-sulfopropyl tetradecyl dimethyl betaine and sodium laureth sulfate in the system also damages the morphology of erythrocytes.

The inventors hypothesize that this might be related to the permeability of the erythrocyte membrane. The diluent prepared by the method can increase the permeability of erythrocyte membranes, so that hemoglobin can permeate out, but the morphology of erythrocytes is not broken due to excessive swelling, and the erythrocytes still keep a good morphology, so that the diluent can be observed on an engine, and the rapid detection of the kit can be realized. However, if the formulation of the present application is destroyed, the balance of the special coordination is destroyed, and hemoglobin can not be released for detection while ensuring the morphology of red blood cells.

In terms of the accuracy detection of the kit, the dilutions of examples 1-5 showed better accuracy than the dilutions of comparative examples 1-3, 5-6, and lower concentrations of hemoglobin could be detected.

Specifically, as can be seen from comparison of the test data of examples 1 and 2 and examples 3 to 5 in Table 2, sodium cholate, 3-sulfopropyl tetradecyl dimethyl betaine and sodium laureth sulfate were mixed in a specific amount and a specific ratio, and the prepared diluent (examples 1 to 2) had higher sensitivity. Although the dilution of example 3 limited the specific ratio of the three, it was not within the optimum dosage range, so the sensitivity was inferior to that of examples 1 and 2; although example 3 still allows for better sensitivity, which is undoubted.

As can be seen from FIGS. 1-4, the positive results were obtained in example 1, example 4, and comparative example 5, while the best effect of example 1 was obtained, the most visible red line, example 4 times, and the least visible red line of comparative example 5. A positive result could not be obtained in comparative example 4.

As can be seen from fig. 5 to 9, the red blood cells using the dilutions of examples 1 and 4 were good and normal in morphology, and could be observed by microscopic examination, and the number and morphology of red blood cells were combined for comprehensive judgment; all red blood cells using the dilutions of comparative examples 1, 5 were disrupted and could not be observed by microscopic examination; the red blood cells using the dilution of comparative example 6 were all disrupted, and the extent of disruption was large, which was not judged.

The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.

Claims

1. The diluent for the fecal occult blood detection kit is characterized by comprising the following components:

05-0.3g/L chelating agent, 0.05-5g/L serum albumin, 0-0.5mL/L preservative, 0.01-0.03g/L sodium cholate, 0.02-0.05 g/L3-sulfopropyl tetradecyl dimethyl betaine, 0.005-0.02g/L sodium lauryl polyoxyethylene ether sulfate;

the buffer was made up for the remainder and maintained at a pH of 7.0-7.8.

2. The diluent for fecal occult blood detection kit according to claim 1, wherein: the diluent comprises the following components: 0.1-0.2g/L chelating agent, 0.05-3g/L serum albumin, 0-0.5mL/L preservative, 0.01-0.02g/L sodium cholate, 0.025-0.035 g/L3-sulfopropyl tetradecyl dimethyl betaine, 0.005-0.01g/L sodium lauryl polyoxyethylene ether sulfate.

3. The diluent for fecal occult blood detection kit according to claim 1, wherein: the mass ratio of the sodium cholate to the 3-sulfopropyl tetradecyl dimethyl betaine to the sodium lauryl polyoxyethylene ether sulfate is 1: (2-3): (0.5-1.0) based on the mass of sodium cholate.

4. The diluent for fecal occult blood detection kit according to claim 1, wherein: the buffer solution is at least one of phosphate buffer solution, borate buffer solution and citrate buffer solution.

5. The diluent for fecal occult blood detection kit according to claim 1, wherein: the chelating agent is at least one or more of EDTA-dipotassium or EDTA-disodium.

6. The diluent for fecal occult blood detection kit according to claim 1, wherein: the serum albumin is at least one or more of bovine serum albumin, horse serum albumin and human serum albumin.

7. A method for preparing a diluent for a fecal occult blood detection kit according to any of claims 1-6, comprising the steps of: mixing and stirring the components uniformly to obtain the finished product.

8. Use of a diluent for a fecal occult blood detection kit according to any of claims 1-6 in fecal analysis instrument detection.