CN106546455A

CN106546455A - Hemolytic agent, the method for the pretreatment of biological sample, the assay method of object content and kit

Info

Publication number: CN106546455A
Application number: CN201510590637.5A
Authority: CN
Inventors: 刘林; 张子千; 左淼; 陈庚文; 崔双
Original assignee: Shenzhen Mindray Bio Medical Electronics Co Ltd
Current assignee: Shenzhen Mindray Bio Medical Electronics Co Ltd
Priority date: 2015-09-16
Filing date: 2015-09-16
Publication date: 2017-03-29
Anticipated expiration: 2035-09-16
Also published as: CN106546455B; CN112858668A; CN112858668B

Abstract

Hemolytic agent, the method for the pretreatment of biological sample, the assay method of object content and kit.The invention provides purposes of the borate buffer system in hemolytic agent is prepared, the hemolytic agent determined for immunoturbidimetry, the method for the pretreatment of biological sample, the assay method of object content and the kit determined for immunoturbidimetry in biological specimen.The hemolytic agent is determined for immunoturbidimetry.Utilize borate buffer system prepare hemolytic agent for immunoturbidimetry determine when, at least one with following advantages：Sensitivity is high, detection process is fast and convenient and high specificity.

Description

Hemolytic agent, the method for the pretreatment of biological sample, the assay method of object content And kit

Technical field

The present invention relates to analysis field.In particular it relates to the method for the pretreatment of hemolytic agent, biological sample, target The assay method and kit of thing content, more specifically, the present invention relates to use of the borate buffer system in hemolytic agent is prepared On the way, for the survey of object content in the hemolytic agent of immunoturbidimetry measure, the method for the pretreatment of biological sample, biological specimen The kit determined method and determine for immunoturbidimetry.

Background technology

Immunoturbidimetry determine principle be：When antigen and antibody are reacted in special dilution system and ratio properly (typically resists Body excess) when, the soluble immune complex for being formed is separated out from liquid phase in the presence of poly- agent (such as polyethylene glycol etc.) is promoted, Particulate is formed, makes reactant liquor turbidity occur.When AC is fixed, the amount of formed immune complex is with anti-in sample The increase of commercial weight and increase, the turbidity of reactant liquor is consequently increased.Therefore, by determine reactant liquor turbidity and with it is a series of Standard control, can calculate the content of antigen in sample.

However, the means that current immunoturbidimetry is determined still have much room for improvement.

The content of the invention

It is contemplated that at least solving one of technical problem present in prior art.

The present invention is completed based on the following discovery of inventor：

First, at present, determined by immunoturbidimetry and the object in blood is detected, in order to avoid blood content pair The interference that immunoturbidimetry is determined, it usually needs blood sample is centrifuged, and then resulting blood plasma or serum are exempted from Epidemic disease turbidimetric assay.Therefore, conventional immunoturbidimetry is determined needs centrifugation apparatus, and high cost, blood sampling volume are big, detection speed compared with Slowly, it is impossible to which enough whole blood test means conventional with other are carried out simultaneously, it is impossible to meet wanting for hospital emergency and outpatient service quick diagnosis Ask.

Second, if product of the existing hemolytic agent after haemolysis process is carried out to whole blood sample directly applies to immunoturbidimetry survey Fixed, the turbidity of usual immune response is not ideal enough, thus it is speculated that this is as the discharged cell component of haemolysis process is to immune response There is certain interference.

In addition, the current hemolytic agent of inventor's discovery is after haemolysis process is carried out to whole blood, it is molten in resulting hemolysate The reaction of antigen and antibody in immunoturbidimetry measure is disturbed in blood agent into branch, in order to avoid this kind of interference effect, it usually needs drop The content of hemolysate components in Low haemolysis agent, can so cause haemolysis process time correspondingly to be extended, or to hemolysate It is diluted, such that immunoturbidimetry is determined to be difficult to obtain relatively low test limit when whole blood is detected.

The present inventor has found after furtheing investigate to hemolytic agent that the buffer system of hemolytic agent can significantly affect haemolysis Hemolysate obtained by agent process whole blood is applied to performance when immunoturbidimetry is determined.Further, inventor is to substantial amounts of slow The system of punching is screened, find using borate buffer system hemolytic agent can realize improve immunoturbidimetry determine specificity and/ Or sensitivity, and can be with the surfactant of higher concentration, particularly nonionic surfactant, preferred saponins surface Activating agent, has good synergy so that the test solution after haemolysis is directly used in immunoturbidimetry measure without the need for dilution.

In view of this, in a first aspect of the present invention, the present invention proposes purposes of the borate buffer system in hemolytic agent is prepared, The hemolytic agent is determined for immunoturbidimetry.

It is surprisingly found by the inventors that, using the hemolytic agent prepared by borate buffer system, biological sample such as whole blood is being carried out Test solution obtained by after haemolysis process may be directly applied to immunoturbidimetry measure, without being diluted to test solution.In addition, Inventor has found, by being effectively prevented the interfering material in test solution and antibody using borate buffer system in hemolytic agent With reference to, and then improve the specificity of immunoturbidimetry measure.Further, inventor also has found, by adopting in hemolytic agent Borate buffer system can effectively strengthen the immune response of antibody and object, and then improve the sensitive of immunoturbidimetry measure Degree.Thus, it is to avoid the centrifugal treating to biological sample such as whole blood, the cost of centrifugation apparatus is saved, shorten detection Time, the collection capacity of biological sample is reduced, such as, for blood sampling volume during whole blood sample, improves detection speed.In addition, The standard biologic inspection project such as blood that need not can be centrifuged with other simultaneously using the immunoturbidimetry assay method of the hemolytic agent Conventional detection project synchronization implementation, for example, can be carried out in the same equipment, so as to meet the requirement of quick diagnosis.

In a second aspect of the present invention, the present invention proposes a kind of hemolytic agent determined for immunoturbidimetry.It is of the invention Embodiment, the hemolytic agent contain：Surfactant；Borate buffer system；And water, wherein, the pH of the hemolytic agent It is worth for 5～9.Thus, using the hemolytic agent, by surfactant and the collective effect of borate buffer system, can be effective Ground is cracked to the haemocyte in biological sample such as whole blood, while the material discharged by cell lysis will not be caused to follow-up Immunoturbidimetry determine adversely affect.Further, since the pH of the hemolytic agent is 5～9, therefore so that biological sample example As the object in whole blood can keep nature, and then, the result obtained by follow-up immunization turbidimetric assay can be more Truly reflect the relevant information of related objective thing.

In a third aspect of the present invention, the present invention proposes a kind of preprocess method of biological sample, and the biological sample is used for Immunoturbidimetry is determined, and embodiments in accordance with the present invention, the preprocess method include：Make the biological sample with it is previously described Hemolytic agent is contacted.As it was previously stated, by hemolytic agent according to embodiments of the present invention is contacted with biological sample, in surface-active Under the collective effect of agent and borate buffer system, the haemocyte in biological sample such as whole blood can be effectively cleaved, it is to avoid Impact of the haemocyte to follow-up immunization turbidimetric assay, while also reducing material that the cracking discharged to follow-up immunoturbidimetry The impact of measure.Further, since the pH of the hemolytic agent is 5～9, therefore so that the object in biological sample such as whole blood It still is able to keep nature after contacting with hemolytic agent, and then, the result obtained by follow-up immunization turbidimetric assay can More realistically reflect the relevant information of related objective thing.

In a fourth aspect of the present invention, the present invention proposes a kind of assay method of object content in biological specimen.According to this Inventive embodiment, the method include：The biological specimen is contacted with hemolytic agent, to obtain test solution, the hemolytic agent Containing surfactant and borate buffer system；And the test solution is incubated with the immunoreagent containing object antibody, with Just carry out immunoturbidimetry and determine the determination object content.Embodiments in accordance with the present invention, determine it immunoturbidimetry is carried out Before, in advance biological specimen such as whole blood is contacted with the hemolytic agent containing surfactant and borate buffer system, can be with profit With surfactant and the collective effect of borate buffer system, effectively the haemocyte in biological sample such as whole blood is split Solution, at the same inventor it was unexpectedly observed that using in the test solution obtained by the hemolytic agent cell lysis cell lysis release material, The negative effect for causing is determined to ensuing immunoturbidimetry less.Further, since the pH of the hemolytic agent is 5～9, therefore, The object in biological sample such as whole blood is enabled to keep nature, and then, obtained by follow-up immunization turbidimetric assay The result for obtaining can more realistically reflect the relevant information of related objective thing.Thus, biological specimen according to embodiments of the present invention The assay method of middle object content has at least one of following advantages：Sensitivity is high, detection process is quick, it is easy and High specificity.

In a fifth aspect of the present invention, the present invention proposes a kind of kit determined for immunoturbidimetry.The kit contains： Surfactant；And borate buffer system.Thus, foregoing hemolytic agent can effectively be provided using the kit. As it was previously stated, the hemolytic agent provided using the kit, effectively can be carried out to the haemocyte in biological sample such as whole blood Cracking, while the material discharged by cell lysis will not be caused to determine follow-up immunoturbidimetry adversely affecting.In addition, Embodiments in accordance with the present invention, the pH of the hemolytic agent can be configured to 5～9, therefore so that in biological sample such as whole blood Object can keep nature, and then, the result obtained by follow-up immunization turbidimetric assay can be more realistically anti- Reflect the real information of related objective thing.

Additionally, embodiments in accordance with the present invention, the purposes of the borate buffer system of the present invention in hemolytic agent is prepared, for exempting from The assay method and use of object content in the hemolytic agent of epidemic disease turbidimetric assay, the preprocess method of biological sample, biological specimen The kit determined in immunoturbidimetry has at least one of following advantages：

1st, embodiments in accordance with the present invention, borate buffer system can be used in detecting object, can be antigen-antibody Reaction provides preferable reaction system, reduces interfering material and is combined with antibody, is effectively facilitated the generation of immune response.

2nd, as C reactive protein is present in blood plasma, so prior art generally needs to gather a large amount of blood and needs to carry out Centrifugal treating so that detection speed is slower.Embodiments in accordance with the present invention, directly can be pre-processed using whole blood, gram The defects such as blood sampling volume is big and detection speed is slow have been taken, the demand of quick inspection has been disclosure satisfy that.

3rd, embodiments in accordance with the present invention, the preprocessed reagent for obtaining of biological sample without the need for dilution by directly and immunoreagent Mixing, saves the time, improves detection efficiency.

4th, embodiments in accordance with the present invention, inventor are surprised to find that borate buffer system can be effectively reduced surfactant To immunoturbidimetry determine interference effect, therefore, borate buffer system can with the surfactant synergy of higher concentration, Haemocylolysis to biological specimen such as whole blood is realized rapidly.Also, as borate buffer system can be effectively reduced table The interference effect that face activating agent is determined to immunoturbidimetry, therefore, even if the large usage quantity of surfactant, haemolysis process gained To test solution in surfactant also immunoturbidimetry will not be determined and interfere, and then after test solution is obtained, can be direct Mixing with immunoreagent carries out immunoturbidimetry measure, without being diluted to test solution, so as to save detection time, carries High detection efficiency.

The additional aspect and advantage of the present invention will be set forth in part in the description, and partly will become bright from the following description It is aobvious, or recognized by the practice of the present invention.

Description of the drawings

Fig. 1 shows calibration curve according to an embodiment of the invention.

Fig. 2 shows calibration curve in accordance with another embodiment of the present invention.

Fig. 3 shows calibration curve in accordance with another embodiment of the present invention.

Fig. 4 shows linear relationship evaluation result in accordance with another embodiment of the present invention.

Fig. 5 shows linear relationship evaluation result in accordance with another embodiment of the present invention.

Fig. 6 shows the schematic flow sheet of the assay method of object content in biological specimen according to an embodiment of the invention.

Specific embodiment

Embodiments of the invention are described below in detail.The embodiments described below is exemplary, is only used for explaining the present invention, And be not considered as limiting the invention.

The present invention proposes purposes of the borate buffer system in hemolytic agent is prepared, the hemolytic agent for immunoturbidimetry measure, life The assay method of object content and the reagent determined for immunoturbidimetry in the method for the pretreatment of thing sample, biological specimen Box, is described in detail below respectively.

Purposes of the borate buffer system in hemolytic agent is prepared

The present invention proposes purposes of the borate buffer system in hemolytic agent is prepared, and the hemolytic agent is determined for immunoturbidimetry.

Embodiments in accordance with the present invention, the object that the hemolytic agent can be used for detecting are not particularly restricted.It is of the invention Some examples, the immunoturbidimetry is determined can include C reactive protein detection, Procalcitonin detection, DDi detection, Guang At least one of chalone C detections, troponin detection and glycosylated hemoglobin detection.It was found by the inventors of the present invention that inciting somebody to action Hemolytic agent according to embodiments of the present invention be applied to immunoturbidimetry determine when, can effectively to C reactive protein, Procalcitonin, DDi, bladder chalone C, troponin and glycosylated hemoglobin detected, especially C reactive protein.And can be with The detection of these objects is effectively further carried out to whole blood sample, the specificity of detection, sensitivity is improve, is saved Testing cost, the collection capacity for shortening detection time, reducing biological sample, such as blood sampling volume during whole blood sample, Improve detection speed.In addition, the immunoturbidimetry assay method using the hemolytic agent need not can be centrifuged with other simultaneously Standard biologic checks project such as routine blood test detection project synchronization implementation, for example, can carry out in the same equipment, so as to full The requirement of sufficient quick diagnosis.

For the hemolytic agent that immunoturbidimetry is determined

In a second aspect of the present invention, the present invention proposes a kind of hemolytic agent determined for immunoturbidimetry.It is of the invention Embodiment, the hemolytic agent contain：Surfactant；Borate buffer system；And water, wherein, the pH value of hemolytic agent is 5～9.

Embodiments in accordance with the present invention, surfactant have amphipathic surfactant, can with haemocyte on equally have There is amphipathic membrane lipid to interact, dissolve membrane lipid and form micella particle, and then surfactant can penetrate into bilayer Between lipid and in lipid layer, increase distance between two-layer lipid, short texture, lipid layer local fracture are final to cause Cell membrane bursts apart, and cytoplasm flows out.Further, when hemolysate is used for immunoturbidimetry reaction as test solution, boric acid delays The interfering material that the system of punching can be effectively prevented in test solution is combined with antibody, and then improves the specificity of immunoturbidimetry measure, And can effectively strengthen the immune response of antibody and object, and then improve the sensitivity of immunoturbidimetry measure.In addition, The standard biologic inspection project such as blood that need not can be centrifuged with other simultaneously using the immunoturbidimetry assay method of the hemolytic agent Conventional detection project synchronization implementation, for example, can be carried out in the same equipment, so as to meet the requirement of quick diagnosis.

Present inventor have further discovered that, using the hemolytic agent, by surfactant and the collective effect of borate buffer system, Effectively the haemocyte in biological sample such as whole blood can be cracked, while the thing discharged by cell lysis will not be caused The follow-up immunoturbidimetry of verifying is determined and is adversely affected, as the pH of the hemolytic agent is 5～9, the preferably hemolytic agent PH is 7, is close to the pH value of human body natural's state, therefore so that the object in biological sample such as whole blood can keep Nature, and then, the phase of related objective thing can more realistically be reflected by the result obtained by follow-up immunization turbidimetric assay Pass information.

Embodiments in accordance with the present invention, the type of the surfactant that can be adopted are not particularly restricted, as long as enabling to The cell membrane of haemocyte bursts apart.Embodiments in accordance with the present invention, preferred surfactant are nonionic surface active agent, Specific example of the invention, nonionic surface active agent include selected from saponin(e, Qula logical (Triton), tween (Tween), At least one of Brij (Brij) and Tetronic.Inventor has found that borate buffer system can be effectively reduced these surfaces and live Property agent immunoturbidimetry measure in interference effect.So as to, the surfactant of higher dosage in hemolytic agent, can be adopted, So as to shorten the time of haemolysis process.

Preferably, in the hemolytic agent, can be using saponin(e as surfactant.Saponin(e another name Escin, saponin, soap Angle, alkali soap body, saponin, saponin or saponarin, are the more complicated glycosides compounds of a class.Saponin(e is distributed very in plant kingdom Extensively, many plants such as ginseng, pseudo-ginseng, the wind-weed, polygala root, Radix Glycyrrhizae, balloonflower root, radix bupleuri, Quillaia saponaria, oil tea, rose etc. be all Containing saponin(e.Saponin(e is made up of with sugar, uronic acid or other organic acids sapogenin, and the sugar for constituting saponin(e common are D- Portugals Grape sugar, L- rhamnoses, D- galactolipins, L-arabinose, L- wood sugars.Common uronic acid has glucuronic acid, galactolipin Aldehydic acid etc..Saponin(e is divided into two classes by the structure of sapogenin：One class is steroidal saponins, and its sapogenin is the derivative of spirostan Thing, it is more constituted (such as Dioscin) by 27 carbon atoms, it is present in Liliaceae and Dioscoreaceae plant mostly；Equations of The Second Kind is Triterpenoid saponin, its sapogenin are the derivatives of triterpene, are made up of 30 carbon atoms mostly.Triterpenoid saponin is divided into tetracyclic triterpene And pentacyclic triterpene, it is present in the plants such as Araliaceae and Umbelliferae mostly.In the present invention, it is preferred to adopt triterpenoid saponin.

Compared with conventional surfactant, saponin(e as surfactant have wide material sources, it is nontoxic, nonirritant, to people Body skin is gentle, it is biodegradable rapid and thoroughly, raw material from renewable resource the advantages of.

Inventor is found through experiments, and saponin(e has that haemolysis is fast, the characteristic that hemolytic is little, can effectively improve object with The immune response of antibody.Additionally, saponin(e itself has stronger stability, it is difficult to be degraded.But, inventor is through grinding Study carefully discovery, in the test solution that hemolytic agent carries out obtained by haemolysis is processed to biological specimen such as whole blood, surfactant such as soap The presence of glycosides can disturb the efficiency that immunoturbidimetry is determined.For this purpose, generally carrying out haemolysis process using surfactants such as saponin(es When, in order to avoid the interference that surfactant such as saponin(e is determined to immunoturbidimetry, generally using the surface-active of low concentration Agent such as saponin(e, or before immunoturbidimetry measure is carried out, need to be diluted process to the test solution obtained by haemolysis process, But the test limit that can so extend haemolysis process time or affect the acquisition that immunoturbidimetry is determined lower.Inventor has found, leads to Cross in hemolytic agent using borate buffer system, can be effectively prevented from what surfactant such as saponin(e was determined to immunoturbidimetry Interference, such that it is able to adopt higher dosage of surfactant, and then can shorten hemolytic agent and carry out the time of haemolysis process, Realize that quick haemolysis is processed.Thus, the according to embodiments of the present invention hemolytic agent determined for immunoturbidimetry can be further Detection process or high specificity with higher sensitivity, faster, easy.

Specific example of the invention, based on the cumulative volume of hemolytic agent, in hemolytic agent, the content of saponin(e is 0.01～30 g/l, Specific example of the invention, the content of saponin(e is 0.1～30 g/l, specific example of the invention, the content of saponin(e For 0.5～30 g/l.Inventor is had found, by the saponin(e using the consumption, effectively can be made with borate buffer system jointly The sensitive of immunoturbidimetry measure is carried out to the test solution obtained by hemolytic agent process biological sample such as whole blood subsequently with further improving Degree and/or specificity.Inventor has found that can significantly speed up hemolytic agent using the surfactant of high concentration is carried out at haemolysis The haemolysis speed of reason, but the test solution for being obtained be directly used in immunoturbidimetry determine when, effect is bad, needs carrying out immune ratio Test solution is diluted before turbid measure, but dilution can increase detection time, and affect sensitivity, in the sample of automation Use on analyzer, it is impossible to meet the requirement of speed and sensitivity.Embodiments in accordance with the present invention, by adopting borate buffer System, can reduce the interference that surfactant is determined to immunoturbidimetry, it is therefore possible to use the saponin(e of higher concentration, from And the time that haemolysis is processed is shortened, and need not dilute, directly test solution can be mixed with immunoreagent so as to be exempted from Epidemic disease turbidimetric assay.Therefore, borate buffer system used in hemolytic agent, enables to the test solution of concentrated surfactant preparation Immunoassays are used directly for, the requirement of the automation sample analyser of high speed is met.

Embodiments in accordance with the present invention, borate buffer system contain boric acid and salt of weak acid.Embodiments in accordance with the present invention, it is weak Hydrochlorate is included selected from borate, phosphate, hydrophosphate, dihydric phosphate, organic sulfonate, organic carboxylate at least It is a kind of.Some examples of the invention, salt of weak acid can be alkali metal salt.Embodiments in accordance with the present invention, alkali metal salt Can be sodium salt or sylvite, it is highly preferred that borate includes at least one selected from sodium tetraborate, Boratex and potassium borate. Embodiments in accordance with the present invention, sodium tetraborate are provided in the form of borax.Embodiments in accordance with the present invention, organic sulfonic acid Salt includes 2- morpholino ethyl sulfonic acids (MES), 3- N-morpholinyls (MOPS), 4- HEPESs (HEPES) and piperazine The sodium salt or sylvite of two ethyl sulfonic acids of -1,4- (PIPES).Inventor by study find, alkali metal ion such as sodium ion or The presence of potassium ion, will not interfere to the immune response that immunoturbidimetry is determined.

It will be appreciated by those skilled in the art that the consumption of boric acid, and be not particularly restricted with the proportioning of salt of weak acid, as long as Suitable buffer capacity is provided, and does not disturb follow-up immunoassays.Embodiments in accordance with the present invention, boric acid concentration can Think 0.01M-1M, most preferably preferred 0.02M-0.5M, 0.05M-0.2M.Embodiments in accordance with the present invention, boric acid Weight ratio with salt of weak acid is 1:0.2～5, preferably 1:0.5～2.

It should be noted that it will be appreciated by those skilled in the art that in the hemolytic agent, other routine groups can also be contained Point, such as osmotic pressure regulator and preservative.

Embodiments in accordance with the present invention, can add osmotic pressure regulator in hemolytic agent, so that osmotic pressure is adjusted appropriate In the range of, such as between 10mOsm to 400mOsm, thus, it is possible to contribute to lysed erythrocyte and blood platelet, and then The consumption of surfactant can be reduced.The type of osmotic pressure regulator is not particularly restricted, can adopt NaCl or KCl, thus, the addition of osmotic pressure regulator, while can improving haemolysis efficiency, can't be to follow-up immunization than turbid survey Surely adversely affect.Also, as it was previously stated, the presence of alkali metal ion such as sodium ion or potassium ion, will not be to exempting from The immune response of epidemic disease turbidimetric assay is interfered.

Embodiments in accordance with the present invention, can add preservative in hemolytic agent, with the anti-corrosion for contributing to reagent and long-term preservation. For the species of preservative does not have special requirement, on market common preservative for example Phenoxyethanol, KF88, gentamicin, Sodium azide.

The preprocess method of biological sample

In a third aspect of the present invention, the present invention proposes a kind of preprocess method of biological sample, and the biological sample is used to exempt from Epidemic disease turbidimetric assay, embodiments in accordance with the present invention, the preprocess method include：Biological sample is made with previously described hemolytic agent Contact.

As it was previously stated, inventor has found, by being effectively prevented in test solution using borate buffer system in hemolytic agent Interfering material is combined with antibody, and then improves the specificity of immunoturbidimetry measure.Further, inventor also has found, passes through The immune response of antibody and object can effectively be strengthened in hemolytic agent using borate buffer system, and then improve immunity The sensitivity of turbidimetric assay.In addition, by adopting hemolytic agent according to embodiments of the present invention, i.e., using surfactant and boron The resulting test solution after haemolysis process is carried out to biological sample such as whole blood of the hemolytic agent of acid buffering system may be directly applied to Immunoturbidimetry is determined, without being diluted to test solution.Further, since the pH of the hemolytic agent is 5～9, therefore so that Object in biological sample such as whole blood can keep nature, and then, obtained by follow-up immunization turbidimetric assay As a result the real information of related objective thing can more realistically be reflected.

Embodiments in accordance with the present invention, the object that the method for the biological specimen pretreatment can be used for detecting are not particularly restricted. Some examples of the invention, the immunoturbidimetry is determined can include C reactive protein detection, Procalcitonin detection, D- bis- At least one of aggressiveness detection, bladder chalone C detection, troponin detection and glycosylated hemoglobin detection.As it was previously stated, this The inventor of invention has found, when hemolytic agent according to embodiments of the present invention is applied to immunoturbidimetry measure, can be effectively C reactive protein, Procalcitonin, DDi, bladder chalone C, troponin and glycosylated hemoglobin are detected, especially Which is C reactive protein.

Embodiments in accordance with the present invention, biological sample include at least selected from whole blood, cerebrospinal fluid, body fluid, serum and blood plasma Kind.Preferred exemplary of the invention, whole blood include at least one selected from Peripheral whole blood and anti-freezing venous whole.Thus, The method of the pretreatment of biological sample according to embodiments of the present invention further can have higher sensitivity, faster, letter Just detection process or high specificity.

Embodiments in accordance with the present invention, the hemolytic agent are 1 with the volume ratio of the whole blood:10～80, preferably 1:20～50.By This, the method for the pretreatment of biological sample according to embodiments of the present invention further can have higher sensitivity, faster, Easy detection process or high specificity.

Embodiments in accordance with the present invention, contact are carried out by whole blood is mixed under 37 degrees Celsius with hemolytic agent, according to The specific example of the present invention, contact are carried out by whole blood is added in hemolytic agent, specific example of the invention, Contact carries out 10～90 seconds, preferably 20～50 seconds.

As previously described, because surfactant such as saponin(e is determined with interference effect for immunoturbidimetry, therefore, in order to be able to Test solution obtained by enough process by haemolysis is used directly for immunoturbidimetry measure, needs the consumption of control surface activating agent can not It is too high, thus haemolysis process time be accomplished by extend.Hemolytic agent according to embodiments of the present invention uses surfactant With the combination of borate buffer system, the interference that surfactant is determined to immunoturbidimetry can be reduced, it is therefore possible to use compared with The saponin(e of high concentration, so as to shorten the time of haemolysis process, preferably 20～50 seconds such as 10～90 seconds, and can be not required to Dilute, directly mix test solution so as to carry out immunoturbidimetry measure with immunoreagent.Thus, it is according to embodiments of the present invention The preprocess method of biological sample can further have higher sensitivity, detection process faster, easy or special Property is strong.

Embodiments in accordance with the present invention, the mode that whole blood is contacted with hemolytic agent are not particularly restricted, and can add whole blood To in hemolytic agent, it is also possible to hemolytic agent is added in whole blood to will pass through mixing so that whole blood is fully contacted with hemolytic agent.Root According to example of the present invention, by whole blood being added in the container for being previously provided with hemolytic agent such as detection cell come real Existing contact of the whole blood with hemolytic agent, thus, it is possible to the situation for avoiding such a unfavorable, will whole blood be added in empty Cause parts whole blood sample adhere on the wall, in turn resulting in whole blood fully can not be contacted with hemolytic agent, subsequent detection knot It is really inaccurate.

It will be appreciated to those of skill in the art that the above-mentioned feature and excellent to described by the hemolytic agent that determines for immunoturbidimetry Point is equally applicable to the preprocess method of the biological sample, will not be described here.

The assay method of object content in biological specimen

In a fourth aspect of the present invention, the present invention proposes a kind of assay method of object content in biological specimen.According to this Inventive embodiment, the method include：

S100：Haemolysis process

In this step, haemolysis process is carried out to biological specimen first, specifically, biological specimen is contacted with hemolytic agent, with Just test solution is obtained, the hemolytic agent contains surfactant and borate buffer system.

Inventor find, by hemolytic agent using borate buffer system can be effectively prevented the interfering material in test solution and Antibody is combined, and then improves the specificity of immunoturbidimetry measure.Further, inventor also has found, by hemolytic agent The immune response of antibody and object can effectively be strengthened using borate buffer system, and then improve immunoturbidimetry measure Sensitivity.In addition, the borate buffer system in hemolytic agent can be coordinated with the surfactant of higher concentration, to biological sample For example after whole blood carries out haemolysis process, resulting test solution may be directly applied to immunoturbidimetry measure, without to test solution It is diluted.Further, since the pH of the hemolytic agent is 5～9, therefore so that the object energy in biological sample such as whole blood Nature is kept enough, and then, related objective can more realistically be reflected by the result obtained by follow-up immunization turbidimetric assay The real information of thing.

Embodiments in accordance with the present invention, the object that the method can be used for detecting are not particularly restricted.Of the invention one A little examples, the immunoturbidimetry is determined can include C reactive protein detection, Procalcitonin detection, DDi detection, Guang suppression At least one of plain C detections, troponin detection and glycosylated hemoglobin detection.As it was previously stated, the present inventor sends out It is existing, when the hemolytic agent containing surfactant and borate buffer system is applied to immunoturbidimetry measure, can be effectively right C reactive protein, Procalcitonin, DDi, bladder chalone C, troponin and glycosylated hemoglobin detected, especially It is C reactive protein.

Embodiments in accordance with the present invention, biological sample include at least selected from whole blood, cerebrospinal fluid, body fluid, serum and blood plasma Kind.Preferred exemplary of the invention, whole blood include at least one selected from Peripheral whole blood and anti-freezing venous whole.Thus, In biological specimen according to embodiments of the present invention the assay method of object content further can have higher sensitivity, compared with Quickly, easy detection process or high specificity.

Embodiments in accordance with the present invention, hemolytic agent are 1 with the volume ratio of whole blood:10～80, preferably 1:20～50.Thus, according to In the biological specimen of the embodiment of the present invention assay method of object content further can have higher sensitivity, faster, Easy detection process or high specificity.

As previously described, because surfactant such as saponin(e is determined with interference effect for immunoturbidimetry, therefore, in order to be able to Test solution obtained by enough process by haemolysis is used directly for immunoturbidimetry measure, needs the consumption of control surface activating agent can not It is too high, thus haemolysis process time be accomplished by extend.Hemolytic agent according to embodiments of the present invention uses surfactant With the combination of borate buffer system, the interference that surfactant is determined to immunoturbidimetry can be reduced, it is therefore possible to use compared with The saponin(e of high concentration, so as to shorten preferably 20～50 seconds such as 10～90 seconds time of haemolysis process, and need not dilute, Directly test solution can be mixed with immunoreagent so as to carry out immunoturbidimetry measure.Thus, biology according to embodiments of the present invention In sample the assay method of object content further can have higher sensitivity, detection process faster, easy or Person's high specificity.

S200：Immunoturbidimetry is determined

In this step, after by haemolysis being carried out to biological specimen processing and obtain test solution, resulting test solution is carried out Immunoturbidimetry is determined, and specifically, after test solution is obtained, test solution is incubated with the immunoreagent containing object antibody, with Just carry out immunoturbidimetry and determine determination object content.

Embodiments in accordance with the present invention, object can for C reactive protein, Procalcitonin, DDi, bladder chalone C, At least one of troponin and glycosylated hemoglobin, preferably C reactive protein, and immunoturbidimetry determine include：

A test solution is mixed by () with immunoreagent, immunoreagent contains the antibody of specific recognition C reactive protein.

Embodiments in accordance with the present invention, in step (a), directly can be mixed test solution not diluted with immunoreagent, and/ Or antibody is supported on carrier, carrier includes at least one selected from latex, magnetic bead and collaurum.Inventors be surprised to learn that, Test solution is needed not move through dilution and then directly can be mixed with immunoreagent, is reduced detecting step, be further shorten detection time, Improve efficiency.Thus, in biological specimen according to embodiments of the present invention such as whole blood sample object content assay method Can further have higher sensitivity, detection process faster, easy or high specificity.

Mixture absorbance change within a predetermined period of time for predetermined wavelength light obtained by (b) determination step (a).

Embodiments in accordance with the present invention, in step (b), predetermined wavelength is 850nm, and/or the length of predetermined amount of time is 120 seconds, specific example of the invention, predetermined amount of time were included the 30th second to the 120th second after mixing.Thus, according to In the biological specimen of the embodiment of the present invention such as whole blood, the assay method of object content further can have higher sensitive Degree, detection process faster, easy or high specificity.

C () determines object content based on absorbance change.

Embodiments in accordance with the present invention, step (c) are further included：Based on absorbance change, the rate of change of biological sample is determined； With the rate of change based on biological sample, using the calibration curve for pre-building, the object content is determined, according to the present invention Specific example, further include：Converted using blood cell volume accounting, the object content is corrected.Thus, In biological specimen according to embodiments of the present invention such as whole blood, the assay method of object content can further have higher spirit Sensitivity, detection process faster, easy or high specificity.Embodiments in accordance with the present invention, can be known by test The object of variable concentrations such as c reactive protein calibration object is reacted with hemolytic agent, immunoreagent, and reaction is read on photometer Reduced turbidity change in time, and reduced turbidity rate of change is calculated, set up the calibration curve of concentration-reduced turbidity rate of change.

Embodiments in accordance with the present invention, rate of change Δ A=(A2-A1)/(T2-T1), wherein A2 refer to mixed in step (b) The reduced turbidity of mixture during T2 seconds, the reduced turbidity of mixture when A1 refers to the mixed T1 seconds in step (b).

It will be appreciated to those of skill in the art that the spy described by above-mentioned preprocess method and hemolytic agent to biological sample Advantage of seeking peace is equally applicable to the assay method of object content in the whole blood sample.

For the kit that immunoturbidimetry is determined

In the fifth aspect of the invention, the present invention proposes a kind of kit determined for immunoturbidimetry.According to the present invention Embodiment, the kit contains：Surfactant；And borate buffer system, wherein, surfactant and boric acid are slow The system of punching is that the hemolytic agent description for being previously used for immunoturbidimetry measure is limited.Thus, it is according to embodiments of the present invention to be used for The kit that immunoturbidimetry is determined has at least one of following advantages：Sensitivity is high, detection process is quick, easy and special It is different in nature strong.

Specifically, embodiments in accordance with the present invention, surfactant have amphipathic surfactant, can be with haemocyte On equally there is amphipathic membrane lipid to interact, dissolving membrane lipid simultaneously forms micella particle, and then surfactant can Penetrate between lipid bilayer and in lipid layer, increase distance between two-layer lipid, short texture, lipid layer local fracture, Cell membrane is finally caused to burst apart, cytoplasm flows out.Further, when hemolysate is used for immunoturbidimetry reaction as test solution, The interfering material that borate buffer system can be effectively prevented in test solution is combined with antibody, and then improves immunoturbidimetry measure Specificity, and can effectively strengthen the immune response of antibody and object, and then improve the sensitive of immunoturbidimetry measure Degree.

Embodiments in accordance with the present invention, borate buffer system contain boric acid and salt of weak acid.Embodiments in accordance with the present invention, it is weak Hydrochlorate is included selected from borate, phosphate, hydrophosphate, dihydric phosphate, organic sulfonate, organic carboxylate at least It is a kind of.Some examples of the invention, salt of weak acid can be alkali metal salt.Embodiments in accordance with the present invention, sodium salt or Sylvite, it is highly preferred that borate includes at least one selected from sodium tetraborate, Boratex and potassium borate.It is of the invention Embodiment, sodium tetraborate are provided in the form of borax.Embodiments in accordance with the present invention, organic sulfonate include 2- morpholines For ethyl sulfonic acid (MES), 3- N-morpholinyls (MOPS), two ethyl sulfonic acid of 4- HEPESs (HEPES) and piperazine -1,4- (PIPES) sodium salt or sylvite.Inventor passes through research and finds, the presence of alkali metal ion such as sodium ion or potassium ion, The immune response that immunoturbidimetry is determined will not be interfered.Thus, it is according to embodiments of the present invention to determine for immunoturbidimetry Kit can further have higher sensitivity, detection process faster, easy or high specificity.

It will be appreciated by those skilled in the art that the consumption of boric acid, and be not particularly restricted with the proportioning of salt of weak acid, as long as Suitable buffer capacity is provided, and does not disturb follow-up immunoassays.Embodiments in accordance with the present invention, boric acid concentration can Think 0.01M-1M, most preferably preferred 0.02M-0.5M, 0.05M-0.2M.Embodiments in accordance with the present invention, it is described Boric acid is 1 with the weight ratio of the salt of weak acid:0.2～5, preferably 1:0.5～2.

Embodiments in accordance with the present invention, when haemolysis system for handling is formed using kit, the salt of weak acid in borate buffer system Addition manner be not particularly restricted, directly salt of weak acid directly can be mixed with other components, it is also possible to add weak acid and Alkali, generates fresh salt of weak acid so as to pass through neutralization reaction.Thus, embodiments in accordance with the present invention, the kit can be entered One step includes：Weak bronsted lowry acids and bases bronsted lowry, weak bronsted lowry acids and bases bronsted lowry are suitable to generate salt of weak acid by neutralization reaction.Thus, facilitate the preservation of kit, Reduce production cost.

Embodiments in accordance with the present invention, the kit may further include：Antibody, the antibody specificity recognize object, Specific example of the invention, object include C reactive protein, and specific example of the invention, antibody are supported on load On body, carrier includes latex, magnetic bead and collaurum.Thus, the according to embodiments of the present invention examination determined for immunoturbidimetry Agent box can further have higher sensitivity, detection process faster, easy or high specificity.

Embodiments in accordance with the present invention, at least a portion of the component of kit are arranged in independent container.Thus, according to The embodiment of the present invention for immunoturbidimetry determine kit further can have higher sensitivity, faster, simplicity Detection process or high specificity.

It will be appreciated to those of skill in the art that embodiments in accordance with the present invention, can be by pre-configured borate buffer body System is arranged in an independent container, it is also possible to respectively boric acid and salt of weak acid are respectively placed in two containers, treat that needs make Used time is mixed in proportion, boric acid, weak bronsted lowry acids and bases bronsted lowry can be respectively placed in three containers again, wait need use when by than Example is mixed.For example, can after the mixing of each component by formula ratio, with water polishing to final volume, such that it is able to It is readily obtained hemolytic agent according to embodiments of the present invention.

It should be noted that it will be appreciated by those skilled in the art that in the kit, other routine groups can also be contained Point, such as osmotic pressure regulator and preservative.

Embodiments in accordance with the present invention, can add osmotic pressure regulator in kit, so that osmotic pressure is adjusted appropriate In the range of, such as between 10mOsm to 400mOsm, thus, it is possible to contribute to lysed erythrocyte and blood platelet, and then The consumption of surfactant can be reduced.The type of osmotic pressure regulator is not particularly restricted, can adopt NaCl or KCl, the thus addition of osmotic pressure regulator, while can improving haemolysis efficiency, can't be to follow-up immunization turbidimetric assay Adversely affect.Also, as it was previously stated, the presence of alkali metal ion such as sodium ion or potassium ion, will not be to immunity The immune response of turbidimetric assay is interfered.

Embodiments in accordance with the present invention, can add preservative in kit, with the anti-corrosion for contributing to reagent and long-term preservation. For the species of preservative does not have special requirement, on market, common preservative such as Phenoxyethanol, KF88, gentamicin are equal Can.

It will be appreciated to those of skill in the art that purposes above for borate buffer system in hemolytic agent is prepared, being used for Hemolytic agent that immunoturbidimetry is determined, the method for the pretreatment of biological sample, in biological specimen object content assay method with And the feature and advantage described by the kit determined for immunoturbidimetry can be mutually to be suitable for, and will not be described here.

The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following enforcement Example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Unreceipted particular technique or condition in embodiment, Carry out according to the technology or condition described by document in the art or according to product description.Agents useful for same or instrument are not noted Bright production firm person, be can pass through city available from conventional products.

Embodiment 1

Whole blood C reactive protein detection hemolytic agent A is prepared according to following formula：

Triton X-114 8.0g

Boric acid 5.6g

4- HEPES sodium 2.5g

Plus deionized water dissolving, appropriate NaOH or HCl adjusted to 7.0, mends deionized water to final volume 1L.

In this embodiment, 8 microlitres of the C reactive protein calibration object (concentration is in the range of 0-320mg/L) of concentration known is added Enter in prepared 0.2mL hemolytic agent A.Under conditions of temperature is kept for 37 DEG C, resulting mixture is stood molten After blood is processed 90 seconds, then 0.2mL C reactive protein immunoreagents are added in the mixture, after mixing, test 850nm's Absorbance signal changes, and after adding so as to Computation immunity reagent, the reduced turbidity rate of change of the 30th second to the 120th second is (also referred to as " anti- Response ") Δ A=(A2-A1)/(T2-T1).

Wherein, Δ A represent reduced turbidity rate of change, T2 and T1 represent respectively immunoreagent add after different time points, A2 For time point T2 when 850nm at absorbance, A1 be time point T1 when 850nm at absorbance.

For a series of c reactive protein calibration object of known variable concentrations, above-mentioned test is carried out, and be than turbid according to ordinate Degree rate of change, abscissa is c reactive protein (CRP) concentration, sets up the calibration curve of hemolytic agent A, is as a result displayed in figure Shown in 1.

Comparative example 1

Whole blood C reactive protein detection hemolytic agent B is prepared according to following formula：

Triton X-114 8.0g

12 water 11.6g of disodium hydrogen phosphate

Two water 1.18g of sodium dihydrogen phosphate

Using deionized water dissolving said components, and adjusted to after 7.0 using appropriate NaOH or HCl, supplement is gone Ionized water is to final volume 1L.

According to method substantially the same manner as Example 1, the calibration curve of hemolytic agent B is set up, is differed only in and is adopted hemolytic agent Hemolytic agent A in B alternative embodiments 1, the calibration curve of hemolytic agent B show in FIG.

From figure 1 it appears that the degree of reaction of 1 hemolytic agent A of embodiment is significantly higher than 1 hemolytic agent B of comparative example, Jin Errong Blood agent A can significantly increase the immune response effect of C reactive protein relative to hemolytic agent B.Thus, it is possible to reach a conclusion, Borate buffer system can effectively strengthen the immune response during immunoturbidimetry is determined relative to other buffer systems, and And Triton X-114 can be effective as the surfactant of hemolytic agent and borate buffer system is used in combination.

Embodiment 2

Whole blood C reactive protein detection hemolytic agent C is prepared according to following formula：

Dodecyldimethylammonium hydroxide inner salt 0.05g

Boric acid 5.6g

4- HEPES sodium 2.5g

According to method substantially the same manner as Example 1, the calibration curve of hemolytic agent C is set up, is differed only in and is adopted hemolytic agent Hemolytic agent A in C alternative embodiments 1, the calibration curve of hemolytic agent C show in fig. 2.

Comparative example 2

Whole blood C reactive protein detection hemolytic agent D is prepared according to following formula：

Dodecyldimethylammonium hydroxide inner salt 0.05g

12 water of disodium hydrogen phosphate, 11.6 g

Two water 1.18g of sodium dihydrogen phosphate

According to method substantially the same manner as Example 1, the calibration curve of hemolytic agent D is set up, is differed only in and is adopted hemolytic agent Hemolytic agent A in D alternative embodiments 1, the calibration curve of hemolytic agent D show in fig. 2.

From figure 2 it can be seen that the degree of reaction of 2 hemolytic agent C of embodiment is significantly higher than 2 hemolytic agent D of comparative example, Jin Errong Blood agent C can significantly increase the immune response effect of C reactive protein relative to hemolytic agent D.Thus, it is possible to reach a conclusion, Borate buffer system can effectively strengthen the immune response during immunoturbidimetry is determined relative to other buffer systems, and And dodecyldimethylammonium hydroxide inner salt can be effective as the surfactant of hemolytic agent and combine with borate buffer system Use.

Embodiment 3

Whole blood C reactive protein detection hemolytic agent E is prepared according to following formula：

Saponin(e 4.0g

Boric acid 5.6g

4- HEPES sodium 2.5g

According to method substantially the same manner as Example 1, the calibration curve of hemolytic agent E is set up, employing is differed only in Hemolytic agent A in hemolytic agent E alternative embodiments 1, and after hemolytic agent is mixed with sample stand haemolysis process when Between be reduced to 30 seconds, the calibration curve of hemolytic agent E shows in figure 3.

Comparative example 3

Whole blood C reactive protein detection hemolytic agent F is prepared according to following formula：

Saponin(e 4.0g

12 water 11.6g of disodium hydrogen phosphate

Two water 1.18g of sodium dihydrogen phosphate

According to method substantially the same manner as Example 1, the calibration curve of hemolytic agent F is set up, is differed only in and is adopted hemolytic agent Hemolytic agent A in F alternative embodiments 1, and after hemolytic agent is mixed with sample stand haemolysis process time be reduced to 30 seconds, The calibration curve of hemolytic agent F shows in figure 3.

From figure 3, it can be seen that for the calibration object of same concentration is tested, the degree of reaction numerical value tested using hemolytic agent E is notable Higher than hemolytic agent F, and then, hemolytic agent E can significantly increase the immune response effect of C reactive protein relative to hemolytic agent F. Thus, it is possible to reach a conclusion, borate buffer system can effectively strengthen immunoturbidimetry survey relative to other buffer systems Immune response in fixed, and saponin(e can be effective as the surfactant of hemolytic agent and borate buffer system joins Close and use, haemolysis process is effectively carried out in 30 seconds, and strengthens the immune response effect of C reactive protein.

Performance test

1st, immune response test：

Using hemolytic agent E and F, according to 3 methods described of embodiment, the clinical anticoagulated whole blood test result of test is compared, as a result It is as shown in the table, as a result show hemolytic agent E the humidification of immune response is made its testing result have higher sensitivity and Test limit.

The immune response test result contrast of hemolytic agent E and hemolytic agent F finds that the low value sample for below 3mg/L is molten Blood agent E test results are significantly higher than hemolytic agent F test results, even for the sample of below 1mg/L, equally can be more effectively Ground detection is distinguished, therefore the borate buffer system in hemolytic agent E, can effectively be strengthened immune response and be improved low value detection Sensitivity.

2nd, test linear

Using hemolytic agent E, according to 3 methods described of embodiment, the sample that known C reactive protein concentration is 310mg/L is taken, Gradient dilution is carried out using physiological saline, each point test three times calculates the recurrence side of test result mean value and theoretical concentration Journey and regression coefficient r square, linear regression curves are as shown in figure 4, test result is as follows：

Theoretical concentration	Test 1	Test 2	Test 3	Mean value
					0.0	0.0	0.1	0.0	0.0
62.5	62.2	62.8	62.0	62.3
					124.6	125.1	124.3	125.6	125.0
188.9	192.7	191.5	190.3	191.5
					254.3	251.2	250.3	252.1	251.2
310.0	319.9	316.4	314.1	316.8

Regression equation：With theoretical concentration as x, the mean value of test concentrations is y, y=1.012x-0.7835, regression coefficient r Square=0.9993, linear test result shows method of the present invention function admirable.

3rd, anti-interference evaluation

Using hemolytic agent E, according to the method described in embodiment 3, check sample and chaff interference sample are tested into 3 respectively It is secondary, with the test average of check sample, the test average of chaff interference sample and the relative deviation of check sample are calculated, as a result such as Shown in following table, test result be less than ± 10%, show that this method interference free performance is excellent.

4th, methodology compares (whole blood-serum)

Using hemolytic agent E, according to the method described in embodiment 3, will be with group sample hemolytic agent E and auspicious super quick C- advanced in years Reactive protein (HS-CRP) determines kit (latex immunoturbidimetry), and (certificate of registry is numbered：Guangdong food medicine prison tool (standard) word 2,011 the No. 2400474) methodology contrastive test is carried out, as a result as shown in figure 5, being determined with stepping auspicious High-sensitivity C reactive protein (HS-CRP) Kit results are x-axis, and the test result of embodiment 3 is y-axis, and equation of linear regression y=0.9716x+0.2966 is returned R square of coefficient=0.9948, illustrates that this method is equivalent with clinical commercialized product.

Comparative example 4

Whole blood C reactive protein detection hemolytic agent G is prepared according to following formula：

Saponin(e 30.0g

12 water 11.6g of disodium hydrogen phosphate

Two water 1.18g of sodium dihydrogen phosphate

According to method substantially the same manner as Example 1, differ only in using the hemolytic agent in hemolytic agent G alternative embodiments 1 A, and stand the time that haemolysis processes after hemolytic agent is mixed with sample and be reduced to 10 seconds, test concentration known calibration object is set up Concentration known quality-control product, quality-control product deviation such as following table, more than ± 10% are tested after calibration curve：

As can be seen here, when the consumption of saponin(e reaches 30 g/l, although rapidly can complete haemolysis in 10 seconds and process, But if directly mixed with immunoreagent after do not dilute test solution, then immunoturbidimetry is determined and can be interfered, relative deviation Than larger.

Embodiment 4

It is formulated as follows whole blood C reactive protein detection buffer solution H：

Disodium hydrogen phosphate. 12 water 11.6g

Sodium dihydrogen phosphate. two water 1.18g

Using deionized water dissolving said components, and adjusted to after 7.0 using appropriate NaOH or HCl, supplement is gone Ionized water is to final volume 1L

According to the method essentially identical with comparative example 4, quality-control product deviation is determined, wherein, difference is to add C- reactions Before protein immunization reagent, increase a step, after addition 0.2mL buffer solutions H mixes 80 seconds, add 0.2mL C Reactive protein immunoreagent, that is, add immunoreagent after diluting test solution.Quality-control product bias contribution such as following table, no more than ± 10%：

It can thus be seen that directly mixing with immunoreagent after do not dilute test solution, then immunoturbidimetry is determined and can be interfered, Relative deviation after dilution is adding immunoreagent by the test solution after haemolysis, can reduce the interference, significantly than larger Reduce relative deviation.

Embodiment 5

Whole blood C reactive protein detection hemolytic agent I is prepared according to following formula：

Saponin(e 30.0g

Boric acid 15.0g

4- HEPES sodium 9.5g

Using deionized water dissolving said components, and adjusted to after 7.0 using appropriate NaOH or HCl, supplement is gone Ionized water is to final volume 1L；

According to method described in embodiment 1, hemolytic agent A used by embodiment 1 is replaced with into hemolytic agent I only, and by hemolytic agent It is kept to 10 seconds with sample incorporation time, other specification is constant, after test concentration known calibration object sets up calibration curve, test is known Concentration quality-control product, quality-control product deviation such as following table, no more than ± 10%：

It can thus be seen that directly mixing with immunoreagent after do not dilute test solution, then immunoturbidimetry can be determined and cause to do Disturb, and by using borate buffer system is adopted in hemolytic agent, the interference can be effectively reduced, phase is significantly reduced It is to deviation, similar with the result of dilution step is added, but lower test limit and faster detection speed can be obtained.

Embodiment 6

Whole blood C reactive protein detection hemolytic agent J is prepared according to following formula：

Saponin(e 0.1g

Boric acid 2.7g

4- HEPES sodium 1.4g

According to method described in embodiment 1, hemolytic agent A used by embodiment 1 is replaced with into hemolytic agent J only, sample volume changes For 4 microlitres, and hemolytic agent and sample incorporation time are extended for 90 seconds, other specification is constant, test concentration known calibration object Concentration known quality-control product, quality-control product deviation such as following table, no more than ± 10% are tested after setting up calibration curve：

Thus, it is possible to find out when the consumption of saponin(e is relatively low, it is only necessary to the time of proper extension haemolysis process, it becomes possible to realize Effectively haemolysis is processed, and resulting hemolysate can't be determined to immunoturbidimetry and be interfered.

Embodiment 7

Whole blood C reactive protein detection hemolytic agent K is prepared according to following formula：

Saponin(e 0.5g

Boric acid 2.7g

4- HEPES sodium 1.4g

According to method described in embodiment 1, hemolytic agent A used by embodiment 1 is replaced with into hemolytic agent K only, and by hemolytic agent It is extended for 80 seconds with sample incorporation time, other specification is constant, test concentration known calibration object is tested after setting up calibration curve Know concentration quality-control product, quality-control product deviation such as following table, no more than ± 10%：

Embodiment 8

Configuration is as shown in the table to prepare whole blood C reactive protein detection hemolytic agent L-Q according to following formula, according to embodiment 1 Described in method, hemolytic agent A used by embodiment 1 is replaced with into hemolytic agent L-Q only, test concentration known calibration object sets up mark Concentration known quality-control product is tested after directrix curve, quality-control product deviation is no more than ± 10%.

Thus, it is possible to it is that comparison is wide to find out using the scope of application of borate buffer system.

In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specific example ", Or the description of " some examples " etc. means the specific features, structure, material or the feature bag that describe with reference to the embodiment or example It is contained at least one embodiment or example of the present invention.In this manual, to the schematic representation of above-mentioned term necessarily It is directed to identical embodiment or example.And, the specific features of description, structure, material or feature can be arbitrary Combined in individual or multiple embodiments or example in an appropriate manner.Additionally, in the case of not conflicting, the skill of this area The feature of the different embodiments or example described in this specification and different embodiments or example can be combined by art personnel And combination.

Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment be it is exemplary, It is not considered as limiting the invention, one of ordinary skill in the art within the scope of the invention can be to above-described embodiment It is changed, changes, replacing and modification.

Claims

1. purposes of the borate buffer system in hemolytic agent is prepared, the hemolytic agent are determined for immunoturbidimetry,

Optionally, the immunoturbidimetry is determined includes C reactive protein detection, Procalcitonin detection, DDi detection, Guang At least one of chalone C detections, troponin detection and glycosylated hemoglobin detection.

2. it is a kind of for immunoturbidimetry determine hemolytic agent, it is characterised in that contain：

Surfactant；

Borate buffer system；And

Water,

Wherein, the pH value of the hemolytic agent is 5～9,

Optionally, the immunoturbidimetry is determined for detecting C reactive protein.

3. hemolytic agent according to claim 2, it is characterised in that the surfactant is non-ionic surfactant Agent,

Optionally, the nonionic surface active agent is included selected from saponin(e, logical Qula, tween, Brij and Tetronic At least one,

Optionally, the surfactant is saponin(e, and the cumulative volume based on the hemolytic agent, the surfactant Content is 0.01～30 g/l,

Optionally, the content of the surfactant is 0.1～30 g/l,

Optionally, the content of the surfactant is 0.5～30 g/l.

4. hemolytic agent according to claim 2, it is characterised in that the borate buffer system contains：

Boric acid；And

Salt of weak acid, the salt of weak acid are included selected from borate, phosphate, hydrophosphate, dihydric phosphate, organic sulfonate With at least one of organic carboxylate,

Optionally, the salt of weak acid be alkali metal salt, particular certain cancers or sylvite,

Optionally, the borate includes at least one selected from sodium tetraborate, Boratex and potassium borate,

Optionally, the sodium tetraborate is provided in the form of borax,

Optionally, the organic sulfonate includes 2- morpholino ethyl sulfonic acids, 3- N-morpholinyls, 4- HEPESs With the sodium salt or sylvite of-two ethyl sulfonic acid of piperazine-Isosorbide-5-Nitrae,

Optionally, the boric acid and the weight ratio of the salt of weak acid are 1:0.2～5.

5. a kind of preprocess method of biological sample, the biological sample are determined for immunoturbidimetry, it is characterised in that included：

The biological sample is made to contact with the hemolytic agent described in any one of claim 2～4,

Optionally, the immunoturbidimetry determine for detect C reactive protein, Procalcitonin, DDi, bladder chalone C, At least one of troponin and glycosylated hemoglobin,

Optionally, the biological sample includes at least one selected from whole blood, cerebrospinal fluid, body fluid, serum and blood plasma,

Optionally, the whole blood includes at least one selected from Peripheral whole blood and anti-freezing venous whole,

Optionally, the contact is carried out by the whole blood is mixed under 37 degrees Celsius with the hemolytic agent,

Optionally, the contact is carried out by the whole blood is added in the hemolytic agent,

Optionally, the contact carries out 10～90 seconds, preferably 20～50 seconds.

6. method according to claim 5, it is characterised in that the hemolytic agent with the volume ratio of the whole blood is 1:10～80, preferably 1:20～50.

7. in a kind of biological specimen object content assay method, it is characterised in that include：

The biological specimen is contacted with hemolytic agent, to obtain test solution, the hemolytic agent contains surfactant and boric acid is slow Rush system；And

By the test solution and the incubation of the immunoreagent containing object antibody, the determination target is determined to carry out immunoturbidimetry Thing content.

8. method according to claim 7, it is characterised in that the biological sample include selected from whole blood, cerebrospinal fluid, At least one of body fluid, serum and blood plasma,

Optionally, the object be C reactive protein, and the immunoturbidimetry determine include：

A the test solution is mixed by () with immunoreagent, the immunoreagent contains the antibody of specific recognition C reactive protein；

Mixture absorbance change within a predetermined period of time for predetermined wavelength light obtained by (b) determination step (a)；And

C () determines the object content based on the absorbance change,

Optionally, in step (a), directly mix the test solution not diluted with the immunoreagent, and/or

The antibody is supported on carrier, and the carrier includes at least one selected from latex, magnetic bead and collaurum,

Optionally, step (c) is further included：

Based on the absorbance change, the rate of change of the biological sample is determined；With

Based on the rate of change of the biological sample, using the calibration curve for pre-building, the object content is determined,

Optionally, further include：

Converted using blood cell volume accounting, the object content is corrected.

9. it is a kind of for immunoturbidimetry determine kit, it is characterised in that contain：

Surfactant；And

Borate buffer system,

Wherein, the surfactant and the borate buffer system be as defined in any one of claim 2～4,

Optionally, further include：

Weak bronsted lowry acids and bases bronsted lowry, the weak bronsted lowry acids and bases bronsted lowry are suitable to generate the salt of weak acid by neutralization reaction.

10. kit according to claim 9, it is characterised in that further include：

Antibody, the antibody specificity recognize object,

Optionally, the object includes C reactive protein,

Optionally, the antibody is supported on carrier, and the carrier includes latex, magnetic bead, collaurum,

Optionally, at least a portion of the component of the kit is arranged in independent container.