CN115236337A

CN115236337A - Improved limulus kit for detecting industrial endotoxin based on dynamic color development method, and use method and application thereof

Info

Publication number: CN115236337A
Application number: CN202211147116.9A
Authority: CN
Inventors: 刘浩宇; 盛长忠; 粟艳; 周泽奇
Original assignee: Dynamiker Biotechnology Tianjin Co Ltd
Current assignee: Dynamiker Biotechnology Tianjin Co Ltd
Priority date: 2022-09-21
Filing date: 2022-09-21
Publication date: 2022-10-25

Abstract

The invention provides an improved industrial endotoxin detection limulus kit based on a dynamic color development method, and a use method and application thereof. The improved limulus kit for detecting industrial endotoxin based on the dynamic color development method comprises a reaction main agent; the reaction main agent comprises a factor C, a factor B, prothrombin, a polypeptide chromogenic substrate, laminarin, a freeze-dried excipient and a permeation stabilizer. The reaction principle of the kit is that the polypeptide chromogenic substrate is digested by coagulase to generate free 2,4-dinitroaniline, and the free 2,4-dinitroaniline is directly detected by an enzyme-labeled instrument, so that the detection route can be shortened and the cost can be reduced by using the kit for detection, compared with the traditional PNA chromogenic substrate method, the kit has the advantages that one more nitro group is added to the structure of the 2,4-dinitroaniline, the color is darker and the sensitivity is higher after the reaction; and the reaction main agent added with the laminarin is not easy to be interfered by protein and medicine in the sample to generate nonspecific turbidity.

Description

Improved limulus kit for detecting industrial endotoxin based on dynamic color development method, and use method and application thereof

Technical Field

The invention belongs to the technical field of manufacturing of detection reagent kits, and particularly relates to an improved limulus kit for detecting industrial endotoxin based on a dynamic color development method, and a use method and application thereof.

Background

Endotoxin is a general term for toxic substances in gram-negative bacteria, is a cell wall component of various gram-negative bacteria, is released after the bacteria are cracked, is also called as pyrogen, and has lipopolysaccharide as a main chemical component. The lipopolysaccharide molecule consists of thallus specific polysaccharide, nonspecific core polysaccharide and lipoid A, and the toxic component is lipoid A. The toxic effects of various bacterial endotoxins are almost the same, and they are mainly manifested as fever, microcirculation disturbance, endotoxin shock, disseminated intravascular coagulation and the like.

Because the endotoxin is generated by bacterial death and lysis or autolysis, a large amount of endotoxin exists in the environment, the endotoxin is harmless when entering a human body through the digestive tract of the body, and a small amount of endotoxin is inactivated by Kupffer Cells (KC) after entering blood through injection, so that the damage to the body can not be caused. When endotoxin enters and accumulates in the blood in large amounts, which exceed the clearance capacity of the body's respective defence systems, varying degrees of endotoxemia can result. Therefore, endotoxin must be detected for injection drugs, biological products, medical instruments and the like, which are easily introduced with endotoxin.

Horseshoe crab blood contains two types of cells, one of which is granulocytes (granulocytes) or so-called amoebocytes (amebacty), which adult horseshoe crabs contain only. Limulus hemagglutination is similar to mammalian hemagglutination and is a cascade of enzymatic reactions. The limulus reagent is sterile lyophilized product prepared from hemocyte lysate of horseshoe crab, and contains factor C, factor B, prothrombin and coagulogen. Under appropriate conditions, bacterial endotoxin activates factor C, causing a series of enzymatic reactions that produce agglutination of the limulus reagents to form a gel.

According to the regulations of endotoxin test method 1143 on the general rule of "Chinese pharmacopoeia" 2020 edition, bacterial endotoxin test methods include two methods, namely, gel method and photometric method, the latter includes turbidity method and chromogenic substrate method.

The gel method is a method for limit detection or semi-quantitative detection of endotoxin by the principle that limulus reagent and endotoxin undergo agglutination reaction, but the method cannot perform quantification and is liable to cause false positive or false negative due to the influence of interfering substances in a sample to be tested.

The turbidity method is a method for measuring the content of endotoxin by using turbidity change in the reaction process of limulus reagent and endotoxin, and can be divided into an end-point turbidity method and a dynamic turbidity method according to the detection principle, but the reaction main agent of the method is easily interfered by protein and medicaments in a sample to be detected to generate nonspecific turbidity.

The chromogenic matrix method is a method for measuring the content of endotoxin by using coagulase generated in the reaction process of detecting limulus reagent and endotoxin to release p-nitrobenzoic acid from a chromogenic substrate, and is divided into an end-point chromogenic method and a dynamic chromogenic method according to a detection principle; the dynamic color development method is a method for detecting the reaction time required for the absorbance or transmittance of a reaction mixture to reach a certain preset detection value or the increase speed of the detection value, does not depend on the polymerization of coagulated protein into gel, and has high sensitivity and strong anti-interference capability.

Therefore, the kit is high in sensitivity and strong in anti-interference capability, is suitable for quantitatively detecting the bacterial endotoxin in samples such as injection medicines, biological products and medical instruments, and has important application prospects in the field of endotoxin detection.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide an improved limulus kit for detecting industrial endotoxin based on a dynamic color development method, and a use method and application thereof. The kit provided by the invention comprises a reaction main agent, a main agent complex solution, pyrogen-free water, a standard substance and a quality control substance; the laminarin is added into the reaction main agent, so that the anti-interference capability can be improved, and the detection sensitivity can be increased; the polypeptide chromogenic substrate is synthesized tripeptide or tetrapeptide of which the tail end is connected with 2,4-dinitroaniline through Boc-Leu-Gly-Arg, the polypeptide chromogenic substrate is digested by coagulase to generate free 2,4-dinitroaniline, and the free 2,4-dinitroaniline is directly detected by an enzyme labeling instrument.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides an improved limulus kit for industrial endotoxin detection based on dynamic color development, wherein the industrial endotoxin detection refers to endotoxin detection in the non-clinical fields of medical instruments, cell culture, biomedicine, vaccines, injections and the like, and the improved limulus kit for industrial endotoxin detection based on dynamic color development comprises a reaction main agent;

the main reaction agent comprises a factor C, a factor B, prothrombin, a polypeptide chromogenic substrate, laminarin, a freeze-dried excipient and a penetration stabilizer.

In the invention, the reaction main agent is added with the G factor inhibitor laminarin, and the laminarin can shield (1, 3)βInterference of the D-glucan by the reaction branches. The limulus blood cells contain factor C, factor G, factor B, prothrombin, coagulogen and the like, the factor C is activated by endotoxin in a sample to be detected to form an activated factor C, the factor C is activated to activate the factor B to form an activated factor B, the factor B is activated to convert the prothrombin into coagulase, the polypeptide chromogenic substrate is digested by the coagulase to generate free 2,4-dinitroaniline, and the 2,4-dinitroaniline has an obvious absorption peak at the wavelength of 405 nm; and the G factor can also be (1, 3)βThe D glucan is activated into an activated factor G, the activated factor G can also convert procoagulant enzyme into coagulase, and then the polypeptide chromogenic substrate is digested by the coagulase to generate free 2,4-dinitroaniline for color development, so that the bypass reaction has interference effect on the endotoxin activated factor C reaction branch. Addition of factor G inhibitors, i.e.(1,3)βthe-D glucan structural analogue laminarin can occupy the G factor activation site in advance, so that the subsequent reaction can not be carried out, thereby achieving the effect of inhibiting the branch reaction.

Preferably, the polypeptide chromogenic substrate comprises Boc-Leu-Gly-Arg-C ₆ H ₅ N ₃ O ₄ (2, 4-dinitroaniline) and/or Boc-Ile-Gly-Arg-C ₆ H ₅ N ₃ O ₄ (2, 4-dinitroaniline).

In the invention, the chromogenic group on the polypeptide chromogenic substrate is 2,4-Dinitroaniline (2, 4-Dinitroaniline), and compared with Paranitroaniline (PNA), the polypeptide chromogenic substrate of the invention has deeper chromogenic process and higher sensitivity, and the 2,4-Dinitroaniline polypeptide compound used in the invention has the structure shown in figure 1.

Preferably, the lyophilization excipient comprises polyvinylpyrrolidone, raffinose and mannitol in a mass ratio of (1.0-3.0): (0.2-0.8): (1.0-5.0), such as 1.0.

Preferably, the penetration stabilizer comprises sucrose, glycerol and sodium chloride in a mass ratio of (1.5-3.0): (3.0-5.0): (1.0-1.5), and may be, for example, 1.5.

According to the invention, the reaction main agent is added with the freeze-drying excipient, mannitol can endow a freeze-drying preparation skeleton, polyvinylpyrrolidone can endow freeze-drying preparation rigidity, raffinose can improve the solubility of the freeze-drying preparation, and in the invention, mannitol, polyvinylpyrrolidone and raffinose are combined and used in a specific proportion, and all components are matched with each other, so that the stability of the form of the freeze-drying preparation can be ensured, and the freeze-dried block is not easy to break and convenient to transport and store. The reaction main agent is also added with a permeation stabilizing agent, and the permeation stabilizing agent can avoid damage to protein active substances in the reaction main agent due to extreme conditions such as low temperature, vacuum and the like in the freeze-drying process.

As a preferred technical scheme of the invention, the improved industrial endotoxin detection limulus kit based on the dynamic color development method comprises the main reaction agents of factor C, factor B and prothrombin、Boc-Leu-Gly-Arg-C ₆ H ₅ N ₃ O ₄ Laminarin, polyvinylpyrrolidone, raffinose, mannitol, sucrose, glycerol and sodium chloride.

As a preferred technical scheme of the invention, the improved industrial endotoxin detection limulus kit based on the dynamic color development method comprises the following main reaction agents of factor C, factor B, proclotting enzyme and Boc-Ile-Gly-Arg-C ₆ H ₅ N ₃ O ₄ Laminarin, polyvinylpyrrolidone, raffinose, mannitol, sucrose, glycerol and sodium chloride.

Preferably, the reaction main agent is prepared by the following preparation method:

(1) Blood collection and cell isolation: placing blood collected from living Limulus cardia into a blood collection bottle containing anticoagulant, centrifuging, removing supernatant, and collecting Limulus blood cells;

(2) Cracking: mixing the limulus blood cells obtained in the step (1) with a RIPA lysate, freezing, and then thawing to obtain a limulus blood cell lysate;

(3) Extraction and separation: firstly, mixing the horseshoe crab hemocyte lysate obtained in the step (2) with a saturated ammonium sulfate solution, carrying out primary centrifugation to collect a supernatant I, discarding a precipitate I, then mixing the supernatant I with the saturated ammonium sulfate solution, carrying out secondary centrifugation to collect a precipitate II, and discarding a supernatant II; redissolving the precipitate, and dialyzing to remove ammonium sulfate to obtain extractive solution containing factor C, factor B and prothrombin;

(4) Preparation: and (4) mixing the extracting solution obtained in the step (3), the polypeptide chromogenic substrate, the laminarin, the freeze-dried excipient and the permeation stabilizer, stirring, and freeze-drying to obtain the reaction main agent.

Preferably, in step (1), the anticoagulant is 0.1-0.5 wt% sodium citrate aqueous solution, which may be 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, or the like, for example.

Preferably, in step (1), the volume ratio of the anticoagulant to the blood is 1 (5-9), and may be, for example, 1.

Preferably, in step (1), the rotation speed of the centrifugation is 600-1500 rpm, such as 600 rpm, 800 rpm, 1000 rpm, 1200 rpm or 1500 rpm, etc., and the time of the centrifugation is 15-25 min, such as 15 min, 18 min, 20 min or 25 min, etc.

In the present invention, the cardia of horseshoe crab is sterilized with 75vol% ethanol solution and/or iodine before blood collection.

In the invention, the specific steps of the step (1) are as follows: placing blood collected from a living body Limulus cardia into a blood collection bottle containing an anticoagulant, wherein the volume ratio of the anticoagulant to the blood is 1 (5-9); centrifuging at 600-1500 rpm for 15-25 min, discarding supernatant, and collecting limulus blood cells.

Preferably, in step (2), the volume ratio of the limulus blood cells to the RIPA lysate is 10 (5-9), and may be, for example, 10.

Preferably, in the step (2), the freezing comprises the following specific steps: freezing at-25 deg.C to-20 deg.C for 12 hr or more, wherein the freezing temperature can be-25 deg.C, -23 deg.C or-20 deg.C, and the freezing time can be 12 hr, 13 hr, 14 hr or 15 hr.

Preferably, in step (2), the RIPA lysate comprises: 20-30 mM Tris-HCl, 100-200 mM NaCl, 0.5-1 wt% NP-40, 0.5-1 wt% sodium deoxycholate and 0.1-0.15 wt% sodium dodecyl sulfate, wherein the solvent is pyrogen-free water, and the pH value of the RIPA lysate is 7.2-8.0.

In the present invention, the concentration of Tris-HCl is 20-30 mM, and may be, for example, 20 mM, 23 mM, 25 mM, 27 mM, 29 mM, or 30 mM.

In the present invention, the concentration of NaCl is 100 to 200 mM, and may be, for example, 100 mM, 130 mM, 150 mM, 170 mM, 190 mM, or 200 mM.

In the present invention, the concentration of NP-40 is 0.5 to 1% by weight, and may be, for example, 0.5%, 0.7%, 0.9%, 1% by weight, or the like.

In the present invention, the concentration of sodium deoxycholate is 0.5 to 1 wt%, and may be, for example, 0.5 wt%, 0.7 wt%, 0.9 wt%, or 1 wt%.

In the present invention, the concentration of the sodium lauryl sulfate is 0.1 to 0.15 wt%, and may be, for example, 0.1 wt%, 0.12 wt%, 0.14 wt%, 0.15 wt%, or the like.

In the present invention, the RIPA lysate has a pH of 7.2 to 8.0, and may be, for example, 7.2, 7.4, 7.6, 7.8, or 8.0. The RIPA lysate has good lysis effect, can accelerate cell disruption and lysate release, improve the extraction efficiency of horseshoe crab blood cells, and ensure the biological activity of horseshoe crab blood cell extracts.

In the invention, the specific steps of the step (2) are as follows: mixing the horseshoe crab blood cells and the RIPA lysate obtained in the step (1), wherein the volume ratio of the horseshoe crab blood cells to the RIPA lysate is 10 (5-9); freezing at-25-20 deg.C for more than 12 h, and thawing to obtain limulus blood cell lysate; wherein, the RIPA lysate comprises: 20-30 mM Tris-HCl, 100-200 mM NaCl, 0.5-1 wt% NP-40, 0.5-1 wt% sodium deoxycholate and 0.1-0.15 wt% sodium dodecyl sulfate, wherein a solvent is pyrogen-free water, and the pH value of a lysate is 7.2-8.0.

Preferably, in step (3), the pH of the saturated ammonium sulfate solution is 7.0-8.0, and may be, for example, 7.0, 7.2, 7.4, 7.5, 7.6, 7.8, or 8.0, and the concentration of the saturated ammonium sulfate solution is 100%.

Preferably, in step (3), the final concentration of ammonium sulfate in the first mixed solution is 20-30%, for example, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or the like.

Preferably, in step (3), the final concentration of ammonium sulfate in the mixed solution II is 55-65%, for example, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, or 65%.

Preferably, in step (3), the centrifugal force of the first centrifugation is 10000-11000 g, such as 10000 g, 10500 g or 11000 g, etc., the time of the first centrifugation is 8-12 min, such as 8 min, 9 min, 10 min, 11 min or 12 min, etc., and the temperature of the first centrifugation is 3-5 ℃, such as 3 ℃,4 ℃ or 5 ℃, etc.

Preferably, in step (3), the centrifugal force of the second centrifugation is 10000-11000 g, such as 10000 g, 10500 g or 11000 g, etc., the time of the second centrifugation is 8-12 min, such as 8 min, 9 min, 10 min, 11 min or 12 min, etc., and the temperature of the second centrifugation is 3-5 ℃, such as 3 ℃,4 ℃ or 5 ℃, etc.

Preferably, in step (3), the centrifugal force of the three-time centrifugation is 10000-11000 g, such as 10000 g, 10500 g or 11000 g, etc., the time of the three-time centrifugation is 8-12 min, such as 8 min, 9 min, 10 min, 11 min or 12 min, etc., and the temperature of the three-time centrifugation is 3-5 ℃, such as 3 ℃,4 ℃ or 5 ℃, etc.

Preferably, in step (3), the molar concentration of the Tris-HCl solution is 0.1-0.12M, such as 0.1M, 0.11M or 0.12M.

Preferably, in step (3), the dialysis time is 8-12 h, such as 8 h, 9 h, 10 h, 11 h or 12 h.

Preferably, in step (3), the dialysis membrane used for dialysis has a molecular weight cut-off of 20000 Da.

The specific steps of the step (3) are as follows: firstly, mixing the limulus blood cell lysate obtained in the step (2) with a saturated ammonium sulfate solution with the pH value of 7.0-8.0 to obtain a mixed solution I, wherein the final concentration of ammonium sulfate in the mixed solution I is 20-30%, centrifuging at 3-5 ℃ and 10000-11000 g for 8-12 min to collect a supernatant I, discarding a precipitate I, then mixing the supernatant I with the saturated ammonium sulfate solution with the pH value of 7.0-8.0 to obtain a mixed solution II, wherein the final concentration of ammonium sulfate in the mixed solution II is 55-65%, centrifuging at 3-5 ℃ and 10000-11000 g for 8-12 min to collect a precipitate II, and discarding the supernatant II; redissolving the precipitate in 0.1-0.12M Tris-HCl solution, removing insoluble protein as denatured protein after redissolving, centrifuging for 10-15 min at 2500-3000 g, dialyzing for 8-12 h to remove ammonium sulfate in the solution, and obtaining the extractive solution containing C factor, B factor and prothrombin with the cut-off molecular weight of dialysis membrane for dialysis being 20000 Da.

Preferably, in the step (4), the stirring is carried out in a specific manner: stirring in ice bath at 0-4 deg.C (such as 0 deg.C, 2 deg.C, 3 deg.C or 4 deg.C) for 3-5 min (such as 3 min, 4 min or 5 min).

Preferably, in step (4), the freeze-drying procedure comprises the following steps in sequence: 45 to-40 ℃ (for example, -45 ℃, -43 ℃, -41 ℃, or-40 ℃) for 8 to 9 h (for example, 8 h, 8.5 h, or 9 h), 35 to-30 ℃ (for example, -35 ℃, -33 ℃, or-30 ℃) for 12 to 14 h (for example, 12 h, 13 h, or 14 h), 20 to-18 ℃ (for example, -20 ℃, -19 ℃, or-18 ℃) for 4.5 to 5 h (for example, 4.5 h, 4.8 h, or 5 h), and 0 to 1 ℃ (for example, 0 ℃ or 1 ℃) for 3 to 4 h (for example, 3 h, 3.5 h, or 4 h).

In the invention, the specific steps of the step (4) are as follows: mixing the extractive solution obtained in step (3), polypeptide chromogenic matrix, laminarin, lyophilized excipient and penetration stabilizer, stirring in ice bath at 0-4 deg.C for 3-5 min to obtain mixed solution; subpackaging the mixed solution into penicillin bottles and/or ampoule bottles, and freeze-drying to obtain a reaction main agent; wherein the freeze drying procedure comprises the following steps in sequence: freezing at-45 to-40 ℃ for 8-9 h, freezing at-35 to-30 ℃ for 12-14 h, freezing at-20 to-18 ℃ for 4.5-5 h, and freezing at 0-1 ℃ for 3-4 h.

Preferably, the concentration of the polypeptide chromogenic substrate in the mixture is 0.5 to 2 mg/mL (e.g., 0.5 mg/mL, 1 mg/mL, 1.5 mg/mL, or 2 mg/mL), the concentration of laminaran in the mixture is 0.5 to 3 mg/mL (e.g., 0.5 mg/mL, 1 mg/mL, 1.5 mg/mL, 2 mg/mL, 2.5 mg/mL, or 3 mg/mL), the concentration of polyvinylpyrrolidone in the mixture is 1 to 3 wt% (e.g., 1 wt%, 2 wt%, or 3 wt%), the concentration of raffinose in the mixture is 0.2 to 0.8 wt% (e.g., 0.2 wt%, 0.4 wt%, 0.6 wt%, or 0.8 wt%), the concentration of mannitol in the mixture is 1 to 5 wt% (e.g., 1 wt%, 2 wt%, 3 wt%, 4 wt%, or 5 wt%), the concentration of sucrose in the mixture is 1.5 to 5 wt% (e.1 wt%, the concentration of glycerol in the mixture is 1.5 wt% (e.5 wt%, or 1.5 wt%), the concentration of sodium chloride in the mixture is 1.5 wt% (e.1 to 3 wt%), or 5 wt%).

As a preferred technical scheme of the invention, the preparation method of the reaction main agent comprises the following steps:

(1) Blood collection and cell isolation: placing blood collected from a living body Limulus cardia into a blood collection bottle containing an anticoagulant, wherein the volume ratio of the anticoagulant to the blood is 1 (5-9); centrifuging at 600-1500 rpm for 15-25 min, discarding supernatant, and collecting limulus blood cells.

(2) Cracking: mixing the horseshoe crab blood cells and the RIPA lysate obtained in the step (1), wherein the volume ratio of the horseshoe crab blood cells to the RIPA lysate is 10 (5-9); freezing at-25 deg.C to-20 deg.C for more than 12 hr, and thawing to obtain limulus blood cell lysate; wherein, the RIPA lysate comprises: 20-30 mM Tris-HCl, 100-200 mM NaCl, 0.5-1 wt% NP-40, 0.5-1 wt% sodium deoxycholate and 0.1-0.15 wt% sodium dodecyl sulfate, wherein the solvent is pyrogen-free water, and the pH value of the RIPA lysate is 7.2-8.0.

(3) Extraction and separation: firstly, mixing the limulus blood cell lysate obtained in the step (2) with a saturated ammonium sulfate solution with the pH value of 7.0-8.0 to obtain a mixed solution I, wherein the final concentration of ammonium sulfate in the mixed solution I is 20-30%, centrifuging at 3-5 ℃ and 10000-11000 g for 8-12 min to collect a supernatant I, discarding a precipitate I, then mixing the supernatant I with the saturated ammonium sulfate solution with the pH value of 7.0-8.0 to obtain a mixed solution II, wherein the final concentration of ammonium sulfate in the mixed solution II is 55-65%, centrifuging at 3-5 ℃ and 10000-11000 g for 8-12 min to collect a precipitate II, and discarding the supernatant II; redissolving the precipitate with 0.1-0.12M Tris-HCl solution, removing insoluble protein after redissolution by centrifuging for 10-15 min at 2500-3000 g, dialyzing for 8-12 h to remove ammonium sulfate in the solution, wherein the cut-off molecular weight of the dialysis membrane used for dialysis is 20000Da, and thus obtaining the extract containing factor C, factor B and prothrombin.

(4) Preparation: mixing the extractive solution obtained in step (3), polypeptide chromogenic matrix, laminarin, lyophilized excipient and penetration stabilizer, and stirring in ice bath at 0-4 deg.C for 3-5 min to obtain mixed solution; wherein the concentration of the polypeptide chromogenic substrate in the mixed solution is 0.5-2 mg/mL, the concentration of the laminaran in the mixed solution is 0.5-3 mg/mL, the concentration of the polyvinylpyrrolidone in the mixed solution is 1-3 wt%, the concentration of the raffinose in the mixed solution is 0.2-0.8 wt%, the concentration of the mannitol in the mixed solution is 1-5 wt%, the concentration of the sucrose in the mixed solution is 1.5-3 wt%, the concentration of the glycerol in the mixed solution is 3-5 wt%, and the concentration of the sodium chloride in the mixed solution is 1-1.5 wt%; subpackaging the mixed solution into penicillin bottles and/or ampoule bottles, and freeze-drying to obtain a reaction main agent; wherein the freeze drying procedure comprises the following steps in sequence: freezing at-45 to-40 ℃ for 8-9 h, freezing at-35 to-30 ℃ for 12-14 h, freezing at-20 to-18 ℃ for 4.5-5 h, and freezing at 0-1 ℃ for 3-4 h.

Preferably, the improved limulus kit for detecting industrial endotoxin based on dynamic color development method further comprises a host double solution, pyrogen-free water, a standard substance and a quality control substance.

Preferably, the main agent redissolution comprises, on a molar basis: tris-HCl 0.01-1 mol/mL, mgCl 0.01-0.3 mol/mL ₂ 、0.01-0.05 mol/mL CaCl ₂ 0.01-0.3 mol/mL NaCl and 0.1-0.3 mol/mL glycine, and the solvent is pyrogen-free water.

In the invention, mgCl is contained in the main agent complex solution ₂ 、CaCl ₂ NaCl and glycine can improve the activity and stability of the main reaction agent.

In the present invention, the concentration of Tris-HCl in the base compound solution is 0.01 to 1 mol/mL, and may be, for example, 0.01 mol/mL, 0.05 mol/mL, 0.1 mol/mL, 0.3 mol/mL, 0.5 mol/mL, 0.9 mol/mL, or 1 mol/mL.

In the invention, mgCl is contained in the main agent complex solution ₂ The concentration of (B) is 0.01 to 0.3 mol/mL, and may be, for example, 0.01 mol/mL, 0.05 mol/mL, 0.08 mol/mL, 0.1 mol/mL, 0.2 mol/mL, or 0.3 mol/mL.

In the invention, caCl in the main agent complex solution ₂ The concentration of (B) is 0.01 to 0.05 mol/mL, and may be, for example, 0.01 mol/mL, 0.02 mol/mL, 0.03 mol/mL, 0.04 mol/mL, or 0.05 mol/mL.

In the present invention, the NaCl concentration in the main agent complex solution is 0.01 to 0.3 mol/mL, and may be, for example, 0.01 mol/mL, 0.05 mol/mL, 0.08 mol/mL, 0.1 mol/mL, 0.2 mol/mL, or 0.3 mol/mL.

In the present invention, the concentration of glycine in the main agent complex solution is 0.1 to 0.3 mol/mL, and may be, for example, 0.1 mol/mL, 0.05 mol/mL, 0.08 mol/mL, 0.1 mol/mL, 0.2 mol/mL, or 0.3 mol/mL.

Preferably, the main agent complex solution is prepared by the following preparation method: mixing Tris-HCl and MgCl ₂ 、CaCl ₂ And NaCl, glycine and pyrogen-free water are stirred and mixed to obtain a mixed solution, the pH value of the mixed solution is adjusted, and filtration is carried out to obtain a main agent complex solution.

Preferably, the time for stirring and mixing is 30-60 min, such as 30 min, 40 min, 50 min or 60 min, etc., and the rotation speed for stirring and mixing is 100-500 rpm, such as 100 rpm, 200 rpm or 500 rpm, etc.

Preferably, the specific steps of adjusting the pH of the mixed solution are: the pH of the mixed solution is adjusted to 6.0 to 8.0 with HCl, and may be, for example, 6.0, 6.5, 7.0, 7.5, or 8.0.

Preferably, the pore size of the filter membrane used for filtration is 0.1-0.22. Mu.m, and may be, for example, 0.1. Mu.m or 0.22. Mu.m.

As a preferred technical scheme of the invention, the main agent complex solution is prepared by the following preparation method:

mixing Tris-HCl and MgCl ₂ 、CaCl ₂ Mixing NaCl, glycine and pyrogen-free water at 100-500 rpm for 30-60 min to obtain mixed solution, adjusting pH of the mixed solution to 6.0-8.0 with HCl, and filtering with filter membrane with pore diameter of 0.1-0.22 μm to obtain main agent compound solution.

Preferably, the titer of the standard is 5-10 EU/branch, for example, 5 EU/branch, 6 EU/branch, 7 EU/branch, 8 EU/branch, 9 EU/branch or 10 EU/branch, etc.

Preferably, the standard also comprises lactose, PEG-8000 and EDTA-2Na in a mass ratio of (1-5): (0.1-0.5): (0.1-0.3), and can be, for example, 1.

Preferably, the titer of the quality control product is 2-4 EU/branch, for example, 2 EU/branch, 3 EU/branch or 4 EU/branch.

Preferably, the quality control product also comprises lactose, PEG-8000 and EDTA-2Na in a mass ratio of (1-5) (0.1-0.5) (0.1-0.3), and can be, for example, 1.

In the invention, PEG-8000 (polyethylene glycol-8000) in the quality control product and the standard product can endow a freeze-dried preparation framework, and lactose can improve the solubility of the freeze-dried preparation. The quality control product is a characteristic component of the improved limulus kit for detecting industrial endotoxin based on the dynamic color development method, and EDTA-2Na is added into the standard product and the quality control product, so that the aggregation and adsorption of lipopolysaccharide can be avoided, the difference between bottles can be reduced, and the accuracy can be improved. Adding a quality control product into the kit to carry out quality control on the kit, calculating the concentration value of the quality control product through a standard curve in the validity period of the kit, and if the concentration value of the quality control product is in the target value range, the kit is valid and the sample test result is valid; if the concentration value of the quality control product is not in the target value range, the kit is invalid, and the sample test result is invalid.

In a second aspect, the present invention provides a method for using the improved limulus kit for detecting industrial endotoxin based on dynamic color development of the first aspect, the method comprising the steps of:

(a) Preparing a detection reagent: respectively preparing a standard sample solution, a test sample solution, a positive test sample solution and a quality control sample solution;

(b) Detection and analysis: respectively adding a standard sample solution, a test sample solution, a positive test sample solution and a quality control sample solution into the microporous plate, respectively adding an equal volume of redissolved reaction main agent into each hole, carrying out kinetic detection, and calculating the concentration of a sample to be detected according to a standard curve.

Preferably, in step (a), the concentration of the standard solution is in the range of 0.005-0.5 EU/mL, and may be, for example, 0.005 EU/mL, 0.05 EU/mL, or 0.5 EU/mL.

Preferably, in step (a), the positive test sample solution is prepared as follows: and diluting the standard substance with the sample solution to be tested to prepare a positive test sample solution.

Preferably, in step (a), the quality control solution is prepared by the following method: re-dissolving the quality control product with pyrogen-free water to obtain a quality control product solution.

In the present invention, in step (b), the parameter setting of the kinetic detection comprises: the plate reading time is 60-120 min (for example, 60 min, 80 min, 100 min, 120 min, etc.), the plate reading interval is 30-60 s (for example, 30 s, 40 s, 50 s, 60 s, etc.), the detection wavelength is 405 nm and 492 nm, the incubation temperature is 36-37 ℃ (for example, 36 ℃ or 37 ℃, etc.), the OnSet OD is set between 0.05 and 0.2 (for example, 0.05, 0.1, 0.2, etc.), the software automatically gives the start time, the measured start time and the logarithm of the endotoxin concentration are subjected to linear regression, and the linear correlation coefficient of the standard curve is calculated.

As a preferred technical scheme of the invention, the improved method for using the limulus kit for detecting the industrial endotoxin based on the dynamic color development method comprises the following steps:

(a) Preparing a detection reagent: preparing a standard substance into a standard substance solution with the concentration range of 0.005-0.5 EU/mL, diluting the standard substance with a sample solution to be detected to prepare a positive sample solution, and re-dissolving a quality control substance with pyrogen-free water to obtain a quality control substance solution;

(b) Detection and analysis: respectively adding a standard sample solution, a test sample solution, a positive test sample solution and a quality control sample solution into a microporous plate, and then respectively adding an equal volume of redissolved reaction main agent into each hole for dynamic detection; the parameter setting of the dynamic detection comprises the following steps: reading the plate for 60-120 min, reading the plate at intervals of 30-60 s, detecting wavelengths of 405 nm and 492 nm, incubating at the temperature of 36-37 ℃, setting OnSet OD between 0.05 and 0.2, automatically giving starting time by software, performing linear regression on the measured starting time and logarithm of endotoxin concentration, and calculating a linear correlation coefficient of a standard curve to obtain the standard curve; and calculating the concentration of the sample to be measured according to the standard curve.

In a third aspect, the present invention provides a limulus kit for detecting industrial endotoxin by dynamic color development in the first aspect and/or a limulus kit for detecting industrial endotoxin by dynamic color development in the second aspect.

The numerical ranges set forth herein include not only the points recited above, but also any points between the numerical ranges not recited above, and are not exhaustive of the particular points included in the ranges for reasons of brevity and clarity.

Compared with the prior art, the invention has the following beneficial effects:

(1) In the invention, 2,4-dinitroaniline is used as a color substrate, so that the color development degree is deepened and the sensitivity is higher; the main reaction agent is added with G factor inhibitor laminarin, and the laminarin can shield (1, 3)β-the bypass of the reaction by the D-glucan; the reaction main agent is added with a freeze-drying excipient, and the components and the proportion of the freeze-drying excipient can ensure that the freeze-drying preparation is blocky and is not easy to break, thereby being convenient for transportation and storage; the reaction main agent is added with a permeation stabilizing agent, and the permeation stabilizing agent can avoid damage to protein active substances in the reaction main agent due to extreme conditions such as low temperature and vacuum in the freeze-drying process.

(2) In the invention, the reaction main agent is extracted and separated by three steps by using a saturated ammonium sulfate solution, so that the impure proteins, interfering substances and the like in the horseshoe crab blood cells can be better removed. The main agent complex solution contains magnesium chloride, calcium chloride, sodium chloride and glycine, and can improve the activity and stability of the main agent.

(3) In the invention, the quality control product is a characteristic component of the improved limulus kit for detecting industrial endotoxin based on the dynamic color development method, and EDTA-2Na is added into the standard product and the quality control product, so that the aggregation and adsorption of lipopolysaccharide can be avoided, the difference between bottles can be reduced, and the accuracy is improved.

Drawings

FIG. 1 is the structure of a 2,4-dinitroaniline polypeptide compound;

FIG. 2 is a standard graph in example 27.

Detailed Description

The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitation of the present invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

The sources of the components used in the following examples and comparative examples are shown in table 1:

TABLE 1 statistical table of experimental materials

Example 1

The embodiment provides an improved limulus kit for detecting industrial endotoxin based on a dynamic color development method, which consists of a reaction main agent, a main agent double solvent, a standard substance, a quality control substance, pyrogen-free water and an enzyme label plate.

The main reaction agent consists of a factor C, a factor B, a clotting zymogen, boc-Leu-Gly-Arg-C ₆ H ₅ N ₃ O ₄ Laminarin, polyvinylpyrrolidone, raffinose, mannitol, sucrose, glycerol and sodium chloride.

The main agent complex solution consists of 0.5 mol/mL Tris-HCl and 0.2 mol/mL MgCl ₂ 、0.025 mol/mL CaCl ₂ 0.2 mol/mL NaCl and 0.2 mol/mL glycine, and the solvent is pyrogen-free water.

The titer of the standard substance is 5 EU/standard, and the standard substance also comprises: 2 wt% lactose, 0.25 wt% PEG-8000 and 0.2 wt% EDTA-2Na.

The titer of the quality control product is 2 EU/count, and the quality control product also comprises: 2 wt% lactose, 0.25 wt% PEG-8000 and 0.2 wt% EDTA-2Na.

The reaction main agent is prepared by the following preparation method:

(1) Blood collection and cell isolation: placing blood collected from a living body Limulus cardia into a blood collection bottle containing an anticoagulant, wherein the volume ratio of the anticoagulant to the blood is 1; centrifuging at 1000 rpm for 20 min, discarding supernatant, and collecting limulus blood cells.

(2) Cracking: mixing the limulus blood cells obtained in the step (1) with the RIPA lysate, wherein the volume ratio of the limulus blood cells to the RIPA lysate is 10; freezing at-20 deg.C for 13 hr, and thawing to obtain limulus blood cell lysate; wherein, the RIPA lysate comprises: 25 mM Tris-HCl, 150 mM NaCl, 0.7 wt% NP-40, 0.7 wt% sodium deoxycholate and 0.12 wt% sodium dodecyl sulfate, wherein a solvent is pyrogen-free water, and the pH value of the lysate is 7.5.

(3) Extraction and separation: firstly, mixing the horseshoe crab blood cell lysate obtained in the step (2) with a saturated ammonium sulfate solution with the pH value of 7.5 to obtain a first mixed solution, wherein the final ammonium sulfate concentration of the first mixed solution is 25%, centrifuging at 4 ℃ for 8 min at 11000 g to collect a first supernatant, discarding a first precipitate, mixing the first supernatant with the saturated ammonium sulfate solution with the pH value of 7.5 to obtain a second mixed solution, wherein the final ammonium sulfate concentration of the second mixed solution is 60%, centrifuging at 4 ℃ for 8 min at 11000 g to collect a second precipitate, and discarding the second supernatant; redissolving the precipitate with 0.1M Tris-HCl solution, removing insoluble protein after redissolving, centrifuging for 10 min at 3000 g, dialyzing for 10 h to remove ammonium sulfate in the solution, wherein the cut-off molecular weight of the dialysis membrane adopted in dialysis is 20000Da, and obtaining the extract containing factor C, factor B and prothrombin.

(4) Preparation: mixing the extractive solution obtained in step (3), polypeptide chromogenic matrix, laminarin, lyophilized excipient and penetration stabilizer, and stirring in ice bath at 0 deg.C for 4 min to obtain mixed solution; wherein, the mixed liquid comprises: 1.5 mg/mL polypeptide chromogenic substrate, 1.5 mg/mL laminarin, 2 wt% polyvinylpyrrolidone, 0.5 wt% raffinose, 2.5 wt% mannitol, 2 wt% sucrose, 4 wt% glycerol, and 1.2 wt% sodium chloride; subpackaging the mixed solution into ampoule bottles by 3 mL/bottle, and freeze-drying to obtain a reaction main agent; wherein, the procedures of freeze drying are as follows in sequence: freezing at-45 deg.C for 8 hr, freezing at-35 deg.C for 12 hr, freezing at-20 deg.C for 4.5 hr, and freezing at 0 deg.C for 3 hr.

The main agent complex solution is prepared by the following preparation method:

reacting Tris-HCl and MgCl ₂ 、CaCl ₂ NaCl, glycine and pyrogen-free water were stirred and mixed at 300 rpmMixing for 50 min to obtain mixed solution, adjusting pH of the mixed solution to 7.5 with HCl, and filtering with filter membrane with pore diameter of 0.1 μm to obtain main agent compound solution.

Example 2

The main reaction agent consists of a factor C, a factor B, a clotting zymogen, boc-Leu-Gly-Arg-C ₆ H ₅ N ₃ O ₄ Laminaran, polyvinylpyrrolidone, raffinose, mannitol, sucrose, glycerol and sodium chloride.

The main agent complex solution consists of 0.5 mol/mL Tris-HCl and 0.3 mol/mL MgCl ₂ 、0.05 mol/mL CaCl ₂ 0.01 mol/mL NaCl and 0.3 mol/mL glycine, and the solvent is pyrogen-free water.

The titer of the standard substance is 10 EU/count, and the standard substance further comprises: 1 wt% lactose, 0.5 wt% PEG-8000 and 0.3 wt% EDTA-2Na.

The titer of the quality control product is 4 EU/count, and the quality control product also comprises: 1 wt% lactose, 0.5 wt% PEG-8000 and 0.3 wt% EDTA-2Na.

The reaction main agent is prepared by the following preparation method:

(1) Blood collection and cell isolation: placing blood collected from a living body Limulus cardia into a blood collection bottle containing an anticoagulant, wherein the volume ratio of the anticoagulant to the blood is 1; centrifuging at 600 rpm for 25 min, discarding supernatant, and collecting Limulus blood cells.

(2) Cracking: mixing the limulus blood cells and the RIPA lysate obtained in the step (1), wherein the volume ratio of the limulus blood cells to the RIPA lysate is 10; freezing at-20 deg.C for 12 hr, and thawing to obtain limulus blood cell lysate; wherein, the RIPA lysate comprises: 20 mM Tris-HCl, 200 mM NaCl, 0.5 wt% NP-40, 0.5 wt% sodium deoxycholate and 0.1 wt% sodium dodecyl sulfate, wherein a solvent is pyrogen-free water, and the pH value of a lysate is 7.2.

(3) Extraction and separation: firstly, mixing the limulus blood cell lysate obtained in the step (2) with a saturated ammonium sulfate solution with the pH value of 7.0 to obtain a mixed solution I, wherein the final concentration of ammonium sulfate in the mixed solution I is 20%, centrifuging at 4 ℃ and 10000 g for 12 min to collect a supernatant I, discarding a precipitate I, mixing the supernatant I with the saturated ammonium sulfate solution with the pH value of 7.0 to obtain a mixed solution II, wherein the final concentration of ammonium sulfate in the mixed solution II is 55%, centrifuging at 4 ℃ and 10000 g for 12 min to collect a supernatant II, and discarding a clear solution II; redissolving the precipitate with 0.12M Tris-HCl solution, removing insoluble protein after redissolving, centrifuging for 15 min at 2500 g, dialyzing for 8 h to remove ammonium sulfate in the solution, and obtaining the extract containing C factor, B factor and prothrombin by the cut-off molecular weight of the dialysis membrane adopted by dialysis being 20000 Da.

(4) Preparation: mixing the extractive solution obtained in step (3), polypeptide chromogenic matrix, laminarin, lyophilized excipient and penetration stabilizer, and stirring in ice bath at 0 deg.C for 5 min to obtain mixed solution; wherein, the mixed liquid comprises: 2 mg/mL of polypeptide chromogenic substrate, 0.5 mg/mL of laminarin, 3 wt% of polyvinylpyrrolidone, 0.2 wt% of raffinose, 1 wt% of mannitol, 1.5 wt% of sucrose, 5 wt% of glycerol and 1.5 wt% of sodium chloride; subpackaging the mixed solution into penicillin bottles by 3 mL/bottle, and freeze-drying to obtain a reaction main agent; wherein, the procedures of freeze drying are as follows in sequence: freezing at-42 deg.C for 8.5 h, freezing at-32 deg.C for 13 h, freezing at-20 deg.C for 4.5 h, and freezing at 0 deg.C for 3 h.

mixing Tris-HCl and MgCl ₂ 、CaCl ₂ And stirring and mixing NaCl, glycine and pyrogen-free water at 500 rpm for 30 min to obtain a mixed solution, adjusting the pH of the mixed solution to 6.0 by adopting HCl, and filtering by adopting a filter membrane with the aperture of 0.1 mu m to obtain a main agent compound solution.

Example 3

The main reaction agent consists of factor C, factor B and coagulantProenzyme, boc-Ile-Gly-Arg-C ₆ H ₅ N ₃ O ₄ Laminarin, polyvinylpyrrolidone, raffinose, mannitol, sucrose, glycerol and sodium chloride.

The main agent complex solution consists of 0.01 mol/mL Tris-HCl and 0.3 mol/mL MgCl ₂ 、0.005 mol/mL CaCl ₂ 0.01 mol/mL NaCl and 0.3 mol/mL glycine, and the solvent is pyrogen-free water.

The titer of the standard substance is 8 EU/standard, and the standard substance further comprises: 5 wt% lactose, 0.1 wt% PEG-8000 and 0.1 wt% EDTA-2Na.

The titer of the quality control product is 4 EU/count, and the quality control product also comprises: 5 wt% lactose, 0.1 wt% PEG-8000 and 0.1 wt% EDTA-2Na.

The reaction main agent is prepared by the following preparation method:

(1) Blood collection and cell isolation: placing blood collected from a living body Limulus cardia into a blood collection bottle containing an anticoagulant, wherein the volume ratio of the anticoagulant to the blood is 1; centrifuging at 1500 rpm for 15 min, discarding supernatant, and collecting limulus blood cells.

(2) Cracking: mixing the limulus blood cells obtained in the step (1) with the RIPA lysate, wherein the volume ratio of the limulus blood cells to the RIPA lysate is 10; freezing at-25 deg.C for 14 hr, and thawing to obtain limulus blood cell lysate; wherein, the RIPA lysate comprises: 30 The lysis solution comprises mM Tris-HCl, 100 mM NaCl, 1 wt% NP-40, 1 wt% sodium deoxycholate and 0.15 wt% sodium dodecyl sulfate, a solvent is pyrogen-free water, and the pH value of the lysis solution is 8.0.

(3) Extraction and separation: firstly, mixing the horseshoe crab blood cell lysate obtained in the step (2) with a saturated ammonium sulfate solution with the pH value of 8.0 to obtain a mixed solution I, wherein the final ammonium sulfate concentration of the mixed solution I is 30%, centrifuging at 4 ℃ for 8 min at 11000 g to collect a supernatant I, discarding a precipitate I, mixing the supernatant I with the saturated ammonium sulfate solution with the pH value of 8.0 to obtain a mixed solution II, centrifuging at 4 ℃ for 8 min at 11000 g to collect a precipitate II, and discarding the supernatant II; redissolving the precipitate with 0.12M Tris-HCl solution, removing insoluble protein after redissolving by centrifuging for 10 min at 3000 g, dialyzing for 12 h to remove ammonium sulfate in the solution, wherein the cut-off molecular weight of the dialysis membrane adopted in dialysis is 20000Da, and thus obtaining the extracting solution containing the factor C, the factor B and the prothrombin.

(4) Preparation: mixing the extractive solution obtained in step (3), polypeptide chromogenic matrix, laminarin, lyophilized excipient and penetration stabilizer, and stirring in ice bath at 0 deg.C for 5 min to obtain mixed solution; wherein, the mixed liquid comprises: 2 mg/mL of a polypeptide chromogenic substrate, 3 mg/mL of laminarin, 1 wt% of polyvinylpyrrolidone, 0.8 wt% of raffinose, 5 wt% of mannitol, 3 wt% of sucrose, 3 wt% of glycerol and 1 wt% of sodium chloride; subpackaging the mixed solution into ampoule bottles by 3 mL/bottle, and freeze-drying to obtain a reaction main agent; wherein the freeze drying procedure comprises the following steps in sequence: freezing at-40 deg.C for 9 hr, freezing at-30 deg.C for 14 hr, freezing at-18 deg.C for 5 hr, and freezing at 0 deg.C for 4 hr.

reacting Tris-HCl and MgCl ₂ 、CaCl ₂ And stirring and mixing NaCl, glycine and pyrogen-free water at 200 rpm for 60 min to obtain a mixed solution, adjusting the pH of the mixed solution to 8 by adopting HCl, and filtering by adopting a filter membrane with the aperture of 0.1 mu m to obtain a main agent compound solution.

Example 4

This example provides an improved limulus kit for industrial endotoxin detection based on the dynamic color development method, which is different from example 1 only in that the concentration of laminarin in the mixed solution is 0.1 mg/mL in the step (4) of preparing the reaction main agent, and the rest of the steps refer to example 1.

Example 5

This example provides an improved limulus kit for industrial endotoxin detection based on the dynamic color development method, which is different from example 1 only in that the concentration of laminarin in the mixed solution is 5 mg/mL in the step (4) of preparing the reaction main agent, and the rest of the steps refer to example 1.

Example 6

This example provides an improved limulus kit for industrial endotoxin detection based on the dynamic color development method, which is different from example 1 only in that in the step (4) of preparing the reaction host, the concentration of the polypeptide color developing substrate in the mixture is 0.1 mg/mL, and the rest of the steps refer to example 1.

Example 7

This example provides an improved limulus kit for industrial endotoxin detection based on the dynamic color development method, which differs from example 1 only in that the concentration of the polypeptide color developing substrate in the mixed solution is 4 mg/mL in the step (4) of the preparation of the reaction base, and the rest of the steps refer to example 1.

Example 8

This example provides an improved limulus kit for the detection of industrial endotoxin based on dynamic color development, which differs from example 1 only in that MgCl is present in the stock solution ₂ Was 0.005 mol/mL, and the rest was according to example 1.

Example 9

This example provides an improved limulus kit for the detection of industrial endotoxin based on dynamic color development, which differs from example 1 only in that MgCl is present in the stock solution ₂ Was 0.5 mol/mL, and the rest was according to example 1.

Example 10

This example provides an improved limulus kit for industrial endotoxin detection based on dynamic color development, which differs from example 1 only in that CaCl is contained in the main agent complex solution ₂ Was 0.005 mol/mL, and the rest was according to example 1.

Example 11

This example provides an improved limulus kit for the detection of industrial endotoxin based on dynamic color development, which differs from example 1 only in that CaCl is contained in the stock solution ₂ Was 0.1 mol/mL, and the rest was according to example 1.

Example 12

This example provides an improved limulus kit for industrial endotoxin detection based on dynamic color development, which differs from example 1 only in that the concentration of NaCl in the main double solution is 0.005 mol/mL, and the rest is referred to example 1.

Example 13

This example provides an improved limulus kit for industrial endotoxin detection based on the dynamic color development method, which differs from example 1 only in that the concentration of NaCl in the main complex solution is 0.5 mol/mL, and the rest of the steps refer to example 1.

Example 14

This example provides an improved limulus kit for industrial endotoxin detection based on dynamic color development, which differs from example 1 only in that the concentration of glycine in the main double solution is 0.05 mol/mL, and the rest is referred to example 1.

Example 15

This example provides an improved limulus kit for industrial endotoxin detection based on dynamic color development, which differs from example 1 only in that the concentration of glycine in the main double solution is 0.5 mol/mL, and the rest of the steps refer to example 1.

Example 16

This example provides an improved limulus kit for the detection of industrial endotoxin based on dynamic color development, which is different from example 1 only in that the substrate for peptide color development in the main double solution is Boc-Leu-Gly-Arg-PNA, and the rest of the procedure is referred to example 1.

Example 17

This example provides an improved limulus kit for industrial endotoxin detection based on dynamic color development, which differs from example 1 only in that the lyophilized excipients are polyvinylpyrrolidone, raffinose and mannitol in a mass ratio of 4.1.

Example 18

This example provides an improved limulus kit for industrial endotoxin detection based on the dynamic color development method, which differs from example 1 only in that the permeation-stabilizing agents are sucrose, glycerol and sodium chloride at a mass ratio of 4.

Example 19

This example provides an improved limulus kit for industrial endotoxin detection based on the dynamic color development method, which is different from example 1 only in that in the preparation method of the reaction main agent, the pH of the saturated ammonium sulfate solution is 6.0 in step (3), and the rest of the steps refer to example 1.

Example 20

This example provides an improved limulus kit for industrial endotoxin detection based on the dynamic color development method, which differs from example 1 only in that in the method for preparing the reaction main agent, the pH of the saturated ammonium sulfate solution is 8.5 in step (3), and the rest of the steps refer to example 1.

Example 21

This example provides an improved limulus kit for industrial endotoxin detection based on the dynamic color development method, which is different from example 1 only in that in the preparation method of the reaction main agent, in step (3), the final concentration of ammonium sulfate is adjusted by directly adding solid ammonium sulfate in each step of extraction and separation, and the rest of the steps refer to example 1.

Example 22

This example provides an improved limulus kit for detecting endotoxin in industry based on dynamic color development, which is different from example 1 only in that in the preparation method of the reaction main agent, in step (3), the final concentration of ammonium sulfate in the mixed solution is 35% during extraction and separation, and the rest steps refer to example 1.

Example 23

This example provides an improved limulus kit for industrial endotoxin detection based on dynamic color development, which is different from example 1 only in that in the preparation method of the reaction primary agent, ammonium sulfate was present at a final concentration of 15% in the mixed solution at the time of extraction and separation in step (3), and the rest of the steps refer to example 1.

Example 24

This example provides an improved limulus kit for detecting endotoxin in industry based on dynamic color development, which is different from example 1 only in that the final concentration of ammonium sulfate in the mixed solution is 50% in the step (3) of the preparation method of the reaction main agent during extraction and separation, and the rest of the steps refer to example 1.

Example 25

This example provides an improved limulus kit for detecting endotoxin in industry based on dynamic color development, which is different from example 1 only in that the final concentration of ammonium sulfate in the mixed solution is 70% in the step (3) of the preparation method of the reaction main agent during extraction and separation, and the rest of the steps refer to example 1.

Example 26

This example provides an improved limulus kit for industrial endotoxin detection based on the dynamic color development method, which differs from example 1 only in that, in the step (3) of preparing the reaction main agent, the extraction and separation steps are:

mixing chloroform and the limulus blood cell lysate obtained in the step (2) according to a mass ratio of 1.

The remaining steps of the preparation of the reaction base refer to example 1.

Comparative example 1

This comparative example provides an improved limulus kit for industrial endotoxin detection based on the dynamic color development method, which is different from example 1 only in that laminarin is not contained in the reaction main agent, and the rest of the steps are referred to example 1.

Comparative example 2

This comparative example provides an improved limulus kit for industrial endotoxin detection based on the dynamic color development method, which differs from example 1 only in that laminarin in the reaction main agent is replaced with curdlan, and the remaining steps are referred to example 1.

Comparative example 3

This comparative example provides an improved limulus kit for industrial endotoxin detection based on the dynamic color development method, which is different from example 1 only in that the reaction host does not contain a lyophilized excipient, and the rest of the steps refer to example 1.

Comparative example 4

This comparative example provides an improved limulus kit for industrial endotoxin detection based on the dynamic color development method, which differs from example 1 only in that the reaction main agent does not contain a permeation stabilizer, and the remaining steps refer to example 1.

Example 27

This example uses the modified dynamic chromogenic-based industrial endotoxin-detecting limulus kit described in example 1 to test a test sample:

(a) Preparing a detection reagent:

preparing a standard solution: sterilizing a packaging bottle of the standard substance by using 75vol% ethanol solution, dissolving the standard substance by using pyrogen-free water to prepare 5 EU/mL standard substance stock solution, uniformly mixing the stock solution in a vortex mixer for 15 min, and then diluting the standard substance stock solution into 0.5 EU/mL, 0.05 EU/mL and 0.005 EU/mL standard substance solutions by using pyrogen-free water in a gradient manner.

Preparation of a test sample: soaking a sample of the gun head to be detected by adopting pyrogen-free water (incubating at 37 ℃ for 1 h), wherein the soaking volume is calculated by a formula: taking the leaching solution for later use according to the calculated volume of 1 mL per branch.

Preparation of a positive test sample: the standard sample is diluted to 0.1 EU/mL by using a sample to be detected for standby.

Preparing a quality control sample: re-dissolving the quality control sample with 1.5 mL pyrogen-free water for later use.

(b) Detection and analysis:

sample adding: the reaction main agent is sterilized by 75vol% ethanol solution, the reaction main agent is redissolved by 3 mL of main agent redissolution (the reaction main agent is obtained by freeze-drying 3 mL mixed solution/ampoule bottle in example 1), 100 muL of standard solution with different concentrations, 100 muL of test sample, 100 muL of positive test sample and 100 muL of quality control sample are respectively added into a pyrogen-free microplate, 100 muL of pyrogen-free water is added into a negative control hole, and 100 muL of redissolved reaction main agent is respectively added into each hole. After the sample is added, the pyrogen-free microplate is put into a microplate reader to be uniformly oscillated and mixed, the kinetic detection is carried out, the plate reading time is 120 min, the plate reading interval is 30 s, the detection wavelength is 405 nm and 492 nm, the incubation temperature is 37 ℃, the OnSet OD is set to be 0.1, the software automatically gives the starting time (T), the measured starting time (T) and the logarithm of the endotoxin concentration (C) are subjected to linear regression, the sample test data are shown in table 2, and the linear correlation coefficient (r) of the standard curve is calculated.

A standard curve was fitted according to the data of table 2: lgT = -5.0236 lgC +16.181, R ² =0.9965, meets the requirements of experimental standards, and the standard curve is made effectively, and is shown in fig. 2.

And calculating the concentration of the sample to be detected according to the standard curve, analyzing the detection data, and calculating the recovery rate of the sample solution of the positive formula, wherein when the recovery rate is within the range of 50-200%, the detection result of the sample to be detected is effective. The recovery rate is calculated as follows:

recovery = (mean of positive test sample-background mean of test sample)/sample addition value x 100%.

TABLE 2 statistical table of test results

And (3) bringing the detection results of the positive test sample and the test sample into a recovery rate calculation formula, and calculating to obtain that the recovery rate of the positive test sample is 103.35 percent and is within the range of 50-200 percent, the detection value of the negative control is not more than the lowest value of a standard curve, the concentration values of the quality control samples are respectively 0.202 EU/mL and 0.196 EU/mL, and the detection result of the experiment is effective within the quality control range of 0.185-0.220 EU/mL.

Example 28

The improved industrial limulus kit for endotoxin detection based on the dynamic color development method described in examples 1 to 26 and comparative examples 1 to 4 was subjected to quality detection including appearance quality test and detection effect test of the kit.

The appearance and quality test results of the modified limulus kit for industrial endotoxin detection based on dynamic color development described in examples 1 to 26 and comparative examples 1 to 4 are shown in Table 3.

TABLE 3 appearance quality inspection results Table

From the quality inspection results in table 3, it can be seen that the addition of a proper amount of excipient to the reaction base can make the reaction base firm and not brittle, and the solubility of the base is affected by an excessive amount; the proper quantity of the penetration stabilizer is added into the reaction main agent to ensure that the reaction main agent has a regular shape and good solubility, and the solubility of the main agent is influenced if the penetration stabilizer is excessive.

The test sample (0.5 EU/mL standard as the test sample) was assayed using the modified dynamic color method-based industrial endotoxin-detecting limulus kit described in examples 1 to 26 and comparative examples 1 to 4, and the detection procedure was carried out with reference to example 27.

Preparation of a standard solution: sterilizing a packaging bottle of the standard substance by using 75vol% ethanol solution, dissolving the standard substance by using pyrogen-free water to prepare 5 EU/mL standard substance storage solution, uniformly mixing the standard substance storage solution in a vortex mixer for 15 min, and then diluting the standard substance storage solution into standard substance solutions of 0.5 EU/mL, 0.05 EU/mL and 0.005 EU/mL by using pyrogen-free water in a gradient manner.

Sample adding: the reaction main agent is disinfected by 75vol% ethanol solution, and 3 mL of main agent compound solution is used for redissolving the reaction main agent. Respectively adding 100 mu L of standard solution into a pyrogen-free microplate, adding 100 mu L of pyrogen-free water into negative control wells, taking 0.5 EU/mL standard solution as a sample to be detected, and simultaneously respectively adding 100 mu L of redissolved reaction main agent into each well.

And (3) detection: the kinetic measurements were carried out with reference to example 27 and the test data are shown in Table 4.

Table 4 test data table for kinetic testing

As can be seen from the results of examples 1, 4-5 and comparative examples 1-2, it was confirmed that addition of laminaran to the reaction base gave a more favorable result, and that the laminaran was (1-3)βThe D glucan structural analogue can occupy the activation site of the G factor in advance, so that the subsequent reaction of the G reaction can not be carried out, and the effect of inhibiting the branch reaction is achieved; root of Kun HaoThe inhibition effect on G reaction is better when the content of the bunsan is 0.5-3 mg/mL, and the inhibition effect on C reaction is also better when the content of the laminaran is about 5 mg/mL.

As can be seen from the test results of example 1 and examples 6 to 7, the coefficient of variation in example 1 is 1.78%, and the coefficients of variation in examples 6 to 7 are as high as 5.71% and 6.50%, and the detection effect is better and the coefficient of variation is lower when the chromogenic group on the polypeptide chromogenic substrate is 2, 4-dinitroaniline; as can be seen from the test results of example 1 and example 16, the start-up time in example 1 was 1839 s, the sensitivity was higher, the start-up time in example 16 was 2204 s, the start-up time in example 16 was longer, and the sensitivity was lower, which indicates that 2,4-dinitroaniline has a deeper color development degree and a higher sensitivity than p-nitroaniline (PNA).

The test results of the examples 1 and 8 to 15 show that the activity and stability of the main agent can be improved by using the mixed solution of magnesium chloride, calcium chloride, sodium chloride and glycine as the main agent complex solution. In a suitable range of ion concentration, mg ²⁺ 、Ca ²⁺ And Na ⁺ The reagent can play a role of an ion activator, and the sensitivity of the limulus reagent is improved, so that the detection range of the reagent is widened; within a suitable glycine concentration range, glycine can function to improve the stability of the limulus reagent and serve as a lyoprotectant.

The test results of the examples 1, 17 to 18 and the comparative examples 3 to 4 show that the freeze-dried preparation is not easy to break in the form of a block, is convenient to transport and store, and can avoid damage to protein active substances caused by extreme conditions such as low temperature and vacuum in the freeze-drying process due to the addition of the freeze-drying excipient and the permeation stabilizer, and when the mass ratio of the freeze-drying excipient is out of the range of (1.0-3.0): (0.2-0.8): (1.0-5.0) and the mass ratio of the permeation stabilizer is out of the range of (1.5-3.0): (3.0-5.0): 1.0-1.5), the activity of the obtained reaction host is reduced, resulting in poor detection effect.

From the test results of example 1 and examples 19-26, it can be seen that, in the extraction separation, the extraction effect is better when the pH of the saturated ammonium sulfate solution used is 7.0-8.0, the protein activity is low or even inactivated when the pH is less than 7.0, and the protein activity is reduced when the pH is higher than 8.0; the final concentration of the ammonium sulfate in the first step is 20 to 30 percent, and the final concentration of the ammonium sulfate in the second step is 55 to 65 percent. When the final concentration of the ammonium sulfate in the first step is less than 20%, macromolecular heteroproteins are precipitated and separated out, interference substances are increased to influence the activity of the reagent, and when the final concentration of the ammonium sulfate in the first step is more than 30%, active proteins such as C factors, G factors and the like are precipitated to cause the activity to be reduced; when the final concentration of ammonium sulfate in the second step is less than 55%, the active substance cannot be completely precipitated, and when the final concentration of ammonium sulfate in the second step is more than 65%, a large amount of interfering proteins are doubly precipitated, resulting in a decrease in activity. When solid ammonium sulfate is added to adjust the final concentration of ammonium sulfate in solution, the pH of the system changes with the addition of ammonium sulfate, resulting in a decrease in protein activity. When other organic reagents are used for one-step extraction, proteins such as B factor, C factor and the like are partially precipitated due to indiscriminate precipitation of the proteins by the organic reagents, so that the activity is reduced.

In conclusion, the improved limulus kit for detecting industrial endotoxin based on the dynamic color development method has the advantages of good detection effect, higher accuracy and sensitivity, shortened detection route and reduced cost, can realize accurate quantitative detection of bacterial endotoxin in samples such as injection drugs, biological products, medical instruments and the like for human and animals by applying the dynamic color development method, has a deeper color and higher sensitivity compared with the traditional PNA color development matrix method because one more nitro group is arranged on the structure of 2,4-dinitroaniline, and is not easy to generate nonspecific turbidity by the interference of proteins and drugs in a sample to be detected after adding laminarin.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention disclosed herein fall within the scope and disclosure of the present invention.

Claims

1. An improved limulus kit for industrial endotoxin detection based on dynamic color development, which is characterized in that the improved limulus kit for industrial endotoxin detection based on dynamic color development comprises a reaction host;

the reaction main agent comprises a factor C, a factor B, prothrombin, a polypeptide chromogenic substrate, laminarin, a freeze-dried excipient and a permeation stabilizer.

2. The improved limulus kit for industrial endotoxin detection based on dynamic color development method according to claim 1, wherein the polypeptide color development substrate comprises Boc-Leu-Gly-Arg-C ₆ H ₅ N ₃ O ₄ And/or Boc-Ile-Gly-Arg-C ₆ H ₅ N ₃ O ₄ (ii) a The freeze-dried excipient comprises polyvinylpyrrolidone, raffinose and mannitol in a mass ratio of (1.0-3.0) to (0.2-0.8) to (1.0-5.0); the permeability stabilizer comprises sucrose, glycerin and sodium chloride in a mass ratio of (1.5-3.0) to (3.0-5.0) to (1.0-1.5).

3. The improved limulus kit for industrial endotoxin detection based on dynamic color development according to claim 1, wherein the reaction host is prepared by the following preparation method:

4. The improved limulus kit for industrial endotoxin detection based on dynamic color development method according to claim 3, wherein the specific step of step (1) is: placing blood collected from a living body Limulus cardia into a blood collection bottle containing an anticoagulant, wherein the volume ratio of the anticoagulant to the blood is 1 (5-9); centrifuging at 600-1500 rpm for 15-25 min, discarding supernatant, and collecting limulus blood cells.

5. The improved limulus kit for industrial endotoxin detection based on dynamic color development according to claim 3, wherein the specific step of step (2) is: mixing the horseshoe crab blood cells and the RIPA lysate obtained in the step (1), wherein the volume ratio of the horseshoe crab blood cells to the RIPA lysate is 10 (5-9); freezing at-25 deg.C to-20 deg.C for more than 12 hr, and thawing to obtain limulus blood cell lysate;

wherein, the RIPA lysate comprises: 20-30 mM Tris-HCl, 100-200 mM NaCl, 0.5-1 wt% NP-40, 0.5-1 wt% sodium deoxycholate and 0.1-0.15 wt% sodium dodecyl sulfate, wherein the solvent is pyrogen-free water, and the pH value of the lysate is 7.2-8.0.

6. The improved limulus kit for industrial endotoxin detection based on dynamic color development method according to claim 3, wherein the step (3) comprises the following specific steps: firstly, mixing the limulus blood cell lysate obtained in the step (2) with a saturated ammonium sulfate solution with the pH value of 7.0-8.0 to obtain a mixed solution I, wherein the final concentration of ammonium sulfate in the mixed solution I is 20-30%, centrifuging at 3-5 ℃ and 10000-11000 g for 8-12 min to collect a supernatant I, discarding a precipitate I, then mixing the supernatant I with the saturated ammonium sulfate solution with the pH value of 7.0-8.0 to obtain a mixed solution II, wherein the final concentration of ammonium sulfate in the mixed solution II is 55-65%, centrifuging at 3-5 ℃ and 10000-11000 g for 8-12 min to collect a precipitate II, and discarding the supernatant II; redissolving the precipitate with 0.1-0.12M Tris-HCl solution, removing insoluble protein after redissolution by centrifuging for 10-15 min at 2500-3000 g, dialyzing for 8-12 h to remove ammonium sulfate in the solution, wherein the cut-off molecular weight of the dialysis membrane used for dialysis is 20000Da, and thus obtaining the extract containing factor C, factor B and prothrombin.

7. The improved limulus kit for industrial endotoxin detection based on dynamic color development according to claim 3, wherein the specific step of step (4) is: mixing the extractive solution obtained in step (3), polypeptide chromogenic matrix, laminarin, lyophilized excipient and penetration stabilizer, stirring in ice bath at 0-4 deg.C for 3-5 min to obtain mixed solution; subpackaging the mixed solution into penicillin bottles and/or ampoule bottles, and freeze-drying to obtain a reaction main agent;

wherein the freeze drying procedure comprises the following steps in sequence: freezing at-45 to-40 ℃ for 8 to 9 hours, freezing at-35 to-30 ℃ for 12 to 14 hours, freezing at-20 to-18 ℃ for 4.5 to 5 hours, and freezing at 0 to 1 ℃ for 3 to 4 hours.

8. The improved limulus kit for industrial endotoxin test based on dynamic color development method according to claim 1, wherein the improved limulus kit for industrial endotoxin test based on dynamic color development method further comprises a host complex solution, pyrogen-free water, a standard and a quality control;

wherein, the main agent redissolution comprises the following components in molar concentration: tris-HCl 0.01-1 mol/mL, mgCl 0.01-0.3 mol/mL ₂ 、0.01-0.05 mol/mL CaCl ₂ 0.01-0.3 mol/mL NaCl and 0.1-0.3 mol/mL glycine, wherein the solvent is pyrogen-free water;

wherein the titer of the standard substance is 5-10 EU/count, and the standard substance also comprises lactose, PEG-8000 and EDTA-2Na with the mass ratio of (1-5) to (0.1-0.5) to (0.1-0.3);

wherein the titer of the quality control product is 2-4 EU/count, and the quality control product also comprises lactose, PEG-8000 and EDTA-2Na with the mass ratio of (1-5) to (0.1-0.5) to (0.1-0.3).

9. The method for using the improved limulus kit for detecting industrial endotoxin based on dynamic color development as claimed in any one of claims 1 to 8, wherein the method for using comprises the steps of:

10. Use of the improved industrial limulus kit for endotoxin detection based on the dynamic color development method of any one of claims 1 to 8 and/or the improved industrial limulus kit for endotoxin detection based on the dynamic color development method of claim 9 in the field of endotoxin detection.