CN103777006B - A kind of immune complex cushions dissociate agent and application thereof - Google Patents
A kind of immune complex cushions dissociate agent and application thereof Download PDFInfo
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Abstract
The present invention relates to medical science, disclose a kind of immune complex buffering to dissociate agent, comprise CIC separating agent, CIC washing agent, CIC double solvents, CIC antigen (or antibody) damping fluid dissociate agent and CIC antigen (or antibody) damping fluid dissociates neutralizing agent.Invention also discloses and utilize the agent of dissociating of this kind of immune complex buffering to detect the method for CIC, by being separated, washing, redissolve, dissociate CIC, neutralization, the antigen finally in detection sample CIC or antibody.The present invention utilizes the agent of dissociating of this kind of immune complex buffering to detect the method for CIC, and establishing the Standard Operating Procedure of antibody in the Standard Operating Procedure of antigen in specific detection CIC and specific detection CIC, is all insurmountable problem of existing CIC detection method.
Description
Technical field
The present invention relates to medical science, particularly relate to a kind of immune complex and cushion agent of dissociating, be also specifically related to this kind of immune complex and cushioned the application that agent of dissociating detects CIC.
Background technology
The pathogen such as bacterium, virus invades after body or sensitizer (antigen, containing autoantigen) stimulate body generation immune response, and then produce specific immunity effector cell or antibody, and with antigen generation specific binding, the compound obtained is called that immune complex is (also known as antigen antibody complex, immunecomplex, IC), mainly contain IgG, IgM, IgA, IgE type immune complex, wherein the most common with IgG and IgM.Because antigen is different from antibody ratios, the IC molecular size formed is different, usually has three kinds of forms: one be antigen-antibody ratio suitable time, form macromolecular insoluble IC (being greater than 19S), easily caught by phagocyte, engulf and remove.Two be antigen amount superfluous time, form micromolecular solubility IC (being less than 6.6S), easily excrete with urine through glomerular filtration.Three is that antigen amount is little over time surplus, form medium sized solubility IC (8.8-19S), it is neither removed by phagocyte, do not discharge by glomerular filtration again, can be free on the long period in blood and other body fluid, also known as CIC ELISA (circulatingimmunocomplex, CIC).
Under normal circumstances, the free antigen in body is combined with corresponding antibodies and forms IC, can be removed by the system of defense of body, as a kind of mode removing foreign matter antigen, favourable to body.But under some pathologic condition, the IC formed in body can not be removed in time, can in local deposits, pass through activating complement, and under blood platelet, neutrophil leucocyte etc. participate in, cause a series of chain reaction and cause tissue damage, occurring clinical symptoms, be called immune complex disease (immunocomplexdisease, ICD).Check tissue in or instrument for circulation of body fluid in IC have the diagnosis helping some disease, pathogenetic research, prognosis evaluation, state of an illness movable observing and Outcome measure etc.Therefore, accurate, special, Sensitive Detection immune complex has important clinical meaning to the diagnosis of disease, treatment and prognosis evaluation, and the detection of CIC comes into one's own day by day.
The World Health Organization (WHO) (WHO) once organized and carried out CIC detection side jurisprudential study, found that there is no any one technology possesses special, sensitive, that batch is efficient, reproducible all features simultaneously.Due to the complicacy of method, susceptibility and detect the limitation of CIC type, current conventional CIC detection technique is all subject to immune globulin classes and subclass, compound size, antigen and antibody ratios in immune complex, the factor such as ability of complement-fixing affects, and is very limited in clinical immunology laboratory applications.
Summary of the invention
The present invention is directed in existing CIC detection technique and testing process, special, sensitive, efficient, reproducible, the interference-free technical matters of batch can not be possessed simultaneously, disclose a kind of immune complex and cushion agent of dissociating, also disclose the application that this kind of immune complex cushions agent of dissociating.And then solve above technical matters.
In order to solve the problems of the technologies described above, the present invention is solved by following technical proposals.
One, for the part of antigen in CIC:
A kind of immune complex cushions agent of dissociating, in following volume ratio proportioning, each component and content is, the dissociate CIC Antigen buffer of agent and 65 μ l-76 μ l of the CIC separating agent of 135 μ l-165 μ l, the CIC washing agent of 290 μ l-315 μ l, the CIC double solvents of 5 μ l-16 μ l, the CIC Antigen buffer of 65 μ l-76 μ l dissociates neutralizing agent.For the HBsAg-Anti-HBs antibody immune complex of preparation, agent prescription of the present invention can make the antigen dissociation yield in CIC reach 76.5%-83.8% (antigen dissociation yield: the number percent referring to antigen amount in the antigen amount that detects after CIC dissociates and actual CIC).And protease digestion method, strong acid dissociate, method, surfactant dissociate the antigen dissociation yield of method all at below 10%-20%.
Wherein, the CIC separating agent of every 1L comprises following component: the polyglycol of the borax of 5.2g-7.7g, the boric acid of 4.1g-6.1g, 70g-90g, the sodium chloride of 6.2g-9.2g, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water.The effect of CIC separating agent is as follows: when the CIC separating agent of every 1L comprise the borax of 5.2g-7.7g, the boric acid of 4.1g-6.1g, the polyglycol of 70g-90g, the sodium chloride of 6.2g-9.2g, the liquid bio antiseptic of 100 μ l-500 μ l, surplus be distilled water time, separated CIC reaches maximum, uses sodium chloride simultaneously instead and also can avoid sodium fluoride in traditional intermediate processing and remain the interference to subsequent detection antigen.The polyglycol that the present invention adopts, as preferably selecting Macrogol 6000; The liquid bio antiseptic mentioned in the present invention is Proclin-300.
The CIC washing agent of every 1L comprises following component: the polyglycol of the borax of 5.2g-7.7g, the boric acid of 4.1g-6.1g, 35g-45g, the sodium chloride of 8.0g-11.7g, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water.The CIC washing agent of more than filling a prescription designs mainly for the formula of CIC separating agent, and its effect washes away foreign protein unnecessary in humoral specimen.The polyglycol that the present invention adopts, as preferably selecting Macrogol 6000; The liquid bio antiseptic mentioned in the present invention is Proclin-300.
The CIC double solvents of every 1L comprises following component: the Triton X-100 of the sodium chloride of 8.0g-9.5g, the NaOH of 0.01g-0.03g, 50 μ l-500 μ l, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water.CIC double solvents designs for buffering capacity size when dissociating, and the degree of injury of CIC double solvents to separation of C IC of above formula is minimum; Adopt isotonic Triton X-100, the Triton X-100 of low concentration assists to dissolve CIC; Triton X-100 of the present invention is TritonX-100 again; The liquid bio antiseptic mentioned in the present invention is Proclin-300.
As preferably, the agent of dissociating of the CIC Antigen buffer of every 1L comprises following component: the glycocoll of 3.5g-5.5g, 9.0ml-15.0ml massfraction are the concentrated hydrochloric acid of 35%-37%, the sodium chloride of 0.35g-0.51g, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water.The agent of dissociating of CIC Antigen buffer makes antigen in CIC reach farthest to dissociate.As with preparation HBsAg-Anti-HBs antibody immune complex for reference, antigen dissociation yield reaches 76.5%-83.8%; The liquid bio antiseptic mentioned in the present invention is Proclin-300.
As preferably, the CIC Antigen buffer of the every 1L neutralizing agent that dissociates comprises following component: the tromethamine of 16g-24g, the sodium chloride of 5.7g-8.6g, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water.The CIC Antigen buffer neutralizing agent that dissociates mainly neutralizes the antigen after CIC dissociates, and makes osmotic pressure, environmental optima that pH reaches antigen, is beneficial to the detection to antigen; The liquid bio antiseptic mentioned in the present invention is Proclin-300.
The agent of dissociating of above-mentioned this kind of immune complex buffering is applied to the detection of antigen in the CIC suffering from infectious diseases, endocrine metabolism disease, autoimmune disease, the serum of tumor disease patient, Pleural effusions, urine, cerebrospinal fluid, arthral fluid.Corresponding CIC whether is there is in the humoral specimen such as serum, Pleural effusions, urine, cerebrospinal fluid, arthral fluid of the patients such as infectious diseases, endocrine metabolism disease, autoimmune disease, Partial tumors disease, we can pass through separation of C IC, thus infer the CIC and content height that whether there is corresponding Specific marker in the body fluid such as patients serum, Pleural effusions, urine, cerebrospinal fluid, arthral fluid, to understand the pathophysiological role of CIC in above-mentioned disease development process and correlativity; If the antigen in corresponding CIC or antibody (under normal circumstances, antigen dissociation yield is higher than antibody dissociation rate) detected in the negative humoral specimen of Research of predicting markers, then can improve the recall rate of this mark and the diagnostic sensitivity to corresponding disease.
Utilize above-mentioned immune complex to cushion to dissociate agent to detect a method of CIC, detect the method for antigen in CIC, for HBsAg-Anti-HBs antibody immune complex, be made up of following step,
A. be separated: the centrifuge tube to 2 1ml adds the sample to be tested that 150 μ l contain CIC respectively, then adds the CIC separating agent of 135 μ l-165 μ l respectively; After mixing, place 30min at 37 DEG C, carry out centrifugal, abandoning supernatant after centrifugal end;
B. wash: in two centrifuge tubes, add the CIC washing agent of 290 μ l-315 μ l respectively, after centrifugal, abandoning supernatant, and CIC sediment is drained;
C. redissolve: respectively to containing in the sedimentary centrifuge tube of the CIC drained, add the CIC double solvents of 5 μ l-16 μ l, centrifuge tube is installed on oscillator, vibrates to solution without lumpy precipitate;
D. blank sample detects: a centrifuge tube is labeled as blank sample pipe, and the Antigen buffer adding 65 μ l-76 μ l dissociates, the Antigen buffer of agent and 65 μ l-76 μ l dissociates neutralizing agent, detects HBsAg, using testing result as blank value after mixing;
E.CIC antigen dissociates: another centrifuge tube is labeled as working sample pipe, the agent and the Antigen buffer adding 65 μ l-76 μ l dissociates, and hatches and places 30min or place 30min 42 DEG C of water-baths, obtain dissociation solution at 56 DEG C of air;
F.CIC antigen dissociates neutralization: in the dissociation solution obtained in step e, adds 65 μ l-76 μ l Antigen buffer and to dissociate neutralizing agent, detect HBsAg, using testing result as measured value after mixing.
Antigen testing result criterion after HBsAg-Anti-HBs antibody immune complex dissociates, as shown in the table:
Antigen testing result criterion after table 1HBsAg-Anti-HBs antibody immune complex dissociates
Note: detect (chemoluminescence method) on Abbott Laboratories of U.S. i2000 immunity analysis instrument, Cutoff value=0.05IU/ml
This kind utilize immune complex cushion dissociate agent detect CIC method by be separated, washing, redissolve, dissociate CIC, neutralization, then (electricity) chemoluminescence method is adopted, western blot test, the clinical immunology laboratory conventional methods such as enzyme linked immunosorbent assay are carried out special sensitive, quantitatively or half-quantitative detection antigen, whole experiment flow fully takes into account temperature, osmotic pressure, pH environment is to CIC, antigen, the impact of antibody, establish the Standard Operating Procedure of antigen in specific detection CIC (after dissociating), this is all insurmountable problem of current existing CIC detection method.
Two, for the part of antibody in CIC:
A kind of immune complex cushions agent of dissociating, in following volume ratio proportioning, each component and content is, the dissociate CIC antibodies buffer of agent and 65-76 μ l of the CIC separating agent of 135-165 μ l, the CIC washing agent of 290-315 μ l, the CIC double solvents of 5-16 μ l, the CIC antibodies buffer of 65-76 μ l dissociates neutralizing agent.For the HBsAg-Anti-HBsAg antibody immune complex of preparation, agent prescription of the present invention can make the antibody dissociation rate in CIC reach 37.5%-51.0% (antibody dissociation rate: the number percent referring to antibody amount in the antibody that detects after CIC dissociates and actual CIC).And existing CIC detection technique is because antibody is destroyed or the existence of some disturbing factors, the antibody in CIC cannot be detected.
Wherein, the CIC separating agent of every 1L comprises following component: the polyglycol of the borax of 5.2g-7.7g, the boric acid of 4.1g-6.1g, 70g-90g, the sodium chloride of 6.2g-9.2g, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water.The effect of CIC separating agent is as follows: when the CIC separating agent of every 1L comprise the borax of 5.2g-7.7g, the boric acid of 4.1g-6.1g, the polyglycol of 70g-90g, the sodium chloride of 6.2g-9.2g, the liquid bio antiseptic of 100 μ l-500 μ l, surplus be distilled water time, separated CIC reaches maximum, uses sodium chloride simultaneously instead and it also avoid sodium fluoride in traditional intermediate processing and remain the interference to subsequent detection antibody.The polyglycol that the present invention adopts, as preferably selecting Macrogol 6000; The liquid bio antiseptic mentioned in the present invention is Proclin-300.
The CIC washing agent of every 1L comprises following component: the polyglycol of the borax of 5.2g-7.7g, the boric acid of 4.1g-6.1g, 35g-45g, the sodium chloride of 8.0g-11.7g, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water.The CIC washing agent of more than filling a prescription designs mainly for the formula of CIC separating agent, and its effect washes away foreign protein unnecessary in humoral specimen.The liquid bio antiseptic mentioned in the present invention is Proclin-300.
The CIC double solvents of every 1L comprises following component: the Triton X-100 of the sodium chloride of 8.0g-9.5g, the NaOH of 0.01g-0.03g, 50 μ l-500 μ l, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water.CIC double solvents designs for buffering capacity size when dissociating, and the degree of injury of CIC double solvents to separation of C IC of above formula is minimum; Adopt isotonic Triton X-100, the Triton X-100 of low concentration assists to dissolve CIC.Triton X-100 of the present invention is TritonX-100 again, and the liquid bio antiseptic mentioned in the present invention is Proclin-300.
As preferably, the agent of dissociating of the CIC antibodies buffer of every 1L comprises following component: the glycocoll of 3.5-5.5g, 4.0ml-6.0ml massfraction are the concentrated hydrochloric acid of 35%-37%, the sodium chloride of 4.4g-6.6g, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water.The agent of dissociating of CIC antibodies buffer makes antibody in CIC reach farthest to dissociate.As with preparation HBsAg-Anti-HBs antibody immune complex for reference, antibody dissociation rate reaches 37.5%-51.0%.The liquid bio antiseptic mentioned in the present invention is Proclin-300.
As preferably, the CIC antibodies buffer of the every 1L neutralizing agent that dissociates comprises following component: the tromethamine of 6.8g-10.0g, the sodium chloride of 6.5g-9.6g, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water.Antibodies buffer that neutralizing agent mainly dissociates to the CIC agent of dissociating of dissociating of CIC antibodies buffer neutralizes, and makes osmotic pressure, environmental optima that pH reaches antibody, is beneficial to the detection of antagonist.The liquid bio antiseptic mentioned in the present invention is Proclin-300.
The agent of dissociating of above-mentioned this kind of immune complex buffering is applied to the detection of antibody in the CIC suffering from infectious diseases, endocrine metabolism disease, autoimmune disease, the serum of tumor disease patient, Pleural effusions, urine, cerebrospinal fluid, arthral fluid.Corresponding CIC whether is there is in the humoral specimen such as serum, Pleural effusions, urine, cerebrospinal fluid, arthral fluid of the patients such as infectious diseases, endocrine metabolism disease, autoimmune disease, Partial tumors disease, we can pass through separation of C IC, thus infer the CIC and content height that whether there is corresponding Specific marker in the body fluid such as patients serum, Pleural effusions, urine, cerebrospinal fluid, arthral fluid, to understand the pathophysiological role of CIC in above-mentioned disease development process and correlativity; If the antigen in corresponding CIC or antibody (under normal circumstances, antigen dissociation yield is higher than antibody dissociation rate) detected in the negative humoral specimen of Research of predicting markers, then can improve the recall rate of this mark and the diagnostic sensitivity to corresponding disease.
Utilize the agent of dissociating of above immune complex buffering to detect a method of CIC, detect the method for antibody in CIC, for HBsAg-Anti-HBs antibody immune complex, be made up of following step,
A. be separated: the centrifuge tube to 2 1ml adds the sample to be tested that 150 μ l contain CIC respectively, then adds the CIC separating agent of 135 μ l-165 μ l respectively; After mixing, place 30min at 37 DEG C, carry out centrifugal, abandoning supernatant after centrifugal end;
B. wash: in two centrifuge tubes, add the CIC washing agent of 290 μ l-315 μ l respectively, after centrifugal, abandoning supernatant, and CIC sediment is drained;
C. redissolve: respectively to containing in the sedimentary centrifuge tube of the CIC drained, add the CIC double solvents of 5 μ l-16 μ l, centrifuge tube is installed on oscillator, vibrates to solution without lumpy precipitate;
D. blank sample detects: a centrifuge tube is labeled as blank sample pipe, and the antibodies buffer adding 65 μ l-76 μ l dissociates, the antibodies buffer of agent and 65 μ l-76 μ l dissociates neutralizing agent, detects Anti-HBs antibody, using testing result as blank value after mixing;
E.CIC antibody dissociation: another centrifuge tube is labeled as working sample pipe, and add 65 μ l-76 μ l antibodies buffer and to dissociate agent, place 30min at 15 DEG C, obtain dissociation solution; (hatch at 15 DEG C of air and place 30min or place 30min 15 DEG C of DEG C of water-baths)
F.CIC antibody dissociation neutralizes: in the dissociation solution obtained in step e, adds 65 μ l-76 μ l antibodies buffer and to dissociate neutralizing agent, detect Anti-HBs antibody, using testing result as measured value after mixing.
Antibody test result criterion after HBsAg-Anti-HBs antibody immune complex dissociates, as shown in the table:
Antibody test result criterion after table 2HBsAg-Anti-HBs antibody immune complex dissociates
Note: detect (chemoluminescence method) on Abbott Laboratories of U.S. i2000 immunity analysis instrument, Cutoff value=10mIU/ml
This kind utilize immune complex cushion dissociate agent detect CIC method by be separated, washing, redissolve, dissociate CIC, neutralization, then (electricity) chemoluminescence method is adopted, western blot test, the clinical immunology laboratory conventional methods such as enzyme linked immunosorbent assay are carried out special sensitive, quantitatively or half-quantitative detection antibody, whole experiment flow fully takes into account temperature, osmotic pressure, pH environment is to CIC, antigen, the impact of antibody, establish the Standard Operating Procedure of antibody in specific detection CIC (after dissociating), this is all insurmountable problem of current existing CIC detection method.
Compared with prior art, beneficial effect of the present invention is:
(1) present invention improves in prior art, the shortcoming of CIC detection technique poor specificity.Antigen in direct-detection CIC or antibody.
(2) present invention improves in prior art, the shortcoming that the sensitivity of CIC detection technique is low, to be dissociated antigen in CIC or antibody by the agent of dissociating of immune complex buffering, again by the antigen in specific detection CIC or antibody, make antigen, level that antibody test result reaches ng/ml and mIU/ml, thus accurately calculate CIC content.
(3) the present invention is reproducible, is applicable to batch and uses, improve work efficiency.Repeatability is suitable with the degree of variation of clinical immunology laboratory conventional sense antigen or antibody, once dissociates and multiple method can be utilized to detect multiple project.
(4) the invention solves existing CIC detection technique and can not possess special, sensitive, that batch is efficient, reproducible problem simultaneously.The present invention both can special sensitive quantitative detection CIC in antigen or antibody; Batch detection (as detected the HBsAg-Anti-HBs antibody immune complex in many parts of serum specimens simultaneously) can be carried out again to same CIC in many parts of humoral specimens; Can also to carrying out efficient detection (as to immune complexs such as the HBsAg-Anti-HBs antibody in a serum specimen, anti-HBe, HCV-cAg-anti-HCV of HBeAg-, HIV-P24-anti-HIV, or IgM, IgG, IgA type immune complex detects) with CIC multiple in a humoral specimen simultaneously.In addition, the present invention does not need special instrument and equipment, is applicable to the antigen of clinical immunology laboratory routine, antibody test project; Really accomplish once to dissociate, detect multiple project simultaneously, repeatability (CV value) reaches below 15%-20%.
(5) this invention removes the interfering material in antigen in existing CIC detection technique or antibody test process.The present invention to dissociate neutralizing agent by using CIC antibodies buffer, has ensured in antigen or antibody test process without any material interference detection results.
(6) present invention improves over protease digestion method, strong acid dissociate method, the shortcoming of surfactant dissociates method only can detect free antigen or negative antibody sample.The present invention no matter be negative to free antigen or antibody or positive sample all applicable, the present invention, by being separated from humoral specimen by CIC, avoids the interference to the antigen in separation of C IC or antibody test of free antigen in humoral specimen or antibody positive.
(7) present invention, avoiding osmotic pressure, pH to the impact of antigen and antibody.The present invention makes the osmotic pressure of detection system, pH value is in antigen or the environmental optima of antibody, thus avoids osmotic pressure, pH to the impact of antigen and antibody.
(8) the present invention is applicable to the detection of low concentration CIC.The present invention can be separated by CIC, adjust the object that the volume that dissociates reaches concentrated CIC, thus substantially increases the recall rate of antigen or antibody in CIC.
(9) above-mentioned immune complex is the present invention also relates to utilize to cushion to dissociate agent to detect the method for CIC, by being separated, washing, redissolve, dissociate CIC, neutralization, the CIC finally in detection sample.Not only establish the Standard Operating Procedure of antigen in specific detection CIC (after dissociating), also establish the Standard Operating Procedure of antibody in specific detection CIC (after dissociating), this is all insurmountable problem of current existing CIC detection method.
(10) the present invention is adapted at clinical immunology laboratory conventional sense.Sample disposal relative ease, be operate in current existing CIC detection technique more convenient, can popularize in clinical labororatory, method to medical diagnosis on disease most worthy.
Embodiment
Embodiment 1
A kind of immune complex cushions agent of dissociating, in following volume ratio proportioning, each component and content is, the dissociate CIC Antigen buffer of agent and 65 μ l of the CIC separating agent of 135 μ l, the CIC washing agent of 290 μ l, the CIC double solvents of 5 μ l, the CIC Antigen buffer of 65 μ l dissociates neutralizing agent.
Wherein, the CIC separating agent of every 1L comprises following component: the polyglycol of the borax of 5.2g, the boric acid of 4.1g, 70g, the sodium chloride of 6.2g, the liquid bio antiseptic of 100 μ l, surplus are distilled water.
The CIC washing agent of every 1L comprises following component: the polyglycol of the borax of 5.2g, the boric acid of 4.1g, 35g, the sodium chloride of 8.0g, the liquid bio antiseptic of 100 μ l, surplus are distilled water.
The CIC double solvents of every 1L comprises following component: the Triton X-100 of the sodium chloride of 8.0g, the NaOH of 0.01g, 50 μ l, the liquid bio antiseptic of 100 μ l, surplus are distilled water.
The agent of dissociating of the CIC Antigen buffer of every 1L comprises following component: the glycocoll of 3.5g, 9.0ml massfraction be 35% concentrated hydrochloric acid, the sodium chloride of 0.35g, the liquid bio antiseptic of 100 μ l, surplus be distilled water.
The CIC Antigen buffer of the every 1L neutralizing agent that dissociates comprises following component: the tromethamine of 16g, the sodium chloride of 5.7g, the liquid bio antiseptic of 100 μ l, surplus are distilled water.
Utilize above-mentioned immune complex to cushion to dissociate agent to detect a method of CIC, detect the method for antigen in CIC, for HBsAg-Anti-HBs antibody immune complex, be made up of following step,
A. be separated: the centrifuge tube to 2 1ml adds 150 μ l Pleural effusions sample to be measured respectively, then adds the CIC separating agent of 135 μ l respectively; After mixing, place 30min at 37 DEG C, carry out centrifugal, abandoning supernatant after centrifugal end;
B. wash: in two centrifuge tubes, add the CIC washing agent of 290 μ l respectively, after centrifugal, abandoning supernatant, and CIC sediment is drained;
C. redissolve: respectively to containing in the sedimentary centrifuge tube of the CIC drained, add the CIC double solvents of 5 μ l, centrifuge tube is installed on oscillator, vibrates to solution without lumpy precipitate;
D. blank sample detects: a centrifuge tube is labeled as blank sample pipe, and the Antigen buffer adding 65 μ l dissociates, the Antigen buffer of agent and 65 μ l dissociates neutralizing agent, detects HBsAg, using testing result as blank value after mixing;
E.CIC antigen dissociates: another centrifuge tube is labeled as working sample pipe, the agent and the Antigen buffer adding 65 μ l dissociates, and hatches and places 30min, obtain dissociation solution at 56 DEG C of air;
F.CIC antigen dissociates neutralization: in the dissociation solution obtained in step e, adds 65 μ l Antigen buffer and to dissociate neutralizing agent, detect HBsAg, using testing result as measured value after mixing.
The test result of Pleural effusions sample to be measured: the blank value <Cutoff recorded, measured value >Cutoff.Namely there is CIC in Pleural effusions sample to be measured, concrete content judges according to measured value size.[note: detect (chemoluminescence method) on Abbott Laboratories of U.S. i2000 immunity analysis instrument, Cutoff value=0.05IU/ml].
Embodiment 2
A kind of immune complex cushions agent of dissociating, in following volume ratio proportioning, each component and content is, the dissociate CIC Antigen buffer of agent and 76 μ l of the CIC separating agent of 165 μ l, the CIC washing agent of 315 μ l, the CIC double solvents of 16 μ l, the CIC Antigen buffer of 76 μ l dissociates neutralizing agent.
Wherein, the CIC separating agent of every 1L comprises following component: the polyglycol of the borax of 7.7g, the boric acid of 6.1g, 90g, the sodium chloride of 9.2g, the liquid bio antiseptic of 500 μ l, surplus are distilled water.
The CIC washing agent of every 1L comprises following component: the polyglycol of the borax of 7.7g, the boric acid of 6.1g, 45g, the sodium chloride of 11.7g, the liquid bio antiseptic of 500 μ l, surplus are distilled water.
The CIC double solvents of every 1L comprises following component: the Triton X-100 of the sodium chloride of 9.5g, the NaOH of 0.03g, 500 μ l, the liquid bio antiseptic of 500 μ l, surplus are distilled water.
The agent of dissociating of the CIC Antigen buffer of every 1L comprises following component: the glycocoll of 5.5g, 15.0ml massfraction be 37% concentrated hydrochloric acid, the sodium chloride of 0.51g, the liquid bio antiseptic of 500 μ l, surplus be distilled water.
The CIC Antigen buffer of the every 1L neutralizing agent that dissociates comprises following component: the tromethamine of 24g, the sodium chloride of 8.6g, the liquid bio antiseptic of 500 μ l, surplus are distilled water.
Utilize above-mentioned immune complex to cushion to dissociate agent to detect a method of CIC, detect the method for antigen in CIC, for HBsAg-Anti-HBs antibody immune complex, be made up of following step,
A. be separated: the centrifuge tube to 2 1ml adds 150 μ l CSF sample to be measured respectively, then adds the CIC separating agent of 165 μ l respectively; After mixing, place 30min at 37 DEG C, carry out centrifugal, abandoning supernatant after centrifugal end;
B. wash: in two centrifuge tubes, add the CIC washing agent of 315 μ l respectively, after centrifugal, abandoning supernatant, and CIC sediment is drained;
C. redissolve: respectively to containing in the sedimentary centrifuge tube of the CIC drained, add the CIC double solvents of 16 μ l, centrifuge tube is installed on oscillator, vibrates to solution without lumpy precipitate;
D. blank sample detects: a centrifuge tube is labeled as blank sample pipe, and the Antigen buffer adding 76 μ l dissociates, the Antigen buffer of agent and 76 μ l dissociates neutralizing agent, detects HBsAg, using testing result as blank value after mixing;
E.CIC antigen dissociates: be labeled as by another centrifuge tube and measure sample pipe, the agent and the Antigen buffer adding 76 μ l dissociates, and places 30min, obtain dissociation solution 42 DEG C of water-baths;
F.CIC antigen dissociates neutralization: in the dissociation solution obtained in step e, adds 76 μ l Antigen buffer and to dissociate neutralizing agent, detect HBsAg, using testing result as measured value after mixing.
The test result of CSF sample to be measured: the blank value <Cutoff recorded, measured value <Cutoff.Namely there is not CIC in CSF sample to be measured.[note: detect (chemoluminescence method) on Abbott Laboratories of U.S. i2000 immunity analysis instrument, Cutoff value=0.05IU/ml].
Embodiment 3
A kind of immune complex cushions agent of dissociating, in following volume ratio proportioning, each component and content is, the dissociate CIC Antigen buffer of agent and 70 μ l of the CIC separating agent of 150 μ l, the CIC washing agent of 300 μ l, the CIC double solvents of 10 μ l, the CIC Antigen buffer of 70 μ l dissociates neutralizing agent.
Wherein, the CIC separating agent of every 1L comprises following component: the polyglycol of the borax of 6.4g, the boric acid of 5.1g, 80g, the sodium chloride of 7.7g, the liquid bio antiseptic of 200 μ l, surplus are distilled water.
The CIC washing agent of every 1L comprises following component: the polyglycol of the borax of 6.4g, the boric acid of 5.1g, 40g, the sodium chloride of 9.9g, the liquid bio antiseptic of 200 μ l, surplus are distilled water.
The CIC double solvents of every 1L comprises following component: the Triton X-100 of the sodium chloride of 9.0g, the NaOH of 0.016g, 300 μ l, the liquid bio antiseptic of 200 μ l, surplus are distilled water.
The agent of dissociating of the CIC Antigen buffer of every 1L comprises following component: the glycocoll of 4.5g, 12ml massfraction be 36% concentrated hydrochloric acid, the sodium chloride of 0.43g, the liquid bio antiseptic of 200 μ l, surplus be distilled water.
The CIC Antigen buffer of the every 1L neutralizing agent that dissociates comprises following component: the tromethamine of 20g, the sodium chloride of 7.3g, the liquid bio antiseptic of 200 μ l, surplus are distilled water.
Utilize above-mentioned immune complex to cushion to dissociate agent to detect a method of CIC, detect the method for antigen in CIC, for HBsAg-Anti-HBs antibody immune complex, be made up of following step,
Utilize the agent of dissociating of immune complex described above buffering to detect a method of CIC, it is characterized in that: the method detecting antigen in CIC, is made up of following step,
A. be separated: the centrifuge tube to 2 1ml adds 150 μ l samples to be tested respectively, then adds the CIC separating agent of 150 μ l respectively; After mixing, place 30min at 37 DEG C, carry out centrifugal, abandoning supernatant after centrifugal end;
B. wash: in two centrifuge tubes, add the CIC washing agent of 300 μ l respectively, after centrifugal, abandoning supernatant, and CIC sediment is drained;
C. redissolve: respectively to containing in the sedimentary centrifuge tube of the CIC drained, add the CIC double solvents of 10 μ l, centrifuge tube is installed on oscillator, vibrates to solution without lumpy precipitate;
D. blank sample detects: a centrifuge tube is labeled as blank sample pipe, and the Antigen buffer adding 70 μ l dissociates, the Antigen buffer of agent and 70 μ l dissociates neutralizing agent, detects HBsAg, using testing result as blank value after mixing;
E.CIC antigen dissociates: another centrifuge tube is labeled as working sample pipe, the agent and the Antigen buffer adding 70 μ l dissociates, and hatches and places 30min, obtain dissociation solution at 56 DEG C of air;
F.CIC antigen dissociates neutralization: in the dissociation solution obtained in step e, adds 70 μ l Antigen buffer and to dissociate neutralizing agent, detect HBsAg, using testing result as measured value after mixing.
The test result of sample to be tested: the blank value >Cutoff recorded, measured value >Cutoff, and measured value <130% blank value.Namely there is not CIC in sample to be tested.[note: detect (chemoluminescence method) on Abbott Laboratories of U.S. i2000 immunity analysis instrument, Cutoff value=0.05IU/ml].
Embodiment 4
A kind of immune complex cushions agent of dissociating, in following volume ratio proportioning, each component and content is, the dissociate CIC antibodies buffer of agent and 65 μ l of the CIC separating agent of 135 μ l, the CIC washing agent of 290 μ l, the CIC double solvents of 5 μ l, the CIC antibodies buffer of 65 μ l dissociates neutralizing agent.
Wherein, the CIC separating agent of every 1L comprises following component: the polyglycol of the borax of 5.2g, the boric acid of 4.1g, 70g, the sodium chloride of 6.2g, the liquid bio antiseptic of 100 μ l, surplus are distilled water.
The CIC washing agent of every 1L comprises following component: the polyglycol of the borax of 5.2g, the boric acid of 4.1g, 35g, the sodium chloride of 8.0g, the liquid bio antiseptic of 100 μ l, surplus are distilled water.
The CIC double solvents of every 1L comprises following component: the Triton X-100 of the sodium chloride of 8.0g, the NaOH of 0.01g, 50 μ l, the liquid bio antiseptic of 100 μ l, surplus are distilled water.
The agent of dissociating of the CIC antibodies buffer of every 1L comprises following component: the glycocoll of 3.5g, 4.0ml massfraction be 35% concentrated hydrochloric acid, the sodium chloride of 4.4g, the liquid bio antiseptic of 100 μ l, surplus be distilled water.
The CIC antibodies buffer of the every 1L neutralizing agent that dissociates comprises following component: the tromethamine of 6.8g, the sodium chloride of 6.5g, the liquid bio antiseptic of 100 μ l, surplus are distilled water.
Utilize the agent of dissociating of above immune complex buffering to detect a method of CIC, detect the method for antibody in CIC, for HBsAg-Anti-HBs antibody immune complex, be made up of following step,
A. be separated: the centrifuge tube to 2 1ml adds 150 μ l samples to be tested respectively, then adds the CIC separating agent of 135 μ l respectively; After mixing, place 30min at 37 DEG C, carry out centrifugal, abandoning supernatant after centrifugal end;
B. wash: in two centrifuge tubes, add the CIC washing agent of 290 μ l respectively, after centrifugal, abandoning supernatant, and CIC sediment is drained;
C. redissolve: respectively to containing in the sedimentary centrifuge tube of the CIC drained, add the CIC double solvents of 5 μ l, centrifuge tube is installed on oscillator, vibrates to solution without lumpy precipitate;
D. blank sample detects: a centrifuge tube is labeled as blank sample pipe, and the antibodies buffer adding 65 μ l dissociates, the antibodies buffer of agent and 65 μ l dissociates neutralizing agent, detects Anti-HBs antibody, using testing result as blank value after mixing;
E.CIC antibody dissociation: another centrifuge tube is labeled as working sample pipe, and add 65 μ l antibodies buffer and to dissociate agent, hatch at 15 DEG C of air and place 30min, obtain dissociation solution;
F.CIC antibody dissociation neutralizes: in the dissociation solution obtained in step e, adds 65 μ l antibodies buffer and to dissociate neutralizing agent, detect Anti-HBs antibody, using testing result as measured value after mixing.
The test result of sample to be tested: the blank value >Cutoff recorded, measured value >Cutoff, and measured value <130% blank value.Namely there is not CIC in sample to be tested.[note: detect (chemoluminescence method) on Abbott Laboratories of U.S. i2000 immunity analysis instrument, Cutoff value=10mIU/ml].
Embodiment 5
A kind of immune complex cushions agent of dissociating, in following volume ratio proportioning, each component and content is, the dissociate CIC antibodies buffer of agent and 76 μ l of the CIC separating agent of 165 μ l, the CIC washing agent of 315 μ l, the CIC double solvents of 16 μ l, the CIC antibodies buffer of 76 μ l dissociates neutralizing agent.
Wherein, the CIC separating agent of every 1L comprises following component: the polyglycol of the borax of 7.7g, the boric acid of 6.1g, 90g, the sodium chloride of 9.2g, the liquid bio antiseptic of 500 μ l, surplus are distilled water.
The CIC washing agent of every 1L comprises following component: the polyglycol of the borax of 7.7g, the boric acid of 6.1g, 45g, the sodium chloride of 11.7g, the liquid bio antiseptic of 500 μ l, surplus are distilled water.
The CIC double solvents of every 1L comprises following component: the Triton X-100 of the sodium chloride of 9.5g, the NaOH of 0.03g, 500 μ l, the liquid bio antiseptic of 500 μ l, surplus are distilled water.
The agent of dissociating of the CIC antibodies buffer of every 1L comprises following component: the glycocoll of 5.5g, 6.0ml massfraction be 37% concentrated hydrochloric acid, the sodium chloride of 6.6g, the liquid bio antiseptic of 500 μ l, surplus be distilled water.
The CIC antibodies buffer of the every 1L neutralizing agent that dissociates comprises following component: the tromethamine of 10.0g, the sodium chloride of 9.6g, the liquid bio antiseptic of 500 μ l, surplus are distilled water.
Utilize the agent of dissociating of above immune complex buffering to detect a method of CIC, detect the method for antibody in CIC, for HBsAg-Anti-HBs antibody immune complex, be made up of following step,
A. be separated: the centrifuge tube to 2 1ml adds 150 μ l samples to be tested respectively, then adds the CIC separating agent of 165 μ l respectively; After mixing, place 30min at 37 DEG C, carry out centrifugal, abandoning supernatant after centrifugal end;
B. wash: in two centrifuge tubes, add the CIC washing agent of 315 μ l respectively, after centrifugal, abandoning supernatant, and CIC sediment is drained;
C. redissolve: respectively to containing in the sedimentary centrifuge tube of the CIC drained, add the CIC double solvents of 16 μ l, centrifuge tube is installed on oscillator, vibrates to solution without lumpy precipitate;
D. blank sample detects: a centrifuge tube is labeled as blank sample pipe, and the antibodies buffer adding 76 μ l dissociates, the antibodies buffer of agent and 76 μ l dissociates neutralizing agent, detects Anti-HBs antibody, using testing result as blank value after mixing;
E.CIC antibody dissociation: another centrifuge tube is labeled as working sample pipe, and add 76 μ l antibodies buffer and to dissociate agent, place 30min 15 DEG C of water-baths, obtain dissociation solution;
F.CIC antibody dissociation neutralizes: in the dissociation solution obtained in step e, adds 76 μ l antibodies buffer and to dissociate neutralizing agent, detect Anti-HBs antibody, using testing result as measured value after mixing.
The test result of sample to be tested: the blank value <Cutoff recorded, measured value >Cutoff.Namely there is CIC in sample to be tested.[note: detect (chemoluminescence method) on Abbott Laboratories of U.S. i2000 immunity analysis instrument, Cutoff value=10mIU/ml].
Embodiment 6
A kind of immune complex cushions agent of dissociating, in following volume ratio proportioning, each component and content is, the dissociate CIC antibodies buffer of agent and 70 μ l of the CIC separating agent of 150 μ l, the CIC washing agent of 300 μ l, the CIC double solvents of 10 μ l, the CIC antibodies buffer of 70 μ l dissociates neutralizing agent.
Wherein, the CIC separating agent of every 1L comprises following component: the polyglycol of the borax of 6.4g, the boric acid of 5.1g, 80g, the sodium chloride of 7.7g, the liquid bio antiseptic of 200 μ l, surplus are distilled water.
The CIC washing agent of every 1L comprises following component: the polyglycol of the borax of 6.4g, the boric acid of 5.1g, 40g, the sodium chloride of 9.9g, the liquid bio antiseptic of 200 μ l, surplus are distilled water.
The CIC double solvents of every 1L comprises following component: the Triton X-100 of the sodium chloride of 9.0g, the NaOH of 0.016g, 300 μ l, the liquid bio antiseptic of 200 μ l, surplus are distilled water.
The agent of dissociating of the CIC antibodies buffer of every 1L comprises following component: the glycocoll of 4.5g, 5.0ml massfraction be 36% concentrated hydrochloric acid, the sodium chloride of 5.5g, the liquid bio antiseptic of 200 μ l, surplus be distilled water.
The CIC antibodies buffer of the every 1L neutralizing agent that dissociates comprises following component: the tromethamine of 8.4g, the sodium chloride of 8.0, the liquid bio antiseptic of 200 μ l, surplus are distilled water.
Utilize the agent of dissociating of above immune complex buffering to detect a method of CIC, detect the method for antibody in CIC, for HBsAg-Anti-HBs antibody immune complex, be made up of following step,
A. be separated: the centrifuge tube to 2 1ml adds 150 μ l samples to be tested respectively, then adds the CIC separating agent of 150 μ l respectively; After mixing, place 30min at 37 DEG C, carry out centrifugal, abandoning supernatant after centrifugal end;
B. wash: in two centrifuge tubes, add the CIC washing agent of 300 μ l respectively, after centrifugal, abandoning supernatant, and CIC sediment is drained;
C. redissolve: respectively to containing in the sedimentary centrifuge tube of the CIC drained, add the CIC double solvents of 10 μ l, centrifuge tube is installed on oscillator, vibrates to solution without lumpy precipitate;
D. blank sample detects: a centrifuge tube is labeled as blank sample pipe, and the antibodies buffer adding 70 μ l dissociates, the antibodies buffer of agent and 70 μ l dissociates neutralizing agent, detects Anti-HBs antibody, using testing result as blank value after mixing;
E.CIC antibody dissociation: another centrifuge tube is labeled as working sample pipe, and add 70 μ l antibodies buffer and to dissociate agent, place 30min 15 DEG C of water-baths, obtain dissociation solution;
F.CIC antibody dissociation neutralizes: in the dissociation solution obtained in step e, adds 70 μ l antibodies buffer and to dissociate neutralizing agent, detect Anti-HBs antibody, using testing result as measured value after mixing.
The test result of sample to be tested: the blank value <Cutoff recorded, measured value <Cutoff.Namely there is not CIC in sample to be tested.[note: detect (chemoluminescence method) on Abbott Laboratories of U.S. i2000 immunity analysis instrument, Cutoff value=10mIU/ml].
Embodiment 7
1. adopt dissociate agent and surfactant method of dissociating of the buffering of the immune complex described in embodiment 3 to detect the results contrast of HCV-Ag-anti-HCV immune complex
In random collecting routine work, ELISA detects serum anti-HCV positive sample totally 22 parts, the method of the detection CIC described in embodiment 3 and surfactant dissociation technique is adopted to dissociate to 22 parts of serum specimens respectively, then adopt ELISA method HCV-Ag kit to detect HCV-Ag simultaneously, the results are shown in Table 3.
Table 3 embodiment 3 is to the HCV-Ag testing result (n=22) in the third hepatopath CIC
2. adopt the buffering of the immune complex described in embodiment 3 dissociate method, the Pepsin digestion method of agent and strong acid that dissociate to detect the results contrast of HBsAg-Anti-HBs antibody immune complex
In random collecting routine work, ELISA detects acute hepatitis b patient and has just entered convalescence (serum HBsAg feminine gender, anti-HBe and anti-HBc is positive, the weak positive or negative of Anti-HBs antibody) sample totally 37 parts, dissociate method, Pepsin digestion method of the method for detection CIC described in embodiment 3 and strong acid is adopted to dissociate to 37 parts of serum specimens respectively, then adopt chemoluminescence method i2000 immunity analysis instrument to detect HBsAg, the results are shown in Table 4.
Table 4 embodiment 3 is to HBsAg testing result (n=37) in the hepatitis B patient CIC of free HBsAg feminine gender
3. adopt dissociate agent and Anti-C1q antibodies bag of the buffering of the immune complex described in embodiment 3 to be detected the results contrast of HBsAg-Anti-HBs antibody immune complex by the anti-Ig immunocapture method of micropore (anti-Ig immunocapture method) by the anti-Ig immunocapture method (C1q immunocapture method) of micropore, anti-Igs antibody bag
In random collecting routine work, ELISA detects chronic hepatitis B patient (serum HBsAg, anti-HBe and anti-HBc are positive) sample totally 52 parts, the method of the detection CIC described in embodiment 3 and C1q immunocapture method, anti-Ig immunocapture method is adopted to dissociate to 52 parts of serum specimens respectively, then adopt ELISA method to detect HBsAg, the results are shown in Table 5.
HBsAg testing result (n=52) in table 5 embodiment 3 couples of Chronic Hepatitis B CIC
4. adopt the buffering of the immune complex described in embodiment 6 agent and Pepsin digestion method, the strong acid Antibody Results that method, surfactant biopsy survey in autoimmune disease CIC that dissociates that dissociates to compare
(active stage and the repose period systemic loupus erythematosus SLE of autoimmune disease patient in random collecting routine work, dry syndrome SS, rheumatoid arthritis RA etc.) each 1 part of sample, the buffering of the immune complex described in embodiment 6 dissociate method and surfactant method of dissociating of agent, Pepsin digestion method, strong acid of dissociating is adopted to dissociate to its serum specimen respectively, then adopt Ou Meng western blot test (WB) to detect autoantibody repertoire, the results are shown in Table 6.
Autoantibody testing result in table 6 embodiment 6 couples of autoimmune disease patient CIC
Note: ± ,+, ++, +++ represent positive degree and increase progressively ,-represent negative findings.* after not being separated by CIC, carry out anti-dsDNA detection, therefore the anti-dsDNA positive detected cannot judge that whether this antibody is from the antibody in CIC or free antibodies.
5. adopt the agent of dissociating of embodiment 3 and the buffering of the immune complex described in embodiment 6 to detect antigen in different body fluid in CIC or Antibody Results
Collect each 13 parts of the urine of Chronic Hepatitis B in routine work, the type ii diabetes patients serum of Long-Time Service insulinize 15 parts, testosterone levels lowly causes the serum 11 parts of infertile patient, rheumatoid arthritis patients arthral fluid 7 parts, embodiment 3 and embodiment 6 is adopted to dissociate to the antigen in above-mentioned humoral specimen CIC or antibody, then ELISA, chemoluminescence method, western blot test is adopted to detect corresponding antigen or antibody, wherein urine carries out 10 times of precipitation concentration, the results are shown in Table 7.
Table 7 embodiment 3 and embodiment 6 are to the antigen in CIC in different body fluid or antibody test result
In a word, the foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to the covering scope of patent of the present invention.
Claims (6)
1. an immune complex cushions agent of dissociating, it is characterized in that: in following volume ratio proportioning, each component and content is, the dissociate CIC Antigen buffer of agent and 65 μ l-76 μ l of the CIC separating agent of 135 μ l-165 μ l, the CIC washing agent of 290 μ l-315 μ l, the CIC double solvents of 5 μ l-16 μ l, the CIC Antigen buffer of 65 μ l-76 μ l dissociates neutralizing agent;
Wherein, the CIC separating agent of every 1L comprises following component: the polyglycol of the borax of 5.2g-7.7g, the boric acid of 4.1g-6.1g, 70g-90g, the sodium chloride of 6.2g-9.2g, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water;
The CIC washing agent of every 1L comprises following component: the polyglycol of the borax of 5.2g-7.7g, the boric acid of 4.1g-6.1g, 35g-45g, the sodium chloride of 8.0g-11.7g, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water;
The CIC double solvents of every 1L comprises following component: the Triton X-100 of the sodium chloride of 8.0g-9.5g, the NaOH of 0.01g-0.03g, 50 μ l-500 μ l, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water.
2. a kind of immune complex buffering according to claim 1 is dissociated agent, it is characterized in that: the agent of dissociating of the CIC Antigen buffer of every 1L comprises following component: the glycocoll of 3.5g-5.5g, 9.0ml-15.0ml massfraction are the concentrated hydrochloric acid of 35%-37%, the sodium chloride of 0.35g-0.51g, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water.
3. a kind of immune complex buffering according to claim 2 is dissociated agent, it is characterized in that: the CIC Antigen buffer of the every 1L neutralizing agent that dissociates comprises following component: the tromethamine of 16g-24g, the sodium chloride of 5.7g-8.6g, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water.
4. an immune complex cushions agent of dissociating, it is characterized in that: in following volume ratio proportioning, each component and content is, the dissociate CIC antibodies buffer of agent and 65 μ l-76 μ l of the CIC separating agent of 135 μ l-165 μ l, the CIC washing agent of 290 μ l-315 μ l, the CIC double solvents of 5 μ l-16 μ l, the CIC antibodies buffer of 65 μ l-76 μ l dissociates neutralizing agent;
Wherein, the CIC separating agent of every 1L comprises following component: the polyglycol of the borax of 5.2g-7.7g, the boric acid of 4.1g-6.1g, 70g-90g, the sodium chloride of 6.2g-9.2g, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water;
The CIC washing agent of every 1L comprises following component: the polyglycol of the borax of 5.2g-7.7g, the boric acid of 4.1g-6.1g, 35g-45g, the sodium chloride of 8.0g-11.7g, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water;
The CIC double solvents of every 1L comprises following component: the Triton X-100 of the sodium chloride of 8.0g-9.5g, the NaOH of 0.01g-0.03g, 50 μ l-500 μ l, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water.
5. a kind of immune complex buffering according to claim 4 is dissociated agent, it is characterized in that: the agent of dissociating of the CIC antibodies buffer of every 1L comprises following component: the glycocoll of 3.5g-5.5g, 4.0ml-6.0ml massfraction are the concentrated hydrochloric acid of 35%-37%, the sodium chloride of 4.4g-6.6g, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water.
6. a kind of immune complex buffering according to claim 5 is dissociated agent, it is characterized in that: the CIC antibodies buffer of the every 1L neutralizing agent that dissociates comprises following component: the tromethamine of 6.8g-10.0g, the sodium chloride of 6.5g-9.6g, the liquid bio antiseptic of 100 μ l-500 μ l, surplus are distilled water.
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