Rapid detection kit for protein residues

Abstract

The invention discloses a rapid detection kit for protein residues. The rapid detection kit comprises a labeled antibody, a coated antibody and a colloidal gold test strip. The labeled antibody is prepared by treating human intestinal epithelial cells and then immunizing a BALB / c mouse through a series of steps; and the coated antibody is prepared by treating human intestinal epithelial cells, immunizing New Zealand white rabbits and purifying in a subsequent process. The colloidal gold test strip is prepared into colloidal gold through a specific method, the labeled antibody and the colloidal gold are incubated, sprayed and dried, a sample pad is processed and then dried, impression and drying are conducted on a PVC bottom plate pasted with an NC film, and then cutting and assembling are conducted. The kit is high in detection speed and meets the instrument turnover requirement; the accuracy is high, and misjudgment is reduced; the method has the advantages that the sensitivity is as low as 100ng / mL, trace protein residues can be accurately detected, the risks of endoscopic infection and the like are reduced, and the method has obvious advantages in the aspect of protein residue detection of medical instruments and has a great application value.

Description

Technical Field

[0001] The invention relates to a rapid detection kit for protein residues. Background Art

[0002] After use, endoscopes come into contact with various contaminants, mainly including blood, sugars, fats, and proteins, especially protein-based mucopolysaccharides. If these contaminants are not cleaned in time, they will dry up on the endoscope and form biofilms, which will not only increase the difficulty of cleaning, but may also lead to disinfection failure. Biofilms contain about 85% polysaccharide matrix and 15% bacterial clusters, which can greatly reduce the effectiveness of disinfectants and are an important source of endoscope-borne infection. Therefore, timely and effective cleaning is the most effective way to prevent the formation of biofilms. The importance of cleaning is reflected in the following aspects: (1) Preventing biofilm formation: Timely cleaning can reduce the formation of biofilms, thereby reducing the risk of endoscope-borne infection. (2) Improving disinfection effect: Cleaned endoscopes are easier to disinfect because cleaning can remove most contaminants, allowing disinfectants to work more effectively. (3) Extending the service life of endoscopes: Regular cleaning and maintenance can reduce damage to endoscopes and extend their service life. (4) Ensuring patient safety: Cleaning and disinfection are important measures to prevent cross-infection and ensure the safety of patients undergoing endoscopic examinations.

[0003] (5) Compliance with medical standards: According to the Technical Specifications for Cleaning and Disinfection of Flexible Endoscopes WS 507-2016, all flexible endoscopes should be thoroughly cleaned and disinfected or sterilized at a high level after each use. (6) Avoiding delays or cancellations of surgery: If the endoscope cleaning step is omitted, any remaining debris may cause the chemical disinfectant or sterilization process to fail or interfere, and delayed treatment may lead to delays or cancellations of surgery. Therefore, cleaning of endoscopes after use is very important, which is not only related to the performance and service life of the endoscope, but also to the safety of patients and the quality of medical care.

[0004] The detection methods of protein residues after endoscope use mainly include the following. (1) Chemical colorimetric method: This is a method that generates colored compounds by the reaction of proteins with specific chemical reagents, and then determines their concentration by colorimetry. It is simple to operate and low in cost, but has low sensitivity and accuracy. (2) Enzyme-linked immunosorbent assay (ELISA): This is a highly sensitive detection method that detects the content of protein residues in medical devices through antigen-antibody reaction. It has high sensitivity and strong specificity, and is suitable for detecting trace amounts of protein residues in medical devices, but the operation steps are complicated and the cost is high. (3) Liquid chromatography: This is a method for efficiently separating and detecting protein residues in medical devices. Proteins are separated by a liquid chromatography column, and then their concentration is determined by a detector. It has the advantages of high sensitivity and high accuracy, but requires professional equipment and technical support, and is relatively expensive. Summary of the invention

[0005] The purpose of the present invention is to provide a protein residue rapid detection kit to solve the problems raised in the above background technology.

[0006] In order to solve the above technical problems, the present invention provides a technical solution: a protein residue rapid detection kit, comprising:

[0007] The labeled antibody is prepared by the following method:

[0008] Human intestinal epithelial cells were obtained, and the red blood cells were washed 2-3 times with ice-cold phosphate buffered saline (PBS), and the supernatant was discarded after each washing by centrifugation. The washed red blood cells were resuspended in an appropriate amount of ice-cold RIPA lysis buffer, and an appropriate amount of protease inhibitor PMSF was added and gently mixed. After incubation on ice for 30 minutes, the lysate was centrifuged at 12000 rpm at 4°C for 5-10 minutes, and the supernatant was collected;

[0009] 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.

[0010] CNBr-Activated Sepharose 4B 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, 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 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;

[0011] The coating antibody is prepared by the following method:

[0012] Human intestinal epithelial cells were taken, and the red blood cells were washed 2-3 times with ice-cold PBS. After each wash, the supernatant was discarded by centrifugation. The washed red blood cells were resuspended in an appropriate amount of ice-cold RIPA lysis buffer, and an appropriate amount of protease inhibitor PMSF was added and gently mixed. After incubation on ice for 30 minutes, the lysate was centrifuged at 12000 rpm at 4°C for 5-10 minutes, and the supernatant was collected;

[0013] 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.

[0014] CNBr-Sepharose 4B gel is used to couple with antigen, 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, the medium is 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, 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, 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, added with 40-50% glycerol and stored at -20°C for a long time;

[0015] A colloidal gold test strip, comprising:

[0016] Colloidal gold, take 100ml ultrapure water and pour it into a 250ml conical flask, add 1.0ml of 1.0% chloroauric acid solution, place it in a magnetic stirrer, stir it continuously and heat it to boiling, quickly add 1.8ml of 1.0% trisodium citrate solution while stirring, the solution changes from light yellow to gray-black and finally to orange-red, continue heating for 15 minutes after the solution turns orange-red, cool to room temperature, adjust the solution pH to about 6.5 with 0.1mol / ml K2CO3, dilute to 100ml with ultrapure water, place it in a brown bottle and store it at 4℃ for later use;

[0017] After incubating the labeled antibody with colloidal gold solution, the gold-labeled complex was sprayed on glass cellulose and dried at 37°C;

[0018] The sample pad treatment solution was dip-coated on the glass cellulose membrane and dried at 37°C;

[0019] 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;

[0020] 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.

[0021] Preferably, the detection sensitivity of the kit for protein residues is as low as 100 ng / mL.

[0022] The advantages of the present invention are: fast detection speed: the detection results can be obtained quickly, meeting the actual needs of rapid turnover of medical equipment and effectively improving the detection efficiency.

[0023] High accuracy: Compared with traditional chemical colorimetric methods, it reduces misjudgments caused by insufficient sensitivity and specificity, and provides more reliable data support for the evaluation of the cleaning effect of medical devices.

[0024] High sensitivity: The minimum detection sensitivity can reach 100ng / mL, which can accurately detect trace amounts of protein residues, greatly reducing medical risks such as endoscopic infection caused by protein residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a photo of the sensitivity test of the colloidal gold test strip. DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] 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.

[0029] Example 1: Preparation of labeled antibodies

[0030] Take human intestinal epithelial cells (purchased from Shanghai Huzhen Industrial 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. This part of the liquid contains the extracted protein. Transfer it to a new centrifuge tube.

[0031] 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.

[0032] Example 2: Preparation of coated antibodies

[0033] Take human intestinal epithelial cells (purchased from Shanghai Huzhen Industrial 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. This part of the liquid contains the extracted protein. Transfer it to a new centrifuge tube.

[0034] 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.

[0035] Example 3: Preparation of colloidal gold test strips

[0036] 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.

[0037] 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.

[0038] Example 4: Sensitivity test of colloidal gold test strips

[0039] Take the extracted epithelial cell protein and dilute it to 500, 400, 300, 200, 100, 50, 0 ng / mL using phosphate buffer. Take 80uL of each and spot it on the prepared colloidal gold test strips. After 10 minutes of rest, observe the results. The results are as follows Figure 1 As shown, except for the 50 and 0 ng / mL test strips, the T lines of the other test strips were able to produce bands, indicating that the test strips were successfully prepared with a sensitivity as low as 100 ng / mL.

[0040] Example 5: Application of colloidal gold test strips for rapid detection of protein residues

[0041] Two methods were used to detect the cleaning results of medical devices in hospitals, namely the chemical method and the colloidal gold test strip method of the present invention. The results are shown in the following table. The two methods each tested 200 cases, of which 16 and 23 were positive results, respectively, indicating that the colloidal gold test strip has a better detection effect.

[0042] method Number of testing equipment Number of positive results Chemical method 200 16 Colloidal gold test strips 200 23

[0043] 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 protein residue rapid detection kit, characterized in that: include: The labeled antibody is prepared by the following method: Human intestinal epithelial cells were obtained, and the red blood cells were washed 2-3 times with ice-cold phosphate buffered saline (PBS), and the supernatant was discarded after each washing by centrifugation. The washed red blood cells were resuspended in an appropriate amount of ice-cold RIPA lysis buffer, and an appropriate amount of protease inhibitor PMSF was added and gently mixed. After incubation on ice for 30 minutes, the lysate was centrifuged at 12000 rpm at 4°C for 5-10 minutes, and the supernatant was collected; 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 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 antibody elution solution to obtain the specific antibody, which is concentrated and dialyzed in PBS for desalting, and 40-50% glycerol is added and stored at -20°C for a long time; The coating antibody is prepared by the following method: Human intestinal epithelial cells were taken, and the red blood cells were washed 2-3 times with ice-cold PBS. After each wash, the supernatant was discarded by centrifugation. The washed red blood cells were resuspended in an appropriate amount of ice-cold RIPA lysis buffer, and an appropriate amount of protease inhibitor PMSF was added and gently mixed. After incubation on ice for 30 minutes, the lysate was centrifuged at 12000 rpm at 4°C for 5-10 minutes, and the supernatant was collected; 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-Sepharose 4B gel is used to couple with antigen, 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, the medium is 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, 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, 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, added with 40-50% glycerol and stored at -20°C for a long time; A colloidal gold test strip, comprising: Colloidal gold, take 100ml ultrapure water and pour it into a 250ml conical flask, add 1.0ml of 1.0% chloroauric acid solution, place it in a magnetic stirrer, stir it continuously and heat it to boiling, quickly add 1.8ml of 1.0% trisodium citrate solution while stirring, the solution changes from light yellow to gray-black and finally to orange-red, continue heating for 15 minutes after the solution turns orange-red, cool to room temperature, adjust the solution pH to about 6.5 with 0.1mol / ml K2CO3, dilute to 100ml with ultrapure water, place it in a brown bottle and store it at 4℃ 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 protein residue rapid detection kit according to claim 1, characterized in that: The detection sensitivity of the kit for protein residues is as low as 100 ng / mL.

Images