CN114924088B

CN114924088B - Strong anti-interference and stable AFU detection kit and preparation method thereof

Info

Publication number: CN114924088B
Application number: CN202210502950.9A
Authority: CN
Inventors: 谭海娟; 朱雨; 张甲波; 王浩; 张金东
Original assignee: Anhui Iprocom Biotechnology Co ltd
Current assignee: Anhui Iprocom Biotechnology Co ltd
Priority date: 2022-05-09
Filing date: 2022-05-09
Publication date: 2023-09-26
Anticipated expiration: 2042-05-09
Also published as: CN114924088A

Abstract

The invention relates to the field of medical immune in-vitro diagnosis, and provides a strong anti-interference and stable AFU detection kit, which comprises: 50-300 mmol/L of buffer solution, 0.01-0.2% of surfactant, 0.05-0.3% of preservative, 0.3-1.0% of stabilizer, 0.75-2% of anti-interference substance and 1-3 mM of substrate; wherein the stabilizer is one of L-cysteine and histidine, and the anti-interference substance is one of alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin and modified cyclodextrin. The invention also provides a preparation method of the AFU detection kit with strong interference resistance and stability. The invention has the advantages that: the heat stability is strong, the heparin interference resistance is strong, the operation is simple, convenient, quick and accurate, and the method is suitable for clinical application.

Description

Strong anti-interference and stable AFU detection kit and preparation method thereof

Technical Field

The invention relates to the field of medical immune in-vitro diagnosis, in particular to a strong anti-interference and stable AFU detection kit and a preparation method thereof.

Background

alpha-L Fucosidase (AFU), a lysosomal acid hydrolase, is classified as alpha-L-Fucosidase (EC32.1.51), and is mainly distributed in various tissues, cells and body fluids in the human body, and has high activity in tissues such as liver, kidney and the like. AFU is capable of hydrolyzing lipids, mucins and glycosaminoglycans containing fucose, and it is this property that is exploited by AFU detection reagents to detect its viability. Several studies have demonstrated that AFU is a novel tumor marker for diagnosing primary liver cancer.

AFU activity in serum of primary liver cancer (PHC) patients is significantly higher than that of normal population, but also metastatic liver cancer, cholangiocellular carcinoma, malignant mesothelioma, malignant vascular endothelial cytoma, cirrhosis, congenital hepatic cyst and other benign hepatic occupancy lesion patients. It is generally considered that: AFU is more sensitive than Alpha Fetoprotein (AFP), and less specific than AFP. AFU and AFP are not obviously related, and the combined monitoring of the AFU and the AFP can improve the detection rate of liver cancer, and particularly has greater diagnostic value on AFP negative and small cell liver cancer.

Serum AFU of PHC patients continues to rise and is of greater magnitude, a property that is more conducive to differential diagnosis. Meanwhile, serum AFU can also be used as an ideal index for PHC postoperative monitoring, tracking and observing, and the change of the serum AFU is related to the disease degree and is 1-2 months earlier than clinical symptoms, so the serum AFU can be used as a PHC curative effect and prognosis judging index.

Currently, methods for determining AFU activity mainly comprise a fluorescence method, an endpoint colorimetric method and a rate method. Wherein, the fluorescence method is to utilize the fucoside compound to release 4-methyl umbrella ketone capable of emitting fluorescence after the fucosidase hydrolysis, and analyze the enzyme activity by checking the fluorescence intensity; the method has high sensitivity, and is applicable to serum and samples with small amounts of biopsy tissue, cells and cerebrospinal fluid; however, the method has high requirements on instruments, gel filtration is needed to remove interfering substances, and a fluorescence spectrophotometer is needed, so that the method cannot be used for automatic analysis and is difficult to popularize. The end point colorimetric method is to take PNP-AFU as a substrate, and generate a color lump after the substrate is decomposed by enzyme, wherein the color lump is yellow under alkaline conditions and quantitatively analyzed by utilizing a spectrophotometry; the method is simple and convenient, has low equipment requirement, but cannot solve the influence of factors such as serum jaundice, hemolysis, lipidemia and the like on a measurement result due to poor anti-interference of an enzyme substrate, and has complex operation and long required reaction time; and AFU is used as enzyme and needs to carry out enzymatic reaction under an acidic environment, and a color lump needs to be colored under an alkaline condition, so that the AFU is not suitable for automatic detection and has great popularization difficulty. The rate method is to catalyze the hydrolysis of a specific substrate containing a chromogenic group by AFU under a proper reaction condition, can increase absorbance at a certain wavelength, continuously monitor the change of absorbance value, calculate the activity of AFU in a sample, has simple operation, short measurement time, improved sensitivity and anti-interference performance, is suitable for various semi/full-automatic analysis instruments, and is convenient for popularization and application in clinical units.

The substrate in the AFU rate method kit is 2-chloro-4-nitrobenzene-alpha-L-fucoside (CNPF), the detection principle is that alpha-L-fucosidase (AFU) in a sample catalyzes the substrate (CNPF) to react to generate 2-chloro-4-nitrophenol (CNP), the substance is yellow, and has an absorption peak at 405nm, so that the concentration of the AFU in the sample can be quantitatively detected by measuring the color development quantity through absorbance. However, there is a problem that the thermal stability of the substrate CNPF on the market is poor, and the substrate accelerates its spontaneous oxidative hydrolysis to naturally develop color at high temperature in aqueous solution, so that the detection result is inaccurate. Secondly, it has been shown that heparin exhibits a positive matrix effect on AFU detection because of the action of the hepatins alpha-L fucosidase, which breaks down the substrate CNPF, thus raising the false nature of the detection results. Thus, there is a need for a stable and heparin interference resistant alpha-L-fucosidase rate assay reagent.

Disclosure of Invention

The invention aims to solve the technical problem of providing the AFU detection kit which has strong thermal stability, strong heparin interference resistance, simple, convenient, quick and accurate operation and is suitable for clinical application and a preparation method thereof.

The invention adopts the following technical scheme to solve the technical problems:

an AFU detection kit that is robust against interference and stable, comprising: 50 to 300mmol/L of buffer solution (pH 4.8 to 5.2), 0.01 to 0.2 percent of surfactant, 0.05 to 0.3 percent of preservative, 0.3 to 1.0 percent of stabilizer, 0.75 to 2 percent of anti-interference substance and 1 to 3mM of substrate; wherein the stabilizer is one of L-cysteine and histidine, and the anti-interference substance is one of alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin and modified cyclodextrin.

In the invention, the buffer solution provides a proper pH environment for the reaction of the substrate and the enzyme, and the proper ion concentration can improve the sensitivity of the reagent; the surfactant can reduce the surface tension of liquid and has better solubilization; the preservative has antibacterial effect and can prevent the reagent from being polluted by long bacteria; the stabilizer can effectively protect the thermal stability of the substrate, so that the effective period of the reagent is effectively prolonged; the anti-interference substance can effectively eliminate the interference of heparin plasma samples and improve the anti-interference capability of the reagent; the substrate is a reactant of alpha-L fucosidase.

As one of preferred embodiments of the present invention, there is provided: buffer solution (pH 5.0) 100mmol/L, surfactant 0.05%, preservative 0.09%, stabilizer 0.5%, anti-interference substance 1%, substrate 2mM; wherein the stabilizer is histidine and the anti-interference substance is modified cyclodextrin.

As a preferred embodiment of the present invention, the buffer is one of acetic acid-sodium acetate buffer (buffer pH range 3.6 to 5.8), citric acid-sodium citrate buffer (buffer pH range 3.0 to 6.6), citric acid-sodium hydroxide-hydrochloric acid buffer (buffer pH range 2.2 to 6.5), disodium hydrogen phosphate-citric acid buffer (buffer pH range 2.2 to 8.0), and potassium hydrogen phthalate-sodium hydroxide buffer (buffer pH range 4.1 to 5.9).

As one of the preferred embodiments of the present invention, the buffer is preferably acetic acid-sodium acetate buffer or citric acid-sodium citrate buffer.

As a preferred embodiment of the present invention, the buffer is more preferably acetic acid-sodium acetate buffer.

As one of the preferable modes of the invention, the surfactant is one of Tween-20, tween-80, tritonX-100 (polyethylene glycol octyl phenyl ether), BRIJ35 (dodecyl polyethylene glycol ether), tetronic1307 and SDS (sodium dodecyl sulfonate).

As one of the preferred modes of the present invention, the surfactant is preferably Triton X-100.

As one of the preferred modes of the invention, the preservative is one of thimerosal, sodium azide, pinocembrin and Proclin300.

As one of the preferred modes of the present invention, the preservative is preferably Proclin300.

As one of the preferable modes of the present invention, the substrate is one of CNPF (2-chloro-4-nitrobenzene-alpha-L-fucoside) and MG-CNPF (M-G-2-chloro-4-nitrobenzene-alpha-L-fucoside).

As one of the preferred embodiments of the present invention, the substrate is preferably MG-CNPF.

As one of the preferable modes of the invention, the preparation method of the modified cyclodextrin comprises the following steps: alkali treatment is carried out on the natural beta cyclodextrin to activate the hydroxyl; then reacts with etherifying agent to break down the intramolecular hydrogen bond and increase the water solubility of cyclodextrin; meanwhile, adding a cross-linking agent to carry out polycondensation with beta cyclodextrin to finally form the beta-cyclodextrin cross-linked polymer.

As one of the preferable modes of the invention, the alkali in the alkali treatment adopts 20-35% sodium hydroxide, the etherifying agent adopts a methylating agent, an ethylating agent or epoxypropane, and the crosslinking agent adopts toluene-2, 4-diisocyanate, epoxychloropropane or epoxy resin.

The preparation method of the strong anti-interference stable AFU detection kit comprises the steps of preparing a buffer solution, sequentially adding a surfactant, a preservative, a stabilizer, an anti-interference substance and a substrate, fully and uniformly mixing and dissolving, and finely adjusting the final pH value to be 5.0+/-0.2 to obtain the target reagent.

Compared with the prior art, the invention has the advantages that:

(1) The alpha-L fucosidase (AFU) reagent is added with the stabilizing agent, so that the spontaneous oxidative hydrolysis of a substrate can be prevented, the effect of protecting the stability of the substrate is achieved, and the thermal stability of the reagent is ensured; when histidine is specifically selected as the stabilizer, the alpha-L fucosidase (AFU) reagent also has better anti-hemoglobin interference capability, so that the anti-interference performance of the reagent is further improved;

(2) The alpha-L fucosidase (AFU) reagent is added with the anti-interference substance, so that the interference of heparin plasma samples can be eliminated, and the detection result of serum is not influenced; wherein, when the anti-interference substance specifically selects modified cyclodextrin, the anti-heparin interference capability is strongest;

(3) The kit can be detected on a full-automatic biochemical analyzer, can be directly used on a machine, is simple, convenient, quick and accurate to operate, and is suitable for clinical application.

Drawings

FIG. 1 is a graph of anti-hemoglobin interference results of groups A, C and E of example 6;

FIG. 2 is a graph showing the correlation between the reagent of the present invention and a well-known marketed reagent in example 7.

Detailed Description

The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples.

Example 1

The embodiment relates to a strong anti-interference and stable AFU detection kit, which comprises: 50mmol/L of citric acid-sodium hydroxide-hydrochloric acid buffer (pH 4.8), 0.01% Tween-20, 0.05% Thiomerosal, 0.3% L-cysteine, 0.75% alpha-cyclodextrin and 1mM CNPF.

The preparation method of the kit comprises the following steps: firstly preparing a citric acid-sodium hydroxide-hydrochloric acid buffer solution, then sequentially adding a surfactant (Tween-20), a preservative (thimerosal), a stabilizer (L-cysteine), an anti-interference substance (alpha-cyclodextrin) and a substrate (CNPF), fully and uniformly mixing and dissolving, and fine-tuning to enable the final pH to be 4.8, thus obtaining the target reagent.

The detection method of the kit in the embodiment comprises the following steps: a rate method; detection equipment: a full-automatic biochemical analyzer; detection conditions: the reaction temperature is 37 ℃, the reaction time is 5min, the main wavelength is 405nm, the auxiliary wavelength is 505nm, the sample size is 25uL, and the reagent size is 250uL.

Example 2

The embodiment relates to a strong anti-interference and stable AFU detection kit, which comprises: disodium hydrogen phosphate-citrate buffer (pH 5.0) 80mmol/L, tween-80.04%, sodium azide 0.08%, histidine 0.4%, beta-cyclodextrin 0.9%, MG-CNPF 1.5mM.

The preparation method of the kit comprises the following steps: firstly preparing disodium hydrogen phosphate-citric acid buffer solution, then sequentially adding a surfactant (Tween-80), a preservative (sodium azide), a stabilizer (histidine), an anti-interference substance (beta-cyclodextrin) and a substrate (MG-CNPF), fully and uniformly mixing and dissolving, and fine-adjusting to enable the final pH value to be 5.0, thus obtaining the target reagent.

Example 3

The embodiment relates to a strong anti-interference and stable AFU detection kit, which comprises: 100mmol/L acetic acid-sodium acetate buffer (pH 5.0), triton X-100.05%, proclin 300.09%, histidine 0.5%, modified cyclodextrin 1%, MG-CNPF 2mM.

The preparation method of the modified cyclodextrin comprises the following steps: the natural beta cyclodextrin (beta-CD) is subjected to chemical structural modification, and the structure of the beta cyclodextrin is changed. The hydroxyl of beta-CD is activated and then reacts with etherifying agent to break the intramolecular hydrogen bond and increase the water solubility of cyclodextrin. And simultaneously adding a cross-linking agent to perform polycondensation with beta-CD, so that the cross-linking agent forms a bridge arm, and performing polycondensation with the other cyclodextrin, and the bridge arm is further prolonged and then connected with the cyclodextrin, so that the beta-cyclodextrin cross-linked polymer is finally formed.

a etherification: firstly, etherifying the alcoholic hydroxyl group on the outer surface of the beta-CD molecular cavity, selecting a proper etherifying agent, converting the-OH part on the beta-CD into-OR under the condition of existence of an alkaline catalyst, destroying the intramolecular hydrogen bond of the beta-CD and increasing the water solubility of cyclodextrin.

The etherifying agent is selected: the etherifying agent may be a methylating agent, ethylating agent, propylene oxide, etc., and is preferably propylene oxide.

Selection of alkaline catalyst and concentration: sodium hydroxide at a concentration of 20% to 35%, preferably 30%.

b crosslinking: meanwhile, the cyclodextrin molecule cavity structure forms certain steric hindrance through polymer crosslinking, so that the inclusion effect has better stereoselectivity, a denser three-dimensional network structure can be constructed to enable the structure to be more embedded with the size of the heparin molecule, the heparin molecule can be better wrapped in hydrogel, the loading capacity of heparin can be further improved, and the release of the heparin molecule can be slowed down.

Selection of a cross-linking agent: toluene-2, 4-diisocyanate, epichlorohydrin, epoxy resin, etc., preferably toluene-2, 4-diisocyanate.

The specific preparation steps of the modified cyclodextrin are (taking optimal selection as an example):

10g of beta-CD are weighed and slowly added to 16mL of 30% (w/w) sodium hydroxide solution, and stirred at constant temperature of 40 ℃ overnight. 5mL of propylene oxide was added rapidly, and after stirring at 40℃for 2 hours, 2mL of toluene-2, 4-diisocyanate was added immediately, and stirring was continued at 40℃for 1 hour. 23mL of acetone was poured and stirred well to terminate the reaction, and acetone was removed after allowing to stand for delamination. The pH was adjusted to 7.0 with 2M hydrochloric acid. Precipitation was performed by adding 4 volumes of absolute ethanol. The lower viscous liquid was centrifuged, diluted with water and poured into a Millipore3kDa ultrafiltration centrifuge tube and centrifuged at 2800g for 10min, and the retentate was collected and centrifuged again (3 replicates). Distilling the trapped fluid under reduced pressure at 45 ℃, and finally obtaining the modified cyclodextrin polymer through freeze drying.

The specific preparation method of the kit comprises the following steps:

firstly preparing an acetic acid-sodium acetate buffer solution, then sequentially adding a surfactant (TritonX-100), a preservative (Proclin 300), a stabilizer (histidine), an anti-interference substance (modified cyclodextrin) and a substrate (MG-CNPF), fully and uniformly mixing and dissolving, and fine-adjusting to enable the final pH to be 5.0, thus obtaining the target reagent.

Example 4

The embodiment relates to a strong anti-interference and stable AFU detection kit, which comprises: 200mmol/L of citric acid-sodium citrate buffer (pH 5.0), BRIJ 35.06%, kathon 0.1%, L-cysteine 1.0%, gamma-cyclodextrin 1.5%, CNPF 2.5mM.

The preparation method of the kit comprises the following steps: firstly preparing a citric acid-sodium citrate buffer solution, then sequentially adding a surfactant (BRIJ 35), a preservative (Kathon), a stabilizer (L-cysteine), an anti-interference substance (gamma-cyclodextrin) and a substrate (CNPF), fully and uniformly mixing and dissolving, and fine-adjusting to enable the final pH to be 5.0, thus obtaining the target reagent.

Example 5

The embodiment relates to a strong anti-interference and stable AFU detection kit, which comprises: potassium hydrogen phthalate-sodium hydroxide buffer (pH 5.2) 300mmol/L, tetronic1307 or SDS 0.2%, procrin3000.3%, histidine 1.0%, gamma-cyclodextrin 2%, MG-CNPF 3mM.

The preparation method of the kit comprises the following steps: firstly preparing potassium hydrogen phthalate-sodium hydroxide buffer solution, then sequentially adding a surfactant (Tetronic 1307 or SDS), a preservative (Proclin 300), a stabilizer (histidine), an anti-interference substance (gamma-cyclodextrin) and a substrate (MG-CNPF), fully and uniformly mixing and dissolving, and fine-adjusting to enable the final pH to be 5.2, thus obtaining the target reagent.

Example 6

The invention is used for explaining the screening and determination of the stabilizing agent and the anti-interference substance in the reagent.

1. Selection of a stabilizer:

the alpha-L fucosidase (AFU) reagent is added with the stabilizing agent, so that the spontaneous oxidative hydrolysis of a substrate can be prevented, the stability of the substrate is protected, and the thermal stability of the reagent is ensured.

(1) Thermal stability screening

The antioxidant is a substance capable of effectively and remarkably delaying or inhibiting oxidation of easily-oxidized substances at a relatively low concentration, and is selected from commonly used natural (nontoxic and harmless) antioxidants mainly comprising amino acids, phenols and vitamin antioxidants aiming at the problem of substrate thermal stability, and antioxidant amino acids are widely applied in aspects of medicines, cosmetics, biochemical research and the like, so that a large class of antioxidant amino acids are focused as stabilizers (including methionine, L-cysteine, arginine, histidine and L-tryptophan) for thermal stability screening data:

methionine, L-cysteine, arginine, histidine and L-tryptophan were selected as stabilizers and subjected to a thermal stability test in six groups of A, B, C, D, E, F, the specific grouping being shown in Table 1.

TABLE 1 thermal stability test grouping case

Serum was tested using a kit (the conditions of the kit are the same except for the stabilizer) to which A, B, C, D, E, F components were added, and the bias between the test values and the test values for day 0 was as follows:

TABLE 2 thermal stability test results

Bias of

A

B

C

D

E

F

Heat stable at 37 ℃ for 1 day

-3.10％

-2.10％

-1.00％

-3.10％

0.60％

-3.10％

Heat stable at 37 ℃ for 3 days

-6.70％

-5.70％

0.50％

-7.20％

-0.30％

-4.60％

Heat stable at 37 ℃ for 5 days

-8.40％

-9.80％

2.10％

-8.00％

1.10％

-9.20％

Heat stable at 37 ℃ for 7 days

-11.80％

-12.40％

-3.00％

-9.90％

-2.30％

-10.70％

Heat stable at 37 ℃ for 15 days

-20.40％

-19.40％

-6.70％

-23.10％

-4.80％

-22.20％

Real-time 1 month

0.40％

1.60％

2.30％

-0.50％

1.44％

-2.10％

Real-time 2 months

-0.70％

-1.00％

2.00％

1.70％

1.00％

0.85％

Real-time 3 months

-5.40％

-4.60％

0.00％

-2.90％

-2.00％

-6.40％

Real-time 6 months

-8.80％

-9.10％

3.00％

-8.20％

-1.00％

-10.00％

Real-time 12 months

-15.40％

-17.20％

-2.00％

-14.20％

-1.00％

-16.70％

From the data in the table above, the stability of groups C and E is better than that of the other groups, indicating that the addition of the stabilizer L-cysteine or histidine can improve the stability of the reagent. The principle is that the substrate in the reagent is easy to oxidize, hydrolyze and develop color spontaneously at high temperature in the solution, and the L-cysteine or histidine has better antioxidation because of stronger free radical scavenging activity, and meanwhile, the antioxidation site is more embedded with the hydrolysis site of the substrate, so that the substrate can be effectively prevented from being oxidized to hydrolyze, thereby playing a role in protecting the stability of the substrate.

(2) Anti-hemoglobin interference screening:

based on the results of tables 1 and 2, it was found that when groups C and E were selected for additional performance evaluations, group C was not resistant to hemoglobin interference, while groups A and E were control. The principle is that L-cysteine has sulfhydryl group, which belongs to soft alkali, heavy metal ion is mostly soft acid, and hemoglobin has iron ion, so that the added L-cysteine combines with iron ion through sulfhydryl group to form insoluble thiolate, thus affecting the detection result, and not resisting the interference of hemoglobin. Thus, when stability and anti-hemoglobin ability are taken into account in combination, group E may be selected, i.e., the stabilizer added is histidine.

The process and the result of screening the data of the anti-hemoglobin interference ability are as follows:

two groups (group C and group E) with obvious effect on the stability of the reagent are primarily screened, and an anti-hemoglobin interference experiment is carried out simultaneously with a control group (group A), wherein the specific steps are as follows: firstly, preparing 9 interference serum samples containing hemoglobin with different gradient concentrations, then, simultaneously detecting the 9 samples by three groups of reagents, and respectively calculating the deviation between the detected value of the serum added with the hemoglobin and the detected value of the serum not added with the hemoglobin, wherein the relative deviation exceeds +/-10%, so that obvious interference is shown.

The results are shown in tables 3, 4, 5 and FIG. 1.

Table 3A group anti-hemoglobin interference test results

Table 4C group anti-hemoglobin interference test results

Table 5E group anti-hemoglobin interference test results

The screening result shows that the L-cysteine is not resistant to hemoglobin because the structure of the L-cysteine is provided with sulfhydryl groups, the sulfhydryl groups are combined with heavy metal ions to form insoluble thiolate, so that the L-cysteine can be combined with iron carried by hemoglobin in a hemolyzed sample, and thus the L-cysteine is not resistant to hemoglobin interference. Histidine is an ideal stabilizer, and has the function of stabilizing a substrate without affecting the performance of the reagent.

(3) Stabilizer concentration screening:

according to the above screening results, histidine was taken as an example, and the content range and the optimal value were further confirmed.

A gradient search was made for the stabilizer (histidine) content range and the results are shown in Table 6. As is clear from Table 6, the concentration range of 0.3% to 1% is more stable (preferably 0.5%), the OD value of less than 0.3% tends to decrease with time, and the OD value of more than 1% tends to increase.

TABLE 6 results of stabilizer (histidine) concentration screening

2. Selection of anti-interference substances:

according to the invention, the interference of the heparin plasma sample is eliminated by adding the anti-interference substance into the reagent, and the detection result of serum is not influenced.

(1) Anti heparin assay

Heparin anticoagulated blood plasma contains heparin, and researches show that heparin presents positive matrix effect on AFU detection to interfere with the AFU detection, and the mechanism is probably that heparin is a mucopolysaccharide containing sulfate groups, has an average molecular weight of 15KD and has an alpha-L like fucosidase action, and can decompose a substrate CNPF, so that the detection result is falsely increased. The judgment scheme for detecting whether the AFU kit is anti-heparin interference is as follows:

(1) respectively and freshly extracting 5mL of venous blood of the same person by using a serum blood collection tube, an EDTA anticoagulation tube and a heparin anticoagulation tube, and respectively centrifuging to obtain serum of the same person (homologous), EDTA anticoagulated plasma and heparin anticoagulated plasma; a total of 10 persons were drawn and 30 samples were obtained.

(2) The 30 samples can be detected simultaneously by using the kit added with different anti-interference substances, and the result is mainly compared with whether the detection values of the three samples, namely the homologous serum, EDTA anticoagulated plasma and heparin anticoagulated plasma, of the different kits have larger deviation (exceeding +/-10%), the concentration of AFU in the serum of the same person and the concentration of AFU in the different anticoagulated plasma are theoretically considered to be free from obvious difference, and if the serum is consistent with the EDTA anticoagulated plasma, the detection value of heparin anticoagulated plasma has larger difference, the reagent is proved to be not resistant to heparin.

The cyclodextrin is a cyclic macromolecule formed by linking six or more glucopyranose units through alpha-1, 4-glycosidic bonds, and has a hollow round table-shaped three-dimensional structure with a hydrophobic cavity and a hydrophilic outer wall, wherein the hydrophobic cavity can be used for wrapping molecules with proper size. Meanwhile, in order to reduce the interference of heparin to a greater extent, the invention further tries to modify the chemical modified cyclodextrin mainly by modifying the space structure, the modified cyclodextrin structure can be more closely attached to the size of heparin molecules, so that the effect that the detection result is not influenced by heparin is achieved, the preparation of the special modified cyclodextrin is shown in (2), and the screening of the special modified cyclodextrin content is shown in (3). In addition, cyclodextrin is a starch derivative with a ring structure, and does not affect the enzyme activity of alpha-L fucosidase in serum, so that the detection result of serum is not affected.

(2) The modified cyclodextrin was prepared as follows: the natural beta cyclodextrin (beta-CD) is subjected to chemical structural modification, and the structure of the beta cyclodextrin is changed. The hydroxyl of beta-CD is activated and then reacts with etherifying agent to break the intramolecular hydrogen bond and increase the water solubility of cyclodextrin. And simultaneously adding a cross-linking agent to perform polycondensation with beta-CD, so that the cross-linking agent forms a bridge arm, and performing polycondensation with the other cyclodextrin, and the bridge arm is further prolonged and then connected with the cyclodextrin, so that the beta-cyclodextrin cross-linked polymer is finally formed.

The etherifying agent is selected: the etherifying agent may be a methylating agent, ethylating agent, propylene oxide, etc.

Selection of alkaline catalyst and concentration: sodium hydroxide with concentration of 20-35%.

Selection of a cross-linking agent: toluene-2, 4-diisocyanate, epichlorohydrin, epoxy resin, and the like.

Wherein, the screening and the determination of the etherifying agent type, the concentration of the alkaline catalyst and the crosslinking agent type in the preparation of the modified cyclodextrin are carried out by an anti-heparin experiment.

Experimental results:

the results of screening the etherifying agent species are shown in Table 7.

The concentration screening results of the basic catalyst are shown in Table 8.

The screening results of the types of the crosslinking agents are shown in Table 9.

TABLE 7 screening results of etherifying agent species

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TABLE 8 concentration screening results of alkaline catalyst

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TABLE 9 screening results of crosslinker species

As can be seen from tables 7 to 9, propylene oxide is the most preferred choice of etherifying agent; in the selection of the alkaline catalyst-sodium hydroxide, the optimal concentration is 30%; the choice of the cross-linking agent is most preferably toluene-2, 4-diisocyanate.

(3) Screening the content of modified cyclodextrin:

a control group and five experimental groups were set up to screen for the content of sexual cyclodextrin.

The results of the modified cyclodextrin content screening are shown in Table 10. In table 10, control group: no modified cyclodextrin is contained; experiment group 1:0.5% modified cyclodextrin; experiment group 2:0.75% modified cyclodextrin; experiment group 3:1.0% modified cyclodextrin; experiment group 4:1.5% modified cyclodextrin; experimental group 5:2.0% modified cyclodextrin.

Table 10 modified cyclodextrin content screening results

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The samples in the table are freshly extracted serum of the same normal person, heparin anticoagulated plasma and EDTA anticoagulated plasma, and the results of simultaneous detection of different experimental groups. As can be seen from the data in the table, the reference group has no anti-interference substance added to detect heparin plasma which is obviously higher than the serum detection value, false positive occurs (AFU reference interval 10-35U/L), and the difference between the result of detecting heparin plasma and the result of detecting serum after the modified cyclodextrin is added to the experimental group is obviously reduced, wherein the experimental groups 3-5 have better anti-heparin effect, and the serum detection value is consistent with the result of the reference example without adding the anti-interference substance, namely the serum detection result is not influenced. Experiment group 3 was optimal in view of cost.

Example 7

This example is used to illustrate the accuracy of the kit of the present invention.

The same sample is detected by the kit of the invention (taking the kit of the example 3 as an example) and a certain known and marketed kit, and the accuracy of the kit of the invention is verified.

The results are shown in FIG. 2. As can be seen from FIG. 2, the reagent of the present invention has excellent correlation with the known reagents on the market and good accuracy.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims

1. An AFU detection kit that is robust against interference and stable, comprising: 50-300 mmol/L of buffer solution, 0.01-0.2% of surfactant, 0.05-0.3% of preservative, 0.3-1.0% of stabilizer, 0.75-2% of anti-interference substance and 1-3 mM of substrate; wherein the stabilizer is histidine and the anti-interference substance is modified cyclodextrin; the preparation method of the modified cyclodextrin comprises the following steps: alkali treatment is carried out on the natural beta cyclodextrin to activate the hydroxyl; then reacts with etherifying agent to break down the intramolecular hydrogen bond and increase the water solubility of cyclodextrin; meanwhile, adding a cross-linking agent to carry out polycondensation with beta cyclodextrin to finally form the beta-cyclodextrin cross-linked polymer.

2. The strong anti-interference and stable AFU detection kit of claim 1, comprising: buffer solution 100mmol/L, surfactant 0.05%, preservative 0.09%, stabilizer 0.5%, anti-interference substance 1%, substrate 2mM.

3. The strong anti-interference and stable AFU detection kit of claim 1 or 2, wherein the buffer is one of acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, disodium hydrogen phosphate-citric acid buffer, potassium hydrogen phthalate-sodium hydroxide buffer.

4. The strong anti-interference and stable AFU detection kit according to claim 1 or 2, wherein the surfactant is one of tween-20, tween-80, triton x-100, BRIJ35, tetronic1307, SDS.

5. The strong anti-interference and stable AFU detection kit according to claim 1 or 2, wherein the preservative is one of thimerosal, sodium azide, pinocembrin, proclin300.

6. The strong anti-interference and stable AFU detection kit according to claim 1 or 2, wherein the substrate is one of CNPF, MG-CNPF.

7. The strong anti-interference and stable AFU detection kit according to claim 1, wherein the alkali in the alkali treatment adopts 20% -35% sodium hydroxide, the etherifying agent adopts a methylating agent, an ethylating agent or epoxypropane, and the crosslinking agent adopts toluene-2, 4-diisocyanate, epoxychloropropane or epoxy resin.

8. The method for preparing the strong anti-interference and stable AFU detection kit according to any one of claims 1 to 7, wherein a buffer solution is prepared, and then a surfactant, a preservative, a stabilizer, an anti-interference substance and a substrate are sequentially added, fully mixed and dissolved, and fine-tuned to make the final pH 5.0+/-0.2, so as to obtain the target reagent.