Rapid Detection Kit for Bacterial Endotoxins from Recombinant Hepatitis B Vaccine and its Usage

Abstract

This invention provides a rapid detection kit for bacterial endotoxins in recombinant hepatitis B vaccine and its usage method. The kit includes the following items: one working standard for bacterial endotoxins, 10 Limulus amebocyte lysate (LAL) reagents, one 5ml vial of 1×PBS buffer solution (Solution A), one 50ml vial of water for bacterial endotoxin testing (BET) solution (Solution B), six pyrogen-free glass test tubes, two test tube racks, one ampoule opener, two sealing films, and one instruction manual. This kit is designed for the bacterial endotoxin test of recombinant hepatitis B vaccine (CHO cells). The solution concentration is pre-set according to the limits specified in the pharmacopoeia, eliminating the need for testing personnel to recalculate the dilution steps and solution concentrations according to the limits. The test can be completed by simply adding samples according to the instructions. The test is easy to repeat and suitable for personnel with or without experience in endotoxin testing.

Description

Technical Field

[0001] This invention belongs to the field of medical testing device technology, specifically relating to a rapid detection kit for bacterial endotoxins from recombinant hepatitis B vaccine and its usage method. Background Technology

[0002] 1. Currently, there are no similar rapid bacterial endotoxin test kits specifically designed for recombinant hepatitis B vaccine (CHO cells) on the domestic market. 2. The bacterial endotoxin standards and water for bacterial endotoxin testing (BET water) used in the gel assay for recombinant hepatitis B vaccine (CHO cells) as specified in the pharmacopoeia are general-purpose standards and solvents, applicable to the detection of bacterial endotoxin content in various drugs, and not specific to recombinant hepatitis B vaccine. 3. Existing detection methods require a high level of operator skill. If operators are not familiar with the bacterial endotoxin detection method, it is difficult to accurately calculate the dilution ratio of standards and solvents, leading to false positives or false negatives and affecting the interpretation of product test results. 4. Currently, the dilution of test samples in bacterial endotoxin detection is mostly done manually. Human error is easily introduced in multi-stage dilution steps, affecting the accuracy of bacterial endotoxin detection. Summary of the Invention

[0003] In view of the above-mentioned defects or deficiencies in the prior art, in order to solve the problems existing in the prior art, the present invention provides a rapid detection kit for bacterial endotoxins of recombinant hepatitis B vaccine and its usage method.

[0004] The main technical solution is: a rapid detection kit for bacterial endotoxins in recombinant hepatitis B vaccine. The kit includes the following items: 1 working standard for bacterial endotoxins, 10 Limulus amebocyte lysate (LAL) reagents, Solution A: 1×PBS buffer solution (5ml / vial), Solution B: water for bacterial endotoxin test (BET) (50ml / bottle), 6 pyrogen-free glass test tubes, 2 test tube racks, 1 ampoule opener, 2 sealing films, and 1 instruction manual.

[0005] Furthermore, the bacterial endotoxin working standard has a specification of 4 EU / vial, the horseshoe crab reagent has a specification of 0.25 EU / ml and 0.1 ml / vial, and the pyrogen-free glass test tube has a specification of 8×150 mm.

[0006] The method for using the rapid bacterial endotoxin detection kit for recombinant hepatitis B vaccine includes the following steps:

[0007] (1) Open 10 ampoules of horseshoe crab reagent and add 0.10 ml of solution B to each ampoule to reconstitute;

[0008] (2) Prepare five groups of solutions: A, B, D, E, and F. Take 0.10 ml of each group and add it to the reconstitution reagent. Prepare two tubes for each group of solutions.

[0009] (3) Place the ampoule test tube racks according to different layouts, cover the test tube racks with sealing film, and incubate them in a thermostat at 37℃±1℃ for 60min±2min.

[0010] (4) After the incubation is complete, gently remove the test tube from the thermostat and slowly invert it 180° to determine the result.

[0011] Furthermore, the prepared solutions include a 1.0 ml:20 μg solution and a 0.5 ml:10 μg solution. The 1.0 ml:20 μg solution was prepared as follows:

[0012] Solution A (negative control 1): 1×PBS buffer (10 mmol / L);

[0013] Solution B (negative control 2): ​​Water for bacterial endotoxin testing (BET water);

[0014] Solution C (standard solution): Add 4.0 ml of solution A to one vial of bacterial endotoxin working standard (4 EU / vial), reconstitute, and vortex for 15 min for later use. Vortex for 30 s before each use.

[0015] Solution D (positive solution for the test sample): 0.10 ml sample + 0.30 ml solution A + 1.6 ml solution B + 2.0 ml solution C. Add the solutions in sequence, mixing for 30 seconds after each step.

[0016] Solution E (test solution): 0.10 ml sample + 0.30 ml solution A + 3.6 ml solution B. Add the solutions sequentially, mixing for 30 seconds after each step.

[0017] Solution F (positive control): 0.20 ml of solution B + 0.20 ml of solution C, mix for 30 seconds.

[0018] Prepare a solution with a sample specification of 0.5 ml: 10 μg.

[0019] Solution D (positive solution for the test sample): 0.05 ml sample + 0.15 ml solution A + 1.8 ml solution B + 2.0 ml solution C. Add the solutions in sequence, mixing for 30 seconds after each step.

[0020] Solution E (test solution): 0.05 ml sample + 0.15 ml solution A + 3.8 ml solution B. Add the solutions in sequence, mixing for 30 seconds after each step.

[0021] The beneficial effects of this invention are as follows:

[0022] This kit is specifically designed for the detection of bacterial endotoxins in recombinant hepatitis B vaccine (CHO cells). While meeting the requirements of General Chapter 1143 "Bacterial Endotoxin Test Method" in the Chinese Pharmacopoeia, it eliminates the calculation steps for testing personnel, reduces the complexity of experimental operations and dilution procedures, and maximally simplifies the detection of bacterial endotoxins while improving detection efficiency. It also uses bacterial endotoxin standards with lower concentrations, reducing consumption by 50 times compared to traditional standards, thus lowering detection costs, and is particularly suitable for single-batch, small-sample testing. This kit reduces environmental pollution and unnecessary actual resource consumption. Detailed Implementation

[0023] The present application will now be described in further detail. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit the invention.

[0024] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The application will now be described in detail.

[0025] Example:

[0026] This kit is designed for the bacterial endotoxin test of recombinant hepatitis B vaccine (CHO cells). The solution concentration is pre-set according to the limits specified in the pharmacopoeia, eliminating the need for laboratory personnel to recalculate the dilution steps and solution concentration according to the limits. The test can be completed by simply adding the sample according to the instructions. The test is easy to repeat and suitable for personnel with or without experience in endotoxin testing.

[0027] reagent kit contents:

[0028] 1. One vial of bacterial endotoxin working standard (specification: 4 EU / vial)

[0029] 2. 10 vials of horseshoe crab reagent (specification: 0.25 EU / ml, 0.1 ml / vial)

[0030] 3. Solution A: 1 vial of 1×PBS buffer (5ml / vial)

[0031] 4. Solution B: One bottle (50ml / bottle) of water for bacterial endotoxin test (BET).

[0032] 5. Six pyrogen-free glass test tubes (size: 8×150mm)

[0033] 6. Two test tube racks

[0034] 7. One bottle opener

[0035] 8. Two sheets of sealing film

[0036] 9. One instruction manual

[0037] manual:

[0038] The "Rapid Detection Kit for Bacterial Endotoxins in Recombinant Hepatitis B Vaccine (CHO Cells)" is specifically designed for the rapid detection of bacterial endotoxins in recombinant hepatitis B vaccine (CHO cell) products. The detection principle and judgment criteria of this kit are based on General Chapter 1143 "Bacterial Endotoxin Test Method - Gel Method" in the 2020 edition of the Chinese Pharmacopoeia, and the detection technology meets relevant requirements and regulations.

[0039] The gel electrophoresis method is a method for the limit detection or semi-quantitative detection of endotoxins based on the principle that horseshoe crab reagent reacts with endotoxins to produce agglutination.

[0040] How to use:

[0041] Prepare AF solution as follows:

[0042] Sample specification (1): 1.0ml:20μg

[0043] Solution A (Negative Control 1): 1×PBS buffer (10 mmol / L)

[0044] Solution B (Negative Control 2): ​​Water for Bacterial Endotoxin Testing (BET Water)

[0045] Solution C (standard solution): Reconstitute one vial (4 EU / vial) of bacterial endotoxin working standard with 4.0 ml of solution A, vortex for 15 min, and set aside. Vortex for 30 s before each use.

[0046] Solution D (positive solution for the test sample): 0.10 ml sample + 0.30 ml solution A + 1.6 ml solution B + 2.0 ml solution C. Add the solutions in sequence, mixing for 30 seconds after each step.

[0047] Solution E (test solution): 0.10 ml sample + 0.30 ml solution A + 3.6 ml solution B. Add the solutions in sequence, mixing for 30 seconds after each step.

[0048] F solution (positive control): 0.20 ml of B solution + 0.20 ml of C solution, mix well for 30 seconds.

[0049] Sample specification (II): 0.5ml:10μg

[0050] Except for solutions D and E, solutions A, B, C, and F are the same as those in sample (I).

[0051] Solution D (positive solution for the test sample): 0.05 ml sample + 0.15 ml solution A + 1.8 ml solution B + 2.0 ml solution C. Add the solutions in sequence, mixing for 30 seconds after each step.

[0052] Solution E (test solution): 0.05 ml sample + 0.15 ml solution A + 3.8 ml solution B. Add the solutions in sequence, mixing for 30 seconds after each step.

[0053] Open 10 ampoules of horseshoe crab reagent. Add 0.10 ml of solution B to each ampoule to reconstitute the solution. For the five groups of solutions A, B, D, E, and F, add 0.10 ml of each group to the reconstituted horseshoe crab reagent. Prepare two tubes for each group of solutions. Place the tubes in an ampoule rack according to the layout in Table 1. Seal the rack with sealing film and incubate at 37℃±1℃ for 60 min±2 min. After incubation, gently remove the tubes from the incubator and slowly invert them 180°. If a gel forms inside the tube and does not deform or slip off the tube wall, the result is positive. If no gel forms or the formed gel is not firm, deformed, or slips off the tube wall, the result is negative. Avoid vibration during the incubation and handling of the tubes to prevent false negative results.

[0054] The judgment criteria are shown in Table 1. If the results differ from those in Table 1, a retest should be considered.

[0055] Table 1 Layout and Judgment Criteria

[0056]

[0057] Note: "+" indicates a positive result, indicating that gel is formed and the gel will not slip when inverted 180°; "-" indicates a negative result, indicating that no gel is formed or the gel does not form and the gel will slip when inverted 180°.

[0058] 1. Precautions:

[0059] (1) Storage conditions for the kit: Store at 2-8℃ away from light and avoid shaking.

[0060] (2) The reagent must be used within 24 hours after opening.

[0061] (3) All equipment that comes into contact with the test sample, standard and diluent must be pyrogen-free.

[0062] (4) The specified solution vortex mixing time must be strictly followed, otherwise it may affect the experimental results.

[0063] The bacterial endotoxin test is the legally mandated quality control test method for pyrogenic substances in the current edition of the Chinese Pharmacopoeia. The gel electrophoresis method is the arbitration method and is widely used for various traditional Chinese medicines, chemical drugs, antibiotics, vaccines, and other biological products. Traditional gel electrophoresis requires technicians with many years of experience; the calculation and dilution steps are complex, and the operation is strictly controlled. It demands a high level of expertise from technicians and a suitable testing environment, making it unsuitable for testing by units or personnel outside of pharmaceutical testing institutions.

[0064] This kit is specifically designed for the detection of bacterial endotoxins in recombinant hepatitis B vaccine (CHO cells). While meeting the requirements of General Chapter 1143 "Bacterial Endotoxin Test Method" in the Chinese Pharmacopoeia, it eliminates the calculation steps for testing personnel, reduces the complexity of experimental operations and dilution procedures, and maximally simplifies the detection of bacterial endotoxins while improving detection efficiency. It also uses bacterial endotoxin standards with lower concentrations, reducing consumption by 50 times compared to traditional standards, thus lowering detection costs, and is particularly suitable for single-batch, small-sample testing. This kit reduces environmental pollution and unnecessary actual resource consumption.

[0065] The implementation plan is based on the "Bacterial Endotoxin Test Method" (General Chapter 1143, Chinese Pharmacopoeia 2020 Edition) - Gel Method. The traditional gel method for bacterial endotoxin detection is as follows:

[0066] The maximum effective dilution (MVD) of this product was calculated according to General Chapter 1143, "Test Method for Bacterial Endotoxins," in the 2020 edition of the Chinese Pharmacopoeia. The maximum effective dilution (MVD) refers to the maximum dilution the test solution is allowed to reach in the test, at which the endotoxin limit is determined. The MVD is determined using the following formula:

[0067] MVD = cL / λ

[0068] In the formula, L represents the bacterial endotoxin limit of the test sample;

[0069] c represents the concentration of the test solution. When L is expressed in EU / mg or EU / U, the unit of c must be mg / ml or U / ml. When L is expressed in EU / ml, c equals 1.0 ml / ml. To calculate the test concentration at MVD, i.e., the minimum effective dilution concentration, the formula L = λ / L can be used. λ is the labeled sensitivity (EU / ml) of the Limulus Amebocyte Lysate (LAL) reagent in the gel electrophoresis method, or the lowest endotoxin concentration on the standard curve used in the photometric assay.

[0070] This product is detected using a 0.25 EU / ml sensitivity Limulus amebocyte lysate (LAL) reagent. The limit for bacterial endotoxin detection should be less than 10 EU / dose. The specifications are test sample A 1.0 ml: 20 μg and test sample B 0.5 ml: 10 μg. Therefore, the MVD is calculated as follows:

[0071] Test sample A MVD = 1.0 ml / ml · 10 EU / dose / 1 ml / dose / 0.25 EU / ml = 40-fold dilution

[0072] The MVD of test sample B is 1.0 ml / ml·10 EU / dose / 0.5 ml / dose / 0.25 EU / ml, which is 80 times dilution. Therefore, the preparation method of the test sample solution is as follows:

[0073] Number A:

[0074] Sample A:

[0075] Take 0.50 ml of the test sample and add 1.5 ml of 1×PBS buffer to obtain a 4-fold diluted solution ①.

[0076] Take 0.50 ml of solution ① and add 2.0 ml of BET water to obtain solution ②, which is diluted 20 times.

[0077] Take 0.50 ml of solution ② and add 0.50 ml of BET water to obtain solution ③, which is diluted 40 times.

[0078] B. Test sample:

[0079] Take 0.40 ml of the test sample and add 1.2 ml of 1×PBS buffer to obtain a 4-fold diluted solution ①.

[0080] Take 0.20 ml of solution ① and add 1.8 ml of BET water to obtain solution ②, which is diluted 40 times.

[0081] Take 0.50 ml of solution ② and add 0.50 ml of BET water to obtain solution ③, which is diluted 80 times.

[0082] Bacterial endotoxin working standard solution:

[0083] Take one vial of bacterial endotoxin working standard (100 EU / vial) and redissolve it with 1.0 ml of BET and water to obtain solution (1) with a concentration of 100 EU / ml.

[0084] Take 0.40 ml of solution (1) and add 3.6 ml of BET water to obtain solution (2) with a concentration of 10 EU / ml.

[0085] Take 1.0 ml of solution (2) and add 9.0 ml of BET water to obtain solution (3) at 1 EU / ml.

[0086] Note: Vortex mixing in the first step should last for at least 15 minutes, and vortex mixing in each subsequent step should last for at least 30 seconds.

[0087] Number B: Positive solution for test sample:

[0088] Take 0.40 ml of solution ② of test sample A or B and add 0.40 ml of bacterial endotoxin standard solution ⑶.

[0089] Solution C: Positive control solution

[0090] Take 0.40 ml of bacterial endotoxin standard solution (3) and add 0.40 ml of BET water.

[0091] Number D: Negative control 1: 1×PBS buffer (10 mmol / L); Negative control 2: BET water.

[0092] Take 0.1 ml of each of the above groups and add it to the horseshoe crab reagent that has been reconstituted with 0.1 ml of BET water. Take 2 tubes of each group, as shown in Table 2.

[0093] Table 2. Preparation of solutions for gel limit test

[0094] serial number Endotoxin concentration / endotoxin solution preparation Number of flat tubes A None / Test solution 2 B 2λ / test solution 2 C 2λ / BET water 2 D No / BET water 2

[0095] Note: A is the test solution; B is the positive control; C is the positive control; D is the negative control.

[0096] Result judgment

[0097] Observe the results after incubation for 60 min ± 2 min. If all parallel tubes of negative control solution D are negative, all parallel tubes of positive control solution B are positive, and all parallel tubes of positive control solution C are positive, the test is valid.

[0098] If both parallel tubes of solution A are negative, the test sample is deemed compliant. If both parallel tubes of solution A are positive, the test sample is deemed non-compliant. If one of the two parallel tubes of solution A is positive and the other is negative, a retest is required. For the retest, four parallel tubes of solution A must be prepared. If all parallel tubes are negative, the test sample is deemed compliant; otherwise, the test sample is deemed non-compliant.

[0099] If the dilution factor of the test sample is less than the MVD and the result of solution A does not meet the requirements, the test sample can be diluted to the MVD and the experiment can be repeated, and the result can be judged again.

[0100] The above description is merely a preferred embodiment of this application and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in this application is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the inventive concept. For example, technical solutions formed by substituting the above features with (but not limited to) technical features with similar functions disclosed in this application.

Claims

1. A method for using a rapid detection kit for bacterial endotoxins in recombinant hepatitis B vaccine, characterized in that... Includes the following steps: (1) Open 10 ampoules of horseshoe crab reagent and add 0.10 ml of solution B to each ampoule to reconstitute; (2) Prepare five groups of solutions: A, B, D, E, and F. Take 0.10 ml of each group and add it to the reconstitution reagent. Prepare two tubes for each group of solutions. (3) Place the ampoule test tube racks according to different layouts, cover the test tube racks with sealing film, and incubate them in a thermostat at 37℃±1℃ for 60min±2min; (4) After the incubation is complete, gently remove the test tube from the thermostat and slowly invert it 180° to determine the result; In step (2), the solution includes a solution with a sample specification of 1.0 ml: 20 μg and a solution with a sample specification of 0.5 ml: 10 μg. The solution with a sample specification of 1.0 ml: 20 μg is prepared as follows: Negative control solution A: 1:1×PBS buffer 10mmol / L; Negative control 2 (Resolution B): Water for bacterial endotoxin testing; C solution standard solution: Add 4.0 ml of solution A to one vial of bacterial endotoxin working standard, reconstitute, and vortex for 15 min for later use. Vortex for 30 s before each use. D solution for positive test sample: 0.10 ml sample + 0.30 ml solution A + 1.6 ml solution B + 2.0 ml solution C, add the solutions in sequence, and mix for 30 seconds after each step; E solution for test sample: 0.10 ml sample + 0.30 ml solution A + 3.6 ml solution B, add the solutions in sequence, and mix for 30 seconds after each step; F solution positive control solution: 0.20 ml of B solution + 0.20 ml of C solution, mix well for 30 seconds; The sample was prepared with a specification of 0.5 ml: 10 μg as follows: D solution is the positive solution for the test sample: 0.05 ml of sample + 0.15 ml of solution A + 1.8 ml of solution B + 2.0 ml of solution C. Add the solutions in sequence and mix for 30 seconds after each step. E solution for test sample: 0.05 ml sample + 0.15 ml solution A + 3.8 ml solution B, add the solutions in sequence, and mix for 30 seconds after each step; The remaining solutions are as follows: Negative control 1 (Solution A): 1×PBS buffer 10 mmol / L; Negative control 2 (Solution B): Water for bacterial endotoxin testing; Standard solution (Solution C): Reconstitute one vial of bacterial endotoxin working standard with 4.0 ml of Solution A, vortex for 15 min, and vortex again for 30 s before each use; Positive control solution (Solution F): Mix 0.20 ml of Solution B with 0.20 ml of Solution C for 30 s. All solutions have a specification of 1.0 ml: 20 μg.

2. The method of using the rapid detection kit for bacterial endotoxins in recombinant hepatitis B vaccine according to claim 1, characterized in that: The kit includes the following items: 1 vial of bacterial endotoxin working standard, 10 vials of horseshoe crab reagent, 1 vial of solution A: 1×PBS buffer, 1 bottle of solution B: water for bacterial endotoxin testing, 6 pyrogen-free glass tubes, 2 test tube racks, 1 ampoule opener, 2 sealing films, and 1 instruction manual.

3. The method of using the rapid detection kit for bacterial endotoxins in recombinant hepatitis B vaccine according to claim 2, characterized in that: The bacterial endotoxin working standard is 4 EU / vial, the horseshoe crab reagent is 0.25 EU / ml and 0.1 ml / vial, and the pyrogen-free glass test tube is 8×150 mm.

Images