CN104714036B - The preparation method of Antithrombin III determination of activity test kit - Google Patents

Abstract

The present invention relates to a kind of clinical detection reagent preparation method, it is specially the preparation method of Antithrombin III determination of activity test kit, Tris-HCl dissolves zymoplasm lyophilized powder and heparin sodium makes S1 reagent, being dissolved by zymoplasm chromophoric substrate S-2238 or Sar-Pro-Arg-P-nitroanilide Tris-HCl and make S2 reagent, S1 reagent and S2 reagent freeze-drying form S1 dried frozen aquatic products and S2 dried frozen aquatic products; It is mixed with S1 freeze-drying redissolution reagent containing Tris-HCl sodium azide solution redissolution S1 dried frozen aquatic products; Ultrapure water dissolves S2 dried frozen aquatic products makes S2 freeze-drying redissolution reagent; Many person-portions pooled plasma carries out freeze-drying, prepares ATIII calibration object; ATIII calibration object is carried out definite value, and definite value scope ATIII% is 90%��110%; Adopting the ATIII calibration object of definite value, S1 freeze-drying to redissolve reagent and reagent production standard curve is redissolved in S2 freeze-drying, linearly dependent coefficient is greater than 0.99; Color reaction is carried out after plasma sample to be measured adds S1 freeze-drying redissolution reagent and S2 freeze-drying redissolution reagent. The test kit cost that the method prepares is low, linearity range width, and good stability is simple to operate.

Description

The preparation method of Antithrombin III determination of activity test kit

Technical field

The present invention relates to a kind of clinical detection reagent preparation method, it is specially the preparation method of Antithrombin III determination of activity test kit.

Background technology

Antithrombin III (antithrombinIII, ATIII) is the inhibitor containing the proteolytic enzyme of Serine such as zymoplasm and factor XI, plasma thromboplastin antecedent I ��, XI ��, IX ��, X ��. It is combined by arginine-Serine peptide bond with zymoplasm. Forming ATIII antithrombin complex and make enzyme-deactivating, heparin can accelerate this reaction and reach more than thousand times. Cause ATIII conformational change after Lysine binding contained by heparin and ATIII, the arginine residues contained by ATIII is more easily combined with the serine residue of zymoplasm. Once heparin-ATIII antithrombin complex is formed, heparin just dissociates from mixture, is again combined and recycled with another molecule ATIII. ATIII-antithrombin complex is then eliminated by reticuloendothelial system. Relative literature proves, in clinical upper blood plasma, the activity change of AT-III and multiple disease are closely related. Increasing or reducing of AT-III is key hemorrhage, thrombus, and it is significant in the diagnosis of the diseases such as hepatopathy, ephrosis, tuberculosis, tumour, DIC, pregnancy induced hypertension syndrome, and the assistance diagnosis aspect of the burst diseases such as cerebral infarction, heart stalk is shown important especially. Therefore, AT-III Activity determination has a good application prospect clinically.

ATIII Chromogenic assay Cleaning Principle adds excessive zymoplasm in blood plasma to be measured, in zymoplasm and blood plasma, ATIII forms 1: 1 mixture, remaining thrombin action is in chromophoric substrate, cracking goes out the group that develops the color, colour developing degree is proportionate with the amount of residue zymoplasm, and with the activity of ATIII in blood plasma in negative correlation. Apply more and more extensive in clinical studies. The existing supply of the test kit of the AT-III Chromogenic assay activity of current import, but expensive. Therefore, the AT-III Activity determination test kit researching and developing reasonable price, specificity and the higher domestic independent brand of sensitivity is significant and be worth for raising AT-III clinical diagnosis level.

Patent CN102690862A, discloses a kind of Antithrombin III and measures test kit (Chromogenic assay), owing to have employed the zymoplasm of heparin derivatives and import, and therefore cost compare height. And the prior art needs change parameter when carrying out ATIII Activity determination on full automatic blood-coagulation instrument, the parameter of change is blood plasma 5 �� l to be measured, get 50 �� l blood plasma and 60 �� lR1 reagent after adding 120 �� l diluents again, then add 60 �� lR2 reagent, finally develop the color. In the design of this external Backend parameter, it is necessary to change parameter light quantity is arranged, and the parameter light quantity value that machine self is arranged in the detection is " 200-400 ", need " 200 " change into " 0 " in the process of detection. This technology is due to cost compare height, and because this reducing the consumption of reagent, reducing of volume may cause the numerical value of last part in the process of colorimetric not show. Therefore, parameter light quantity value is arranged adjustment in order to " 0 ". This technical costs is higher, and inconvenient operation, also easily there is error in result.

Summary of the invention

For above-mentioned technical problem, the present invention provides a kind of method preparing ATIII Activity determination test kit. The product cost prepared is low, good stability, can supporting use for multiple Blood coagulation instrument, meets the clinical requirement for ATIII Activity determination.

For achieving the above object, the present invention provides following technical scheme,

The preparation method of Antithrombin III determination of activity test kit, comprises the following steps:

(1) using 10��50mmol/LTris-HCl solution of pH8.4 zymoplasm lyophilized powder and heparin sodium to be dissolved, be mixed with S1 reagent, the concentration of thrombin of S1 reagent is 5��20IU/mL, and heparin concentration is 0.2��10IU/mL;

(2) 10��50mmol/LTris-HCl of chromophoric substrate S-2238 or Sar-Pro-Arg-P-nitroanilide pH7.0 is dissolved, it is mixed with the solution that concentration of substrate is 2��5 ��m of ol/mL, be S2 reagent;

(3) in S1 reagent and S2 reagent, add auxiliary agent respectively and carry out freeze-drying, form S1 dried frozen aquatic products and S2 dried frozen aquatic products;

The content of the auxiliary agent that S1 reagent adds in S1 reagent is: BSA to be 0.5��5%, KCl concentration be 15��250mmol/L, EDTA-2K concentration are 1��10mmol/L, Trypsin inhibitor,Trasylol 0.1��5IU/mL, polyethylene glycol 6000 concentration is 1��5mg/ml;

The mass ratio of the auxiliary agent that S2 reagent adds in S2 reagent is: BSA is 0.5��5%, N.F,USP MANNITOL is 1��5%;

(4) use the S1 reagent redissolution liquid redissolution S1 dried frozen aquatic products of 10��50mmol/LTris-HCl containing pH8.4,0.1��0.5mg/ml sodiumazide, it is mixed with S1 freeze-drying redissolution reagent; Use ultrapure water to dissolve S2 dried frozen aquatic products, it is mixed with S2 freeze-drying redissolution reagent;

(5) take fresh many person-portions pooled plasma, add stablizer, sanitas material carry out freeze-drying, prepare ATIII calibration object;

Adopting WHO/BS/10.2146 standard substance, on full automatic blood-coagulation instrument CA-1500, ATIII calibration object is carried out definite value, definite value scope ATIII% is 90%��110%;

(6) on full automatic blood-coagulation instrument CA-1500, adopting the ATIII calibration object of definite value, S1 freeze-drying to redissolve reagent and reagent production standard curve is redissolved in S2 freeze-drying, linearly dependent coefficient is greater than 0.99;

(7) plasma sample to be measured is used to detect on full automatic blood-coagulation instrument CA-1500, detection obtains the OD value of plasma sample to be measured, machine calculates the activity value of ATIII in plasma sample to be measured automatically, trace routine is as follows: carrying out color reaction after adding 175 �� lS1 freeze-drying redissolution reagent and 33 �� lS2 freeze-drying redissolution reagent in 16 �� l plasma samples to be measured, whole process does not adjust any parameter of instrument self.

The present invention provides the preparation method of Antithrombin III determination of activity test kit, the test kit cost prepared is low, linearity range is wide reaches 200%��3.1%, good stability, full automatic blood-coagulation instrument detects and does not need to change any parameter, simple to operate, supporting can use for multiple Blood coagulation instrument, meet the clinical requirement for ATIII Activity determination.

Embodiment

In conjunction with the embodiments, the present invention is set forth further:

Embodiment is to prepare ATIII Activity determination reagent and is specifically detected as example on full automatic blood-coagulation instrument CA-1500.

(1) 10��50mmol/LTris-HCl, pH8.4 solution is used zymoplasm lyophilized powder and heparin sodium to be dissolved, the zymoplasm of different concns and heparin sodium aqua are mixed, being mixed with S1 reagent, concentration of thrombin is 5��20IU/mL, and heparin concentration is 0.2��10IU/mL.

(2) 10��50mmol/LTris-HCl of chromophoric substrate S-2238 or Sar-Pro-Arg-P-nitroanilide pH7.0 is dissolved, it is mixed with the solution that concentration of substrate is 2��5 ��m of ol/mL, be S2 reagent.

(3) in S1 reagent and S2 reagent, add stablizer respectively and vehicle carries out freeze-drying, S1 dried frozen aquatic products and S2 dried frozen aquatic products can be prepared into. The content of the auxiliary agent that S1 reagent adds in S1 reagent is: 0.5%��5%BSA, 20��50mmol/LTris-HCl, 15��250mmol/LKCl, 1��10mmol/LEDTA-2K, 0.1��5IU/mL Trypsin inhibitor,Trasylol, 1��5mg/ml polyethylene glycol 6000; The mass ratio of the auxiliary agent that S2 reagent adds in S2 reagent is: BSA is 0.5��5%, N.F,USP MANNITOL is 1��5%.

(4) use the S1 reagent redissolution liquid redissolution S1 dried frozen aquatic products of 10��50mmol/LTris-HCl containing pH8.4,0.1��0.5mg/ml sodiumazide, it is mixed with S1 freeze-drying redissolution reagent; Use ultrapure water to dissolve S2 dried frozen aquatic products, it is mixed with S2 freeze-drying redissolution reagent.

(5) on CA-1500, it may also be useful to the ATIII calibration object production standard curve that definite value is good, the typical curve linearly dependent coefficient of making is greater than 0.99.

(6) plasma sample to be measured is used to detect on full automatic blood-coagulation instrument CA-1500, detection obtains the OD value of sample, machine calculates the activity value of ATIII in sample to be tested automatically, trace routine is as follows: carrying out color reaction after adding 175 �� lS1 freeze-drying redissolution reagent and 33 �� lS2 freeze-drying redissolution reagent in 16 �� l blood plasma, whole process does not adjust any parameter of instrument self.

Test of product performance is tested:

(1) detected result on full automatic blood-coagulation instrument

With SysmexCA-1500 Automatic coagulometer and supporting Antithrombin III detection reagent (SIEMENS) and the present embodiment ATIII detection reagent production standard curve, the results are shown in Table 1.

Table 1ATIII standard curve making result

ATIII (%)	Embodiment reagent	SIEMENS reagent
			150	0.407	0.407
100	0.713	0.750
			50	1.154	1.154
25	1.360	1.272
			12.5	1.452	1.390
R2	-0.998	-0.998

As seen from the above table, the typical curve that the present embodiment products obtained therefrom makes is good R linearly²> 0.99, detected result is consistent with import SIEMENS company product.

(2) product redissolution Detection of Stability

ATIII embodiment prepared is divided into 3 parts after redissolving, and is placed in 37 DEG C, 2 DEG C-8 DEG C and room temperature (20 DEG C-25 DEG C) preservation respectively, and sampling at regular intervals carries out typical curve preparation, and result is such as table 2 and table 3:

Table 2SIEMENS company reagent redissolves rear 37 DEG C of study on the stability results

ATIII (%)	Before placement	2 hours	4 hours	6 hours	8 hours	24 hours
							150	0.483	0.507	0.450	0.462	0.442	0.053
100	0.804	0.789	0.759	0.739	0.752	0.321
							50	1.098	1.117	1.059	1.067	1.081	0.629
25	1.270	1.232	1.266	1.241	1.251	0.806
							12.5	1.322	1.304	1.456	1.305	1.279	0.898
R2	-1.000	-0.999	-0.999	-0.998	-0.997	-0.998

The reagent rear 37 DEG C of study on the stability results of redissolution that table 3 embodiment is obtained

By the data of table 2 and table 3 it will be seen that the equal < 10% of fluctuation range of self-control reagent detected result in 8 hours of 37 DEG C of insulations, linearly dependent coefficient >=0.99. 37 DEG C of insulations are after 24 hours, the numerical value of self-control reagent and SIEMENS reagent all has reduction, but compare import SIEMENS reagent, it is obvious that SIEMENS reagent is placed in the reduction of the numerical value after 37 DEG C, therefore makes reagent by oneself at 37 DEG C of good stabilities being incubated 24 hours in import SIEMENS reagent.

Reagent rear 2 DEG C-8 DEG C study on the stability results of redissolution that table 4 embodiment is obtained

ATIII (%)	Before placement	2 hours	4 hours	8 hours	48 hours	96 hours
							150	0.540	0.537	0.502	0.516	0.486	0.503
100	0.782	0.733	0.723	0.787	0.752	0.788
							50	1.084	1.165	1.038	1.077	1.024	1.091
25	1.241	1.304	1.219	1.145	1.184	1.242
							12.5	1.288	1.350	1.286	1.230	1.265	1.333
R2	-0.999	-0.998	-0.996	-0.998	-0.998	-1.000

Table 4 places each detection in front and back numerical fluctuations range L EssT.LTssT.LT 10% at 2 DEG C��8 DEG C, linearly dependent coefficient >=0.99 after proving embodiment redissolution, it is possible to think that latter 2 DEG C��8 DEG C of self-control reagent redissolution prepared by the present invention has good stability.

After the reagent redissolution that table 5 embodiment is obtained, room temperature stability investigates result

ATIII (%)	Before placement	2 hours	4 hours	8 hours	48 hours	96 hours
							150	0.540	0.563	0.537	0.528	0.532	0.562
100	0.782	0.784	0.774	0.788	0.753	0.797
							50	1.084	1.085	1.089	1.092	1.038	1.092
25	1.241	1.250	1.253	1.248	1.219	1.234
							12.5	1.288	1.306	1.312	1.303	1.286	1.296
R2	-0.997	-0.998	-0.996	-0.998	-0.996	-0.999

Table 5 experimental result proves that ATIII Activity determination reagent stability prepared by the present invention is better, the equal < 10% of the fluctuation range of detected result, linearly dependent coefficient >=0.99, it is possible to think that the rear room temperature of this product redissolution is deposited and can keep stable in 96 hours.

(3) freeze-drying prods Detection of Stability

ATIII freeze-dried reagent embodiment prepared, is placed in 37 DEG C respectively, and room temperature (20 DEG C-25 DEG C) preserves, and sampling at regular intervals carries out standard curve determination preparation, and to investigate reagent stability, result is as follows:

Table 6 freeze-drying prods 37 DEG C of study on the stability results

ATIII (%)	Before placement	1 day	2 days	3 days	4 days	7 days
							150	0.477	0.435	0.423	0.489	0.410	0.452
100	0.742	0.713	0.702	0.728	0.712	0.707
							50	1.037	0.994	0.956	0.993	0.937	0.967
25	1.175	1.135	1.109	1.139	1.091	1.115
							12.5	1.248	1.219	1.168	1.219	1.150	1.204
R2	-1.000	-0.999	-0.999	-0.999	-0.996	-0.999

Table 6 result display self-control reagent dried frozen aquatic products is at the equal < 10% of fluctuation range of 37 DEG C of insulation detected results after 7 days, and the good > 0.99 of linear preservers, show that self-control freeze-drying prods is incubated 7 days rear stabilities at 37 DEG C good.

Table 7 freeze-drying prods room temperature stability investigates result

ATIII (%)	Before placement	1 day	2 days	3 days	4 days	7 days
							150	0.477	0.505	0.486	0.462	0.477	0.484
100	0.742	0.783	0.742	0.745	0.740	0.722
							50	1.037	1.051	1.022	1.015	1.001	1.012
25	1.175	1.215	1.149	1.170	1.153	1.162
							12.5	1.248	1.265	1.233	1.243	1.213	1.232
R2	-1.000	-0.999	-1.000	-0.999	-1.000	-0.999

Table 7 result display self-control reagent dried frozen aquatic products is at the equal < 10% of fluctuation range of incubation at room temperature detected result after 7 days, and linearly dependent coefficient >=0.99, shows that self-control freeze-drying prods is good at incubation at room temperature 7 days rear stabilities.

Claims (1)

1. the preparation method of Antithrombin III determination of activity test kit, it is characterised in that, comprise the following steps:

(1) using 10��50mmol/LTris-HCl solution of pH8.4 zymoplasm lyophilized powder and heparin sodium to be dissolved, be mixed with S1 reagent, the concentration of thrombin of S1 reagent is 5��20IU/mL, and heparin concentration is 0.2��10IU/mL;

(2) 10��50mmol/LTris-HCl of chromophoric substrate S-2238 or Sar-Pro-Arg-P-nitroanilide pH7.0 is dissolved, it is mixed with the solution that concentration of substrate is 2��5 ��m of ol/mL, be S2 reagent;

(3) in S1 reagent and S2 reagent, add auxiliary agent respectively and carry out freeze-drying, form S1 dried frozen aquatic products and S2 dried frozen aquatic products;

The content of the auxiliary agent that S1 reagent adds in S1 reagent is: BSA to be 0.5��5%, KCl concentration be 15��250mmol/L, EDTA-2K concentration are 1��10mmol/L, Trypsin inhibitor,Trasylol 0.1��5IU/mL, polyethylene glycol 6000 concentration is 1��5mg/ml; The mass ratio of the auxiliary agent that S2 reagent adds in S2 reagent is: BSA is 0.5��5%, N.F,USP MANNITOL is 1��5%;

(4) use the S1 reagent redissolution liquid redissolution S1 dried frozen aquatic products of 10��50mmol/LTris-HCl containing pH8.4,0.1��0.5mg/ml sodiumazide, it is mixed with S1 freeze-drying redissolution reagent; Use ultrapure water to dissolve S2 dried frozen aquatic products, it is mixed with S2 freeze-drying redissolution reagent;

(5) take fresh many person-portions pooled plasma, add stablizer, sanitas material carry out freeze-drying, prepare ATIII calibration object;

Adopting WHO/BS/10.2146 standard substance, on full automatic blood-coagulation instrument CA-1500, ATIII calibration object is carried out definite value, definite value scope ATIII% is 90%��110%;

(6) on full automatic blood-coagulation instrument CA-1500, adopting the ATIII calibration object of definite value, S1 freeze-drying to redissolve reagent and reagent production standard curve is redissolved in S2 freeze-drying, linearly dependent coefficient is greater than 0.99;

(7) plasma sample to be measured is used to detect on full automatic blood-coagulation instrument CA-1500, detection obtains the OD value of plasma sample to be measured, machine calculates the activity value of ATIII in plasma sample to be measured automatically, trace routine is as follows: carrying out color reaction after adding 175 �� lS1 freeze-drying redissolution reagent and 33 �� lS2 freeze-drying redissolution reagent in 16 �� l plasma samples to be measured, whole process does not adjust any parameter of instrument self.