Blood residue rapid detection kit

Abstract

The invention discloses a rapid detection kit for blood residues. The rapid detection kit comprises a labeled antibody, a coated antibody and a colloidal gold test strip. The labeled antibody is obtained by treating human red blood cells, immunizing mice and purifying through a series of steps; the coated antibody is prepared by treating human red blood cells, immunizing a rabbit and then purifying through a specific process. The preparation process of the colloidal gold test strip comprises the following steps: preparing colloidal gold, incubating a labeled antibody and the colloidal gold, spraying and drying, treating a sample pad, drying, impression on a PVC (Polyvinyl Chloride) bottom plate pasted with a nitrocellulose membrane, drying, and finally cutting and assembling into a detection strip. The kit is rapid in detection and can meet the rapid turnover requirement of instruments; the accuracy is high, and subjective misjudgment is reduced; the sensitivity is as low as 50ng / mL, trace blood residues can be effectively detected, the risks of cross infection and medical negligence are reduced, and the method has remarkable advantages and application value in the field of blood residue detection of medical instruments.

Description

Technical Field

[0001] The invention relates to a rapid detection kit for blood residue. Background Art

[0002] In the medical field, the cleaning quality of medical devices is of vital importance to ensure the health of patients and medical staff and the smooth development of medical services. Medical devices will inevitably come into contact with the patient's blood during use. If there is still blood residue after cleaning, it may cause serious consequences.

[0003] At present, there are many defects in the methods of detecting blood residues in medical devices. The traditional visual observation method is one of the most basic and widely used detection methods, but it relies on the subjective judgment of the operator, which is greatly affected by subjective factors such as the operator's attitude, meticulousness, and grasp of the judgment criteria. It is easy to make misjudgments, resulting in the incomplete cleaning of medical devices cannot be accurately identified, thereby increasing the potential risk of cross infection.

[0004] The occult blood test method uses the activity of iron-containing heme peroxidase in hemoglobin to produce a color change reaction under the action of acid and hydrogen peroxide to check whether there is residual blood on the instrument. However, this method has low sensitivity and is often unable to effectively detect trace amounts of residual blood, allowing some residual blood to escape detection, which also poses a hidden danger to medical safety.

[0005] ATP test is a rapid test method that indirectly reflects organic substances (including blood residues) by measuring ATP levels. Although its detection speed is fast, it is an indirect test of blood residues and requires specific equipment and reagents. The cost is relatively high and the skill requirements for operators are also high. This has limited its wide application in routine testing to a certain extent, and the accuracy of its test results is also affected by many factors.

[0006] Compared with the present invention, the existing detection methods have obvious deficiencies in detection speed, accuracy and sensitivity. The present invention aims to overcome these shortcomings and develop a new blood residue rapid detection kit, which realizes rapid, accurate and highly sensitive detection of blood residues in medical devices through a unique preparation method of labeled antibodies and coated antibodies, combined with a carefully designed colloidal gold test strip, to provide more reliable protection for medical safety, meet the urgent needs of hospitals and other medical institutions for efficient and accurate detection of medical devices, fill the gap in the existing technology in this regard, and promote the further development of medical device detection technology. Summary of the invention

[0007] The object of the present invention is to provide a rapid blood residue detection kit to solve the problems raised in the above-mentioned background technology.

[0008] In order to solve the above technical problems, the present invention provides a technical solution: a blood residue rapid detection kit, comprising:

[0009] The labeled antibody is prepared by the following method:

[0010] Take human erythrocytes, wash them 2-3 times with ice-cold phosphate buffered saline (PBS), centrifuge and discard the supernatant after each wash, resuspend the washed erythrocytes in an appropriate amount of ice-cold RIPA lysis buffer, add an appropriate amount of protease inhibitor PMSF and gently mix, incubate on ice for 30 minutes, centrifuge the lysate at 12000 rpm at 4°C for 5-10 minutes, and collect the supernatant;

[0011] Select 6-8 week old purebred BALB / c female mice, weighing 18-20 g, and perform primary immunization by intraperitoneal injection and subcutaneous injection of 0.3 mL of a mixture of antigen and adjuvant. Subsequent immunizations are performed every other week for a total of 6 immunizations. Finally, 0.2 mL is boosted by tail vein immunization 3 days before fusion. After the immunization cycle, blood is collected from the ear vein to collect serum, and the antibody titer is detected by indirect ELISA. Pre-immunization rabbit serum is used as a negative control.

[0012] CNBr-Activated Sepharose 4B gel is used to couple with antigens. The gel and antigen are mixed in a coupling buffer, shaken at room temperature for 2-4 hours or at 4°C overnight, incubated with 1% BSA solution at room temperature for 2 hours or at 4°C overnight to block unreacted active groups, and the medium is washed with PBS for more than 3 times, more than 15 ml each time, and the antiserum is diluted with an equal volume of PBS, centrifuged to obtain the supernatant, and the diluted antiserum is passed through the antigen affinity column, and the specific antibody will be adsorbed. The column is eluted with an antibody elution solution to obtain the specific antibody, which is then concentrated and dialyzed in PBS for desalination, and 40-50% glycerol is added and stored at -20°C for a long time;

[0013] The coating antibody is prepared by the following method:

[0014] Take human red blood cells, wash the red blood cells with ice-cold PBS 2-3 times, centrifuge and discard the supernatant after each wash, resuspend the washed red blood cells in an appropriate amount of ice-cold RIPA lysis buffer, add an appropriate amount of protease inhibitor PMSF and gently mix, incubate on ice for 30 minutes, centrifuge the lysate at 12000 rpm at 4°C for 5-10 minutes, and collect the supernatant;

[0015] Healthy 6-week-old New Zealand white rabbits weighing about 2 kg were selected. After adapting to the new environment, about 10 ml of blood was drawn from the ear vein, and serum was collected as a negative control. For the initial immunization, 400 ug of antigen was mixed with Freund's complete adjuvant (CFA) for each rabbit, and then subcutaneously injected under the skin around the shoulders of the rabbit and intramuscularly injected into the hind thigh. For subsequent immunizations, 100 ug was injected each time after mixing with Freund's incomplete adjuvant (IFA) and antigen. The immunization cycle was 20 days. Blood was drawn 7-10 days after the immunization, and a total of 4-5 immunizations were performed. One week after the last immunization, blood was drawn from the ear vein to collect serum, and the antibody titer was detected by indirect ELISA method. At the same time, the rabbit serum before immunization was used as a negative control.

[0016] CNBr-Sepharose4B gel is used to couple with antigens. The gel and antigen are mixed in coupling buffer, shaken at room temperature for 2-4 hours or at 4°C overnight, incubated with 1% BSA solution at room temperature for 2 hours or at 4°C overnight to block unreacted active groups, washed with PBS for more than 3 times, more than 15 ml each time, the antiserum is diluted with an equal volume of PBS, centrifuged to obtain the supernatant, and the antigen affinity column is washed with 10 times the volume of PBS to balance the column, the diluted antiserum is added to the balanced column, gently mixed and shaken at room temperature for 2-4 hours, or at 4°C overnight, the antigen column is washed with 10 times the volume of PBS to wash away the foreign proteins bound to the column, and the column is washed with 2 times the volume of antibody elution solution to obtain specific antibodies, concentrated with sucrose or polyethylene glycol, dialyzed in PBS for desalting, and added with 40-50% glycerol and stored at -20°C for a long time;

[0017] A colloidal gold test strip, comprising:

[0018] Colloidal gold, by taking 100ml ultrapure water and pouring it into a 250ml conical flask, adding 1.0ml of 1.0% chloroauric acid solution, placing it in a magnetic stirrer and stirring it continuously and heating it to boiling, and quickly adding 1.8ml of 1.0% trisodium citrate solution while stirring, the solution changes from light yellow to gray-black and finally to orange-red. After the solution changes to orange-red, continue heating for 15 minutes, cool to room temperature, adjust the solution pH to about 6.5 with 0.1mol / mlK2CO3, dilute it to 100ml with ultrapure water, and place it in a brown bottle and store it at 4°C for later use;

[0019] After incubating the labeled antibody with colloidal gold solution, the gold-labeled complex was sprayed on glass cellulose and dried at 37°C;

[0020] The sample pad treatment solution was dip-coated on the glass cellulose membrane and dried at 37°C;

[0021] Stick the nitrocellulose membrane (NC membrane) on the PVC base plate, adjust the C line (sheep anti-mouse lgM) and T line (coated antibody) of the impression machine to a suitable distance, and then make impressions, and dry them at 37°C;

[0022] Cut the absorbent pad, prepared gold pad and sample pad to the appropriate width, and stick them on the PVC board together with the nitrocellulose membrane with printed C and T lines. Cut the assembled large board into test strips of a certain width according to the width of the test card slot. The cutting width should be uniform, without indentations or scratches.

[0023] Preferably, the detection sensitivity of the kit for blood residues is as low as 50 ng / mL.

[0024] The advantages of the present invention are: rapid detection: the detection results can be obtained in a short time, the detection efficiency is improved, and the needs of hospitals and other institutions for rapid turnover of medical equipment are met.

[0025] High accuracy: Compared with traditional visual observation and other methods, it reduces the influence of subjective factors, provides more reliable test results, and effectively avoids the problem of incomplete cleaning of medical devices due to misjudgment.

[0026] High sensitivity: The sensitivity is as low as 50ng / mL, which can detect trace amounts of blood residue, greatly reducing the risk of cross infection and medical accidents caused by residual blood. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a photo of the sensitivity test of the colloidal gold test strip. DETAILED DESCRIPTION

[0028] For the purpose of the following detailed description, it should be understood that the present invention may adopt various alternative changes and step sequences, unless expressly specified to the contrary. In addition, except in any operating examples, or otherwise indicated, all numbers representing the amount of ingredients used in the specification and claims should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the attached claims are approximate values ​​that vary according to the desired performance to be obtained by the present invention. At least it is not intended to limit the application of the doctrine of equivalents to the scope of the claims, and each numerical parameter should at least be interpreted according to the number of reported significant figures and by applying ordinary rounding techniques.

[0029] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0030] In addition, it should be understood that any numerical range described herein is intended to include all subranges subsumed therein. For example, a range of "1 to 10" is intended to include all subranges between (and including) the stated minimum value of 1 and the stated maximum value of 10, i.e., having a minimum value equal to or greater than 1 and a maximum value equal to or less than 10.

[0031] Example 1: Preparation of labeled antibodies

[0032] Take human red blood cells (purchased from Huizhi Heyuan Biotechnology (Suzhou) Co., Ltd.), wash the red blood cells with ice-cold PBS (phosphate buffered saline), and repeat 2-3 times. Centrifuge after each wash and discard the supernatant. Resuspend the washed red blood cells in an appropriate amount of ice-cold RIPA lysis buffer, add an appropriate amount of protease inhibitor PMSF, and mix gently. Incubate on ice for 30 minutes to ensure that the cells are fully lysed. Transfer the lysate to a centrifuge tube and centrifuge at 12000rpm for 5-10 minutes at 4°C. This step precipitates the cell membrane and insoluble substances to obtain the supernatant. Carefully collect the supernatant after centrifugation, which contains the extracted protein. Transfer it to a new centrifuge tube.

[0033] Select purebred BALB / c mice aged 6-8 weeks, weighing 18-20g, female. : The primary immunization was performed by intraperitoneal injection and subcutaneous injection of 0.3mL of a mixture of antigen and adjuvant. Subsequent immunizations were performed every other week for a total of 6 immunizations, and finally 0.2mL was boosted through the tail vein 3 days before fusion. After the immunization cycle, blood was collected from the ear vein to collect serum to detect antibody titer. One week after the last immunization, blood was collected from the ear vein to collect serum, and the antibody titer was detected by indirect ELISA, and pre-immunization rabbit serum was used as a negative control. CNBr-Activated Sepharose 4B gel was used to couple with antigen for antibody purification. The gel and antigen were mixed in the coupling buffer and shaken at room temperature for 2-4 hours or at 4°C overnight. Incubate with 1% BSA solution at room temperature for 2 hours or at 4°C overnight to block unreacted active groups. Wash the medium with PBS for more than 3 times, more than 15ml each time. Dilute the antiserum with an equal volume of PBS and centrifuge to obtain the supernatant. The diluted antiserum is passed through the antigen affinity column, and the specific antibody will be adsorbed. Elution: The column is eluted with antibody eluent to obtain the specific antibody. After the eluted antibody is obtained, it is concentrated and dialyzed in PBS to remove salt, and 40-50% glycerol is added and placed at -20°C for long-term storage.

[0034] Example 2: Preparation of coated antibodies

[0035] Take human red blood cells (purchased from Huizhi Heyuan Biotechnology (Suzhou) Co., Ltd.), wash the red blood cells with ice-cold PBS (phosphate buffered saline), and repeat 2-3 times. Centrifuge after each wash and discard the supernatant. Resuspend the washed red blood cells in an appropriate amount of ice-cold RIPA lysis buffer, add an appropriate amount of protease inhibitor PMSF, and mix gently. Incubate on ice for 30 minutes to ensure that the cells are fully lysed. Transfer the lysate to a centrifuge tube and centrifuge at 12000rpm for 5-10 minutes at 4°C. This step precipitates the cell membrane and insoluble substances to obtain the supernatant. Carefully collect the supernatant after centrifugation, which contains the extracted protein. Transfer it to a new centrifuge tube.

[0036] Select healthy 6-week-old New Zealand white rabbits weighing about 2Kg, and let them adapt to the new living environment. After at least a few days of stabilization, the first blood draw is performed. Carefully place the rabbit in a fixed rack and shave the hair on the rabbit's ears to make the blood vessels clearly visible. Use a syringe to draw about 10ml of blood from the ear vein and collect serum as a negative control. It is more suitable for each rabbit to be immunized with 400ug of antigen for the first time. After the antigen is fully mixed with Freund's complete adjuvant (CFA), it is subcutaneously injected under the skin around the rabbit's shoulders and intramuscularly injected into the hind thigh. Subsequent immunizations are 100ug each time, and Freund's incomplete adjuvant (IFA) is used to mix with the antigen for injection. The immunization cycle is 20 days, and blood is drawn 7-10 days after immunization, for a total of 4-5 immunizations. One week after the last immunization, blood is drawn from the ear vein to collect serum, and the antibody titer is detected by indirect ELISA, while the rabbit serum before immunization is used as a negative control. CNBr-Sepharose 4B gel is used to couple with the antigen for antibody purification. Mix the gel and antigen in the coupling buffer and shake at room temperature for 2-4 hours or at 4°C overnight. Incubate with 1% BSA solution at room temperature for 2 hours or at 4°C overnight to block unreacted active groups. Wash the medium with PBS for more than 3 times, more than 15 ml each time. Dilute the antiserum with an equal volume of PBS and centrifuge to obtain the supernatant. Wash the antigen affinity column with 10 volumes of PBS to balance the column. Add the diluted antiserum to the balanced column and gently mix and shake at room temperature for 2-4 hours or at 4°C overnight. Wash the antigen column with 10 volumes of PBS to wash away the foreign proteins bound to the column. Wash the column with 2 volumes of antibody elution buffer to obtain specific antibodies. Equilibrate the column with 10 volumes of PBS, seal the column with 20% alcohol, and store at 4°C. After obtaining the eluted antibody, concentrate it with sucrose or polyethylene glycol, dialyze it in PBS to desalt it, add 40-50% glycerol and place it at -20°C for long-term storage.

[0037] Example 3: Preparation of colloidal gold test strips

[0038] First, prepare colloidal gold as follows. Take 100 ml of ultrapure water and pour it into a 250 ml conical flask. Add 1.0 ml of 1.0% chloroauric acid solution, place it in a magnetic stirrer, stir continuously and heat to boiling. While stirring, quickly add 1.8 ml of 1.0% trisodium citrate solution, at which time the solution gradually changes from light yellow to gray-black and finally to orange-red. After the solution turns orange-red, continue heating for 15 minutes. Cool to room temperature, and adjust the pH of the solution to about 6.5 with 0.1 mol / ml K2CO3. After the volume is made up to 100 ml with ultrapure water, place it in a brown bottle and store it at 4°C for later use.

[0039] The prepared labeled antibody is incubated with the colloidal gold solution. After completion, the gold-labeled complex is sprayed on the glass cellulose and dried in a drying room or oven at 37°C. The sample pad treatment solution is dip-coated on the glass cellulose membrane and dried in a drying room or oven at 37°C. The nitrocellulose membrane (NC membrane) is attached to the PVC bottom plate, and the C line (sheep anti-mouse lgM) and T line (prepared coated antibody) of the impression machine are adjusted to the appropriate distance, and then the impression is made. Finally, the printed membrane is placed in a drying room or oven at 37°C to dry it. The absorbent pad, the prepared gold pad, and the sample pad are cut to the appropriate width, and the nitrocellulose membrane with the C and T lines printed is attached to the position of the PVC board. According to the width of the test card slot, the assembled large plate is cut into test strips of a certain width, and the cutting width is uniform, without indentations or scratches.

[0040] Example 4: Sensitivity test of colloidal gold test strips

[0041] Take the extracted red blood cell protein and dilute it to 500, 400, 300, 200, 100, and 50 ng / mL using phosphate buffer. Take 80uL of each and apply it to the prepared colloidal gold test strips. After 10 minutes of rest, observe the results. Figure 1 As shown, the T lines of all test strips were able to produce bands, indicating that the test strips were successfully prepared with a sensitivity as low as 50 ng / mL.

[0042] Example 5: Application of colloidal gold test strips for rapid detection of blood residues

[0043] Three methods were used to detect the cleaning results of medical devices in hospitals, namely visual inspection, occult blood test paper method and colloidal gold test paper method of the present invention. The results are shown in the following table. The three methods tested 100 cases each, and the number of positive results was 5, 8 and 14 cases respectively, indicating that the colloidal gold test paper has a better detection effect.

[0044] method Number of testing equipment Number of positive results Visual inspection 100 5 Occult blood test strips 100 8 Colloidal gold test strips 100 14

[0045] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A rapid test kit for blood residue, characterized in that: include: The labeled antibody is prepared by the following method: Take human erythrocytes, wash them 2-3 times with ice-cold phosphate buffered saline (PBS), centrifuge and discard the supernatant after each wash, resuspend the washed erythrocytes in an appropriate amount of ice-cold RIPA lysis buffer, add an appropriate amount of protease inhibitor PMSF and gently mix, incubate on ice for 30 minutes, centrifuge the lysate at 12000 rpm at 4°C for 5-10 minutes, and collect the supernatant; Select 6-8 week old purebred BALB / c female mice, weighing 18-20 g, and perform primary immunization by intraperitoneal injection and subcutaneous injection of 0.3 mL of a mixture of antigen and adjuvant. Subsequent immunizations are performed every other week for a total of 6 immunizations. Finally, 0.2 mL is boosted by tail vein immunization 3 days before fusion. After the immunization cycle, blood is collected from the ear vein to collect serum, and the antibody titer is detected by indirect ELISA. Pre-immunization rabbit serum is used as a negative control. CNBr-Activated Sepharose 4B gel is used to couple with antigens. The gel and antigen are mixed in a coupling buffer, shaken at room temperature for 2-4 hours or at 4°C overnight, incubated with 1% BSA solution at room temperature for 2 hours or at 4°C overnight to block unreacted active groups, and the medium is washed with PBS for more than 3 times, more than 15 ml each time, and the antiserum is diluted with an equal volume of PBS, centrifuged to obtain the supernatant, and the diluted antiserum is passed through the antigen affinity column, and the specific antibody will be adsorbed. The column is eluted with an antibody elution solution to obtain the specific antibody, which is then concentrated and dialyzed in PBS for desalination, and 40-50% glycerol is added and stored at -20°C for a long time; The coating antibody is prepared by the following method: Take human red blood cells, wash the red blood cells with ice-cold PBS 2-3 times, centrifuge and discard the supernatant after each wash, resuspend the washed red blood cells in an appropriate amount of ice-cold RIPA lysis buffer, add an appropriate amount of protease inhibitor PMSF and gently mix, incubate on ice for 30 minutes, centrifuge the lysate at 12000 rpm at 4°C for 5-10 minutes, and collect the supernatant; Healthy 6-week-old New Zealand white rabbits weighing about 2 kg were selected. After adapting to the new environment, about 10 ml of blood was drawn from the ear vein, and serum was collected as a negative control. For the initial immunization, 400 ug of antigen was mixed with Freund's complete adjuvant (CFA) for each rabbit, and then subcutaneously injected under the skin around the shoulders of the rabbit and intramuscularly injected into the hind thigh. For subsequent immunizations, 100 ug was injected each time after mixing with Freund's incomplete adjuvant (IFA) and antigen. The immunization cycle was 20 days. Blood was drawn 7-10 days after the immunization, and a total of 4-5 immunizations were performed. One week after the last immunization, blood was drawn from the ear vein to collect serum, and the antibody titer was detected by indirect ELISA method. At the same time, the rabbit serum before immunization was used as a negative control. CNBr-Sepharose4B gel is used to couple with antigens. The gel and antigen are mixed in coupling buffer, shaken at room temperature for 2-4 hours or at 4°C overnight, incubated with 1% BSA solution at room temperature for 2 hours or at 4°C overnight to block unreacted active groups, washed with PBS for more than 3 times, more than 15 ml each time, the antiserum is diluted with an equal volume of PBS, centrifuged to obtain the supernatant, and the antigen affinity column is washed with 10 times the volume of PBS to balance the column, the diluted antiserum is added to the balanced column, gently mixed and shaken at room temperature for 2-4 hours, or at 4°C overnight, the antigen column is washed with 10 times the volume of PBS to wash away the foreign proteins bound to the column, and the column is washed with 2 times the volume of antibody elution solution to obtain specific antibodies, concentrated with sucrose or polyethylene glycol, dialyzed in PBS for desalting, and added with 40-50% glycerol and stored at -20°C for a long time; A colloidal gold test strip, comprising: Colloidal gold, by taking 100ml ultrapure water and pouring it into a 250ml conical flask, adding 1.0ml of 1.0% chloroauric acid solution, placing it in a magnetic stirrer and stirring it continuously and heating it to boiling, and quickly adding 1.8ml of 1.0% trisodium citrate solution while stirring, the solution changes from light yellow to gray-black and finally to orange-red. After the solution changes to orange-red, continue heating for 15 minutes, cool to room temperature, adjust the solution pH to about 6.5 with 0.1mol / mlK2CO3, dilute it to 100ml with ultrapure water, and place it in a brown bottle and store it at 4°C for later use; After incubating the labeled antibody with colloidal gold solution, the gold-labeled complex was sprayed on glass cellulose and dried at 37°C; The sample pad treatment solution was dip-coated on the glass cellulose membrane and dried at 37°C; Stick the nitrocellulose membrane (NC membrane) on the PVC base plate, adjust the C line (sheep anti-mouse lgM) and T line (coated antibody) of the impression machine to a suitable distance, and then make impressions, and dry them at 37°C; Cut the absorbent pad, prepared gold pad and sample pad to the appropriate width, and stick them on the PVC board together with the nitrocellulose membrane with printed C and T lines. Cut the assembled large board into test strips of a certain width according to the width of the test card slot. The cutting width should be uniform, without indentations or scratches.

2. The rapid test kit for residual blood according to claim 1, characterized in that: The detection sensitivity of the kit for blood residues is as low as 50 ng / mL.

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