CN103777005A - CIC (circulating immune complex) antibody buffer solution dissociation neutralizer for immune complex buffer dissociation agent - Google Patents
CIC (circulating immune complex) antibody buffer solution dissociation neutralizer for immune complex buffer dissociation agent Download PDFInfo
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Abstract
The invention relates to the field of medical detection, and discloses a CIC (circulating immune complex) antibody buffer solution dissociation neutralizer for an immune complex buffer dissociation agent. Every 1L of the CIC antibody buffer solution dissociation neutralizer comprises 6.8-10.0g of trometamol, 6.5-9.6g of sodium chloride, 100-500 mu l of liquid biological preservative and the balance of distilled water. The method for detecting CIC by using the immune complex buffer dissociation agent establishes a standard operation process for specifically detecting the antibody in the CIC, which is the problem that can not be solved by the existing CIC detection methods at present. The immune complex buffer dissociation agent has the technical characteristics of high specificity and sensitivity, high efficiency and favorable repetitiveness, is free from interference, and can perform quantitative or qualitative detection. The immune complex buffer dissociation agent is used for detecting CIC in body fluid samples of patients with infectious diseases, incretion metabolic diseases, autoimmune diseases and part of neoplastic diseases.
Description
Technical field
The present invention relates to medical science detection field, relate in particular to a kind of for the immune complex buffering CIC antibody damping fluid of the agent neutralizing agent that dissociates that dissociates.
Background technology
The pathogen such as bacterium, virus is invaded 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 obtaining is called immune complex and (claims again antigen antibody complex, immune complex, IC), mainly contain IgG, IgM, IgA, IgE type immune complex, wherein the most common with IgG and IgM.Because antigen is different from antibody ratio, the IC molecular size forming is different, conventionally has three kinds of forms: the one, when antigen-antibody ratio is suitable, form macromolecular insoluble IC (being greater than 19S), and easily caught, engulf and remove by phagocyte.The 2nd, when antigen amount is superfluous, form micromolecular solubility IC (being less than 6.6S), easily see through glomerular filtration and excrete with urine.The 3rd, when antigen amount is slightly superfluous, form medium sized solubility IC (8.8-19S), it is neither removed by phagocyte, can not discharge by glomerular filtration again, can be free on the long period in blood and other body fluid, claim again CIC ELISA (circulating immuno complex, CIC).
Under normal circumstances, the free antigen in body is combined with corresponding antibodies and is formed IC, can be removed by the system of defense of body, as a kind of mode of removing foreign matter antigen, favourable to body.But under some pathologic condition, the IC forming in body can not be removed in time, can be in local deposits, pass through activating complement, and under blood platelet, neutrophil leucocyte etc. participate in, cause a series of chain reactions and cause tissue damage, there is clinical symptoms, be called immune complex disease (immuno complex disease, ICD).Check that in tissue or in instrument for circulation of body fluid, having of IC helps the diagnosis of some disease, pathogenetic research, prognosis evaluation, state of an illness movable observing and curative effect judgement etc.Therefore, accurate, special, diagnosis, treatment and the prognosis evaluation of Sensitive Detection immune complex to disease has important clinical meaning, and the detection of CIC comes into one's own day by day.
The World Health Organization (WHO) (WHO) once organized and carried out the research of the CIC detection side science of law, found there is no any technology and possessed all features special, sensitive, that batch is efficient, reproducible simultaneously.Due to the limitation of complicacy, susceptibility and the CIC type that detects of method, current conventional CIC detection technique is all subject to the factors such as the ability of immunoglobulin (Ig) kind and subclass, compound size, antigen and antibody ratio in immune complex, complement-fixing to be affected, 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, can not possess special, sensitive, efficient, reproducible, the technical matters that is not interfered in batches simultaneously, disclose a kind of solved above technical matters for the immune complex buffering CIC antibody damping fluid of the agent neutralizing agent that dissociates that dissociates.
In order to solve the problems of the technologies described above, the present invention is solved by following technical proposals.
A kind of immune complex cushions the agent of dissociating, in following volume ratio proportioning, each component and content is, the CIC double solvents of the CIC separating agent of 135-165 μ l, the CIC washing agent of 290-315 μ l, 5-16 μ l, the CIC antibody damping fluid of the 65-76 μ l CIC antibody damping fluid of agent and the 65-76 μ l neutralizing agent that dissociates that dissociates.Take the anti-HBs immune complex of HBsAg-of preparation as example, 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 that refers to antibody amount in the antibody amount 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, cannot detect the antibody in CIC.
Wherein, the CIC separating agent of every 1L comprises that the borax of following component: 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, liquid bio antiseptic, the surplus of 100 μ l-500 μ l are distilled water.The effect of CIC separating agent is as follows: in the time that the CIC of every 1L separating agent comprises that liquid bio antiseptic, the surplus of sodium chloride, the 100 μ l-500 μ l of polyglycol, the 6.2g-9.2g of boric acid, the 70g-90g of borax, the 4.1g-6.1g of 5.2g-7.7g are distilled water, separated CIC reaches maximum, uses sodium chloride simultaneously instead and also can avoid the residual interference to subsequent detection antibody of sodium fluoride in traditional intermediate processing.The polyglycol that the present invention adopts, as preferably selecting Macrogol 6000; The liquid bio antiseptic of mentioning in the present invention is Proclin-300.
The CIC washing agent of every 1L comprises that the borax of following component: 5.2g-7.7g, the boric acid of 4.1g-6.1g, the polyglycol of 35g-45g, the sodium chloride of 8.0g-11.7g, liquid bio antiseptic, the surplus of 100 μ l-500 μ l are distilled water.More than the CIC washing agent of formula designs mainly for the formula of CIC separating agent, and its effect is to wash away foreign protein unnecessary in humoral specimen.The liquid bio antiseptic of mentioning in the present invention is Proclin-300.
The CIC double solvents of every 1L comprises that the sodium chloride of following component: 8.0g-9.5g, the NaOH of 0.01g-0.03g, the Triton X-100 of 50 μ l-500 μ l, liquid bio antiseptic, the surplus of 100 μ l-500 μ l are distilled water.The buffering capacity size of CIC double solvents when dissociating and design the degree of injury minimum of the CIC double solvents of above formula to separation of C IC; Adopt and wait the Triton X-100 oozing, the Triton X-100 of low concentration assists to dissolve CIC.Triton X-100 of the present invention is again Triton X-100, and the liquid bio antiseptic of mentioning in the present invention is Proclin-300.
The agent of dissociating of the CIC antibody damping fluid of every 1L comprises that the glycocoll of following component: 3.5-5.5g, the concentrated hydrochloric acid that 4.0ml-6.0ml massfraction is 35%-37%, the sodium chloride of 4.4g-6.6g, liquid bio antiseptic, the surplus of 100 μ l-500 μ l are distilled water.The agent of dissociating of CIC antibody damping fluid makes in CIC antibody reach farthest to dissociate.If the anti-HBs immune complex of HBsAg-take preparation is as reference, antibody dissociation rate reaches 37.5%-51.0%.The liquid bio antiseptic of mentioning in the present invention is Proclin-300.
As preferably, the CIC antibody damping fluid of the every 1L neutralizing agent that dissociates comprises that the tromethamine of following component: 6.8g-10.0g, the sodium chloride of 6.5g-9.6g, liquid bio antiseptic, the surplus of 100 μ l-500 μ l are distilled water.The CIC antibody damping fluid neutralizing agent that dissociates is mainly that the antibody damping fluid that CIC is dissociated neutralizes, and the CIC antibody damping fluid of this component neutralizing agent that dissociates makes osmotic pressure, pH reach the comparatively desirable environment of antibody, is beneficial to the detection of antagonist.The liquid bio antiseptic of mentioning in the present invention is Proclin-300.
As preferably, the CIC antibody damping fluid of the every 1L neutralizing agent that dissociates comprises that the tromethamine of following component: 7.8g-9.5g, the sodium chloride of 6.8g-9.0g, liquid bio antiseptic, the surplus of 120 μ l-490 μ l are distilled water.The CIC antibody damping fluid of this component neutralizing agent that dissociates makes osmotic pressure, pH reach the comparatively desirable environment of antibody, is beneficial to the detection of antagonist.The liquid bio antiseptic of mentioning in the present invention is Proclin-300.
As preferably, the CIC antibody damping fluid of the every 1L neutralizing agent that dissociates comprises that the tromethamine of following component: 8.4g-9.5g, the sodium chloride of 8.0g-9.0g, liquid bio antiseptic, the surplus of 200 μ l-490 μ l are distilled water.The CIC antibody damping fluid of this component neutralizing agent that dissociates makes osmotic pressure, pH reach the more preferably environment of antibody, is beneficial to the detection of antagonist.The liquid bio antiseptic of mentioning in the present invention is Proclin-300.
As preferably, the CIC antibody damping fluid of the every 1L neutralizing agent that dissociates comprises that tromethamine, the sodium chloride of 8.0g, the liquid bio antiseptic of 200 μ l, the surplus of following component: 8.4g are distilled water.The CIC antibody damping fluid of this component neutralizing agent that dissociates can make osmotic pressure, pH reach the environmental optima of antibody, is beneficial to the detection of antagonist most.The liquid bio antiseptic of mentioning in the present invention is Proclin-300.
Above-mentioned this kind of immune complex buffering dissociate agent be applied to suffer from infectious diseases, the detection of CIC in the serum of endocrine metabolism disease, autoimmune disease, Partial tumors Disease, chest ascites, urine, cerebrospinal fluid, arthral fluid.In the humoral specimens such as the patients' such as infectious diseases, endocrine metabolism disease, autoimmune disease, Partial tumors disease serum, chest ascites, urine, cerebrospinal fluid, arthral fluid, whether there is corresponding CIC, we can pass through separation of C IC, thereby infer the CIC and the content height that in the body fluid such as patients serum, chest ascites, urine, cerebrospinal fluid, arthral fluid, whether there are corresponding Specific marker, to understand pathophysiological role and the correlativity of CIC in above-mentioned disease development process; If antigen or the antibody (under normal circumstances, antigen dissociation yield is higher than antibody dissociation rate) in corresponding CIC detected in the negative humoral specimen of Research of predicting markers, can improve the recall rate of this mark and the diagnostic sensitivity to corresponding disease.
A method of utilizing above immune complex buffering to dissociate agent detection CIC, in detection CIC, the method for antibody, take the anti-HBs immune complex of HBsAg-as example, is made up of following step,
A. separate: the sample to be tested that adds respectively 150 μ l to contain CIC to the centrifuge tube of 2 1ml, then add respectively the CIC separating agent of 135 μ l-165 μ l; After mixing, 37 ℃ place 30min, carry out centrifugal, abandoning supernatant after centrifugal end;
B. washing: in two centrifuge tubes, add respectively the CIC washing agent of 290 μ l-315 μ l, centrifugal after, abandoning supernatant, and CIC sediment is drained;
C. redissolve: respectively to containing in the sedimentary centrifuge tube of the CIC draining, add the CIC double solvents of 5 μ l-16 μ l, centrifuge tube is installed on oscillator, vibrate in solution without block sediment;
D. blank sample detects: a centrifuge tube is labeled as to blank sample pipe, and adds the antibody damping fluid of the 65 μ l-76 μ l antibody damping fluid of agent and the 65 μ l-76 μ l neutralizing agent that dissociates that dissociates, mix the anti-HBs of rear detection, using testing result as blank value;
E.CIC antibody dissociation: another centrifuge tube is labeled as to working sample pipe, and adds the agent of dissociating of 65 μ l-76 μ l antibody damping fluids, place 30min(at 15 ℃ and hatch and place 30min or place 30min 15 ℃ of ℃ of water-baths at 15 ℃ of air), obtain dissociation solution;
F.CIC antibody dissociation neutralization: in the dissociation solution obtaining in step e, add the 65 μ l-76 μ l antibody damping fluids neutralizing agent that dissociates, mix the anti-HBs of rear detection, using testing result as measured value.
Antibody test result criterion after the anti-HBs immune complex of HBsAg-dissociates, as shown in the table:
Antibody test result criterion after the anti-HBs immune complex of table 1 HBsAg-dissociates
Note: detect (chemoluminescence method), Cutoff value=10mIU/ml on the i2000 of Abbott Laboratories of U.S. immunity analysis instrument
This kind utilize immune complex buffering to dissociate method that agent detects CIC is by separating, washing, redissolve, CIC dissociates, neutralization, then adopt (electricity) chemoluminescence method, 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, set up the Standard Operating Procedure of antibody in specific detection CIC (after dissociating), this is all insurmountable problems of current existing CIC detection method.
Compared with prior art, beneficial effect of the present invention is:
(1) the present invention has improved in prior art, the shortcoming of CIC detection technique poor specificity.Antibody in direct-detection CIC.
(2) the present invention has improved in prior art, the shortcoming that the sensitivity of CIC detection technique is low, cushion by immune complex the antibody that the agent of dissociating is dissociated in CIC, again by the antibody in specific detection CIC, make antibody test result reach the level of ng/ml and mIU/ml, thereby accurately calculate CIC content.
(3) the present invention is reproducible, is applicable to using in batches, has improved work efficiency.Repeatability is suitable with the degree of variation of clinical immunology laboratory conventional sense antibody, once dissociates and can utilize several different methods to detect multiple projects.
(4) the invention solves existing CIC detection technique and can not possess problem special, sensitive, that batch is efficient, reproducible simultaneously.Antibody in both can the be special sensitive quantitative detection CIC of the present invention; Can carry out batch detection (as detected the anti-HBs immune complex of HBsAg-in many parts of serum specimens simultaneously) to same CIC in many parts of humoral specimens again; Can also be to carry out efficient detection (immune complex such as HBs as anti-in the HBsAg-to in a serum specimen, the anti-HBe of HBeAg-, HCV-cAg-anti-HCV, HIV-P24-anti-HIV, or IgM, IgG, IgA type immune complex detect) with multiple CIC in a humoral specimen simultaneously.In addition, the present invention does not need special instrument and equipment, is applicable to the antibody test project of clinical immunology laboratory routine; Really accomplish once to dissociate, detect multiple projects simultaneously, repeatability (CV value) reaches below 15%-20%.
(5) the present invention has eliminated the interfering material in antibody test process in existing CIC detection technique.The present invention, by using the CIC antibody damping fluid neutralizing agent that dissociates, has ensured in antibody test process without any material interference detection results.
(6) no matter the present invention is that feminine gender or positive sample are all suitable for to free antibodies.The present invention, by CIC is separated from humoral specimen, has avoided the interference of free antibody positive to the antibody test in separation of C IC in humoral specimen.
(7) the present invention has avoided the impact that osmotic pressure, pH antagonist detect.The present invention makes osmotic pressure, the environmental optima of pH value in antibody of detection system, thus the impact of having avoided osmotic pressure, pH antagonist to detect.
(8) the present invention is applicable to the detection of low concentration CIC.The present invention can separate by CIC, adjust the volume that dissociates and reach the object of concentrated CIC, thereby has greatly improved the recall rate of antibody in CIC.
(9) the present invention also relates to utilize the agent of dissociating of above-mentioned immune complex buffering to detect the method for CIC, by separating, wash, redissolve, dissociate CIC, neutralization, finally detect the CIC in sample.Set up the Standard Operating Procedure of antibody in specific detection CIC (after dissociating), this is all insurmountable problems of current existing CIC detection method.
(10) the present invention is adapted at clinical immunology laboratory conventional sense.Sample disposal is relatively easy, be in current existing CIC detection technique, operate more convenient, can be universal in clinical labororatory, method to medical diagnosis on disease most worthy.
Embodiment
Embodiment 1
A kind of immune complex cushions the agent of dissociating, in following volume ratio proportioning, each component and content is, the CIC double solvents of the CIC separating agent of 135 μ l, the CIC washing agent of 290 μ l, 5 μ l, the CIC antibody damping fluid of the 65 μ l CIC antibody damping fluid of agent and the 65 μ l neutralizing agent that dissociates that dissociates.
Wherein, the CIC separating agent of every 1L comprises that borax, the boric acid of 4.1g, the polyglycol of 70g, the sodium chloride of 6.2g, the liquid bio antiseptic of 100 μ l, the surplus of following component: 5.2g are distilled water.
The CIC washing agent of every 1L comprises that borax, the boric acid of 4.1g, the polyglycol of 35g, the sodium chloride of 8.0g, the liquid bio antiseptic of 100 μ l, the surplus of following component: 5.2g are distilled water.
The CIC double solvents of every 1L comprises that sodium chloride, the NaOH of 0.01g, the Triton X-100 of 50 μ l, the liquid bio antiseptic of 100 μ l, the surplus of following component: 8.0g are distilled water.
The agent of dissociating of the CIC antibody damping fluid of every 1L comprises that the glycocoll of following component: 3.5g, concentrated hydrochloric acid, the sodium chloride of 4.4g, the liquid bio antiseptic of 100 μ l, the surplus that 4.0ml massfraction is 35% are distilled water.
The CIC antibody damping fluid of the every 1L neutralizing agent that dissociates comprises that the tromethamine of following component: 6.8g-10.0g, the sodium chloride of 6.5g-9.6g, liquid bio antiseptic, the surplus of 100 μ l-500 μ l are distilled water.
A method of utilizing above immune complex buffering to dissociate agent detection CIC, in detection CIC, the method for antibody, take the anti-HBs immune complex of HBsAg-as example, is made up of following step,
A. separate: add respectively 150 μ l chest ascites to be measured sample to the centrifuge tube of 2 1ml, then add respectively the CIC separating agent of 135 μ l; After mixing, 37 ℃ place 30min, carry out centrifugal, abandoning supernatant after centrifugal end;
B. washing: in two centrifuge tubes, add respectively the CIC washing agent of 290 μ l, centrifugal after, abandoning supernatant, and CIC sediment is drained;
C. redissolve: to containing in the sedimentary centrifuge tube of the CIC draining, add the CIC double solvents of 5 μ l respectively, centrifuge tube is installed on oscillator, vibrate in solution without block sediment;
D. blank sample detects: a centrifuge tube is labeled as to blank sample pipe, and adds the antibody damping fluid of the 65 μ l antibody damping fluid of agent and the 65 μ l neutralizing agent that dissociates that dissociates, mix the anti-HBs of rear detection, using testing result as blank value;
E.CIC antibody dissociation: another centrifuge tube is labeled as to working sample pipe, and adds the agent of dissociating of 65 μ l antibody damping fluids, hatch and place 30min at 15 ℃ of air, obtain dissociation solution;
F.CIC antibody dissociation neutralization: in the dissociation solution obtaining in step e, add the 65 μ l antibody damping fluids neutralizing agent that dissociates, mix the anti-HBs of rear detection, using testing result as measured value.
The test result of chest ascites sample to be measured: the blank value >Cutoff recording, measured value >Cutoff, and measured value <130% blank value.Be not have CIC in chest ascites sample to be measured.[note: detect (chemoluminescence method), Cutoff value=10mIU/ml on the i2000 of Abbott Laboratories of U.S. immunity analysis instrument].
Embodiment 2
A kind of immune complex cushions the agent of dissociating, in following volume ratio proportioning, each component and content is, the CIC double solvents of the CIC separating agent of 165 μ l, the CIC washing agent of 315 μ l, 16 μ l, the CIC antibody damping fluid of the 76 μ l CIC antibody damping fluid of agent and the 76 μ l neutralizing agent that dissociates that dissociates.
Wherein, the CIC separating agent of every 1L comprises that borax, the boric acid of 6.1g, the polyglycol of 90g, the sodium chloride of 9.2g, the liquid bio antiseptic of 500 μ l, the surplus of following component: 7.7g are distilled water.
The CIC washing agent of every 1L comprises that borax, the boric acid of 6.1g, the polyglycol of 45g, the sodium chloride of 11.7g, the liquid bio antiseptic of 500 μ l, the surplus of following component: 7.7g are distilled water.
The CIC double solvents of every 1L comprises that sodium chloride, the NaOH of 0.03g, the Triton X-100 of 500 μ l, the liquid bio antiseptic of 500 μ l, the surplus of following component: 9.5g are distilled water.
The agent of dissociating of the CIC antibody damping fluid of every 1L comprises that the glycocoll of following component: 5.5g, concentrated hydrochloric acid, the sodium chloride of 6.6g, the liquid bio antiseptic of 500 μ l, the surplus that 6.0ml massfraction is 37% are distilled water.
The CIC antibody damping fluid of the every 1L neutralizing agent that dissociates comprises that tromethamine, the sodium chloride of 9.6g, the liquid bio antiseptic of 500 μ l, the surplus of following component: 10.0g are distilled water.
A method of utilizing above immune complex buffering to dissociate agent detection CIC, in detection CIC, the method for antibody, take the anti-HBs immune complex of HBsAg-as example, is made up of following step,
A. separate: add respectively 150 μ l urine specimen to be measured to the centrifuge tube of 2 1ml, then add respectively the CIC separating agent of 165 μ l; After mixing, 37 ℃ place 30min, carry out centrifugal, abandoning supernatant after centrifugal end;
B. washing: in two centrifuge tubes, add respectively the CIC washing agent of 315 μ l, centrifugal after, abandoning supernatant, and CIC sediment is drained;
C. redissolve: to containing in the sedimentary centrifuge tube of the CIC draining, add the CIC double solvents of 16 μ l respectively, centrifuge tube is installed on oscillator, vibrate in solution without block sediment;
D. blank sample detects: a centrifuge tube is labeled as to blank sample pipe, and adds the antibody damping fluid of the 76 μ l antibody damping fluid of agent and the 76 μ l neutralizing agent that dissociates that dissociates, mix the anti-HBs of rear detection, using testing result as blank value;
E.CIC antibody dissociation: another centrifuge tube is labeled as to working sample pipe, and adds the agent of dissociating of 76 μ l antibody damping fluids, place 30min 15 ℃ of ℃ of water-baths, obtain dissociation solution;
F.CIC antibody dissociation neutralization: in the dissociation solution obtaining in step e, add the 76 μ l antibody damping fluids neutralizing agent that dissociates, mix the anti-HBs of rear detection, using testing result as measured value.
The test result of urine specimen to be measured: the blank value <Cutoff recording, measured value >Cutoff.Be to have CIC in urine specimen to be measured.[note: detect (chemoluminescence method), Cutoff value=10mIU/ml on the i2000 of Abbott Laboratories of U.S. immunity analysis instrument].
Embodiment 3
A kind of immune complex cushions the agent of dissociating, in following volume ratio proportioning, each component and content is, the CIC double solvents of the CIC separating agent of 150 μ l, the CIC washing agent of 300 μ l, 10 μ l, the CIC antibody damping fluid of the 70 μ l CIC antibody damping fluid of agent and the 70 μ l neutralizing agent that dissociates that dissociates.
Wherein, the CIC separating agent of every 1L comprises that borax, the boric acid of 5.1g, the polyglycol of 80g, the sodium chloride of 7.7g, the liquid bio antiseptic of 200 μ l, the surplus of following component: 6.4g are distilled water.
The CIC washing agent of every 1L comprises that borax, the boric acid of 5.1g, the polyglycol of 40g, the sodium chloride of 9.9g, the liquid bio antiseptic of 200 μ l, the surplus of following component: 6.4g are distilled water.
The CIC double solvents of every 1L comprises that sodium chloride, the NaOH of 0.016g, the Triton X-100 of 300 μ l, the liquid bio antiseptic of 200 μ l, the surplus of following component: 9.0g are distilled water.
The agent of dissociating of the CIC antibody damping fluid of every 1L comprises that the glycocoll of following component: 4.5g, concentrated hydrochloric acid, the sodium chloride of 5.5g, the liquid bio antiseptic of 200 μ l, the surplus that 5.0ml massfraction is 36% are distilled water.
The CIC antibody damping fluid of the every 1L neutralizing agent that dissociates comprises that the tromethamine of following component: 8.4g, 8.0 sodium chloride, the liquid bio antiseptic of 200 μ l, surplus are distilled water.
A method of utilizing above immune complex buffering to dissociate agent detection CIC, in detection CIC, the method for antibody, take the anti-HBs immune complex of HBsAg-as example, is made up of following step,
A. separate: add respectively 150 μ l CSF sample to be measured to the centrifuge tube of 2 1ml, then add respectively the CIC separating agent of 150 μ l; After mixing, 37 ℃ place 30min, carry out centrifugal, abandoning supernatant after centrifugal end;
B. washing: in two centrifuge tubes, add respectively the CIC washing agent of 300 μ l, centrifugal after, abandoning supernatant, and CIC sediment is drained;
C. redissolve: to containing in the sedimentary centrifuge tube of the CIC draining, add the CIC double solvents of 10 μ l respectively, centrifuge tube is installed on oscillator, vibrate in solution without block sediment;
D. blank sample detects: a centrifuge tube is labeled as to blank sample pipe, and adds the antibody damping fluid of the 70 μ l antibody damping fluid of agent and the 70 μ l neutralizing agent that dissociates that dissociates, mix the anti-HBs of rear detection, using testing result as blank value;
E.CIC antibody dissociation: another centrifuge tube is labeled as to working sample pipe, and adds the agent of dissociating of 70 μ l antibody damping fluids, place 30min 15 ℃ of ℃ of water-baths, obtain dissociation solution;
F.CIC antibody dissociation neutralization: in the dissociation solution obtaining in step e, add the 70 μ l antibody damping fluids neutralizing agent that dissociates, mix the anti-HBs of rear detection, using testing result as measured value.
The test result of CSF sample to be measured: the blank value <Cutoff recording, measured value <Cutoff.Be in CSF sample to be measured, not have CIC.[note: detect (chemoluminescence method), Cutoff value=10mIU/ml on the i2000 of Abbott Laboratories of U.S. immunity analysis instrument].
Embodiment 4
A kind of immune complex cushions the agent of dissociating, in following volume ratio proportioning, each component and content is, the CIC double solvents of the CIC separating agent of 150 μ l, the CIC washing agent of 300 μ l, 10 μ l, the CIC antibody damping fluid of the 70 μ l CIC antibody damping fluid of agent and the 70 μ l neutralizing agent that dissociates that dissociates.
Wherein, the CIC separating agent of every 1L comprises that borax, the boric acid of 5.1g, the polyglycol of 80g, the sodium chloride of 7.7g, the liquid bio antiseptic of 200 μ l, the surplus of following component: 6.4g are distilled water.
The CIC washing agent of every 1L comprises that borax, the boric acid of 5.1g, the polyglycol of 40g, the sodium chloride of 9.9g, the liquid bio antiseptic of 200 μ l, the surplus of following component: 6.4g are distilled water.
The CIC double solvents of every 1L comprises that sodium chloride, the NaOH of 0.016g, the Triton X-100 of 300 μ l, the liquid bio antiseptic of 200 μ l, the surplus of following component: 9.0g are distilled water.
The agent of dissociating of the CIC antibody damping fluid of every 1L comprises that the glycocoll of following component: 4.5g, concentrated hydrochloric acid, the sodium chloride of 5.5g, the liquid bio antiseptic of 200 μ l, the surplus that 5.0ml massfraction is 36% are distilled water.
The CIC antibody damping fluid of the every 1L neutralizing agent that dissociates comprises that tromethamine, the sodium chloride of 6.8g, the liquid bio antiseptic of 120 μ l, the surplus of following component: 7.8g are distilled water.
A method of utilizing above immune complex buffering to dissociate agent detection CIC, in detection CIC, the method for antibody, take the anti-HBs immune complex of HBsAg-as example, is made up of following step,
A. separate: add respectively 150 μ l samples to be tested to the centrifuge tube of 2 1ml, then add respectively the CIC separating agent of 150 μ l; After mixing, 37 ℃ place 30min, carry out centrifugal, abandoning supernatant after centrifugal end;
B. washing: in two centrifuge tubes, add respectively the CIC washing agent of 300 μ l, centrifugal after, abandoning supernatant, and CIC sediment is drained;
C. redissolve: to containing in the sedimentary centrifuge tube of the CIC draining, add the CIC double solvents of 10 μ l respectively, centrifuge tube is installed on oscillator, vibrate in solution without block sediment;
D. blank sample detects: a centrifuge tube is labeled as to blank sample pipe, and adds the antibody damping fluid of the 70 μ l antibody damping fluid of agent and the 70 μ l neutralizing agent that dissociates that dissociates, mix the anti-HBs of rear detection, using testing result as blank value;
E.CIC antibody dissociation: another centrifuge tube is labeled as to working sample pipe, and adds the agent of dissociating of 70 μ l antibody damping fluids, place 30min 15 ℃ of ℃ of water-baths, obtain dissociation solution;
F.CIC antibody dissociation neutralization: in the dissociation solution obtaining in step e, add the 70 μ l antibody damping fluids neutralizing agent that dissociates, mix the anti-HBs of rear detection, using testing result as measured value.
The test result of sample to be tested: the blank value <Cutoff recording, measured value <Cutoff.Be in sample to be tested, not have CIC.[note: detect (chemoluminescence method), Cutoff value=10mIU/ml on the i2000 of Abbott Laboratories of U.S. immunity analysis instrument].
Embodiment 5
A kind of immune complex cushions the agent of dissociating, in following volume ratio proportioning, each component and content is, the CIC double solvents of the CIC separating agent of 150 μ l, the CIC washing agent of 300 μ l, 10 μ l, the CIC antibody damping fluid of the 70 μ l CIC antibody damping fluid of agent and the 70 μ l neutralizing agent that dissociates that dissociates.
Wherein, the CIC separating agent of every 1L comprises that borax, the boric acid of 5.1g, the polyglycol of 80g, the sodium chloride of 7.7g, the liquid bio antiseptic of 200 μ l, the surplus of following component: 6.4g are distilled water.
The CIC washing agent of every 1L comprises that borax, the boric acid of 5.1g, the polyglycol of 40g, the sodium chloride of 9.9g, the liquid bio antiseptic of 200 μ l, the surplus of following component: 6.4g are distilled water.
The CIC double solvents of every 1L comprises that sodium chloride, the NaOH of 0.016g, the Triton X-100 of 300 μ l, the liquid bio antiseptic of 200 μ l, the surplus of following component: 9.0g are distilled water.
The agent of dissociating of the CIC antibody damping fluid of every 1L comprises that the glycocoll of following component: 4.5g, concentrated hydrochloric acid, the sodium chloride of 5.5g, the liquid bio antiseptic of 200 μ l, the surplus that 5.0ml massfraction is 36% are distilled water.
The CIC antibody damping fluid of the every 1L neutralizing agent that dissociates comprises that tromethamine, the sodium chloride of 9.0g, the liquid bio antiseptic of 490 μ l, the surplus of following component: 9.5g are distilled water.
A method of utilizing above immune complex buffering to dissociate agent detection CIC, in detection CIC, the method for antibody, take the anti-HBs immune complex of HBsAg-as example, is made up of following step,
A. separate: add respectively 150 μ l samples to be tested to the centrifuge tube of 2 1ml, then add respectively the CIC separating agent of 150 μ l; After mixing, 37 ℃ place 30min, carry out centrifugal, abandoning supernatant after centrifugal end;
B. washing: in two centrifuge tubes, add respectively the CIC washing agent of 300 μ l, centrifugal after, abandoning supernatant, and CIC sediment is drained;
C. redissolve: to containing in the sedimentary centrifuge tube of the CIC draining, add the CIC double solvents of 10 μ l respectively, centrifuge tube is installed on oscillator, vibrate in solution without block sediment;
D. blank sample detects: a centrifuge tube is labeled as to blank sample pipe, and adds the antibody damping fluid of the 70 μ l antibody damping fluid of agent and the 70 μ l neutralizing agent that dissociates that dissociates, mix the anti-HBs of rear detection, using testing result as blank value;
E.CIC antibody dissociation: another centrifuge tube is labeled as to working sample pipe, and adds the agent of dissociating of 70 μ l antibody damping fluids, place 30min 15 ℃ of ℃ of water-baths, obtain dissociation solution;
F.CIC antibody dissociation neutralization: in the dissociation solution obtaining in step e, add the 70 μ l antibody damping fluids neutralizing agent that dissociates, mix the anti-HBs of rear detection, using testing result as measured value.
The test result of sample to be tested: the blank value <Cutoff recording, measured value <Cutoff.Be in sample to be tested, not have CIC.[note: detect (chemoluminescence method), Cutoff value=10mIU/ml on the i2000 of Abbott Laboratories of U.S. immunity analysis instrument].
Embodiment 6
1. adopt immune complex buffering described in embodiment 3 to dissociate Antibody Results comparison that agent and Pepsin digestion method, strong acid dissociates in method, surfactant biopsy survey autoimmune disease CIC
(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, adopt respectively immune complex buffering described in embodiment 6 dissociate method and the surfactant method of dissociating of agent, Pepsin digestion method, strong acid of dissociating to dissociate to its serum specimen, then adopt Ou Meng western blot test (WB) to detect autoantibody repertoire, the results are shown in Table 2.
Table 2 embodiment 3 is to the autoantibody testing result in autoimmune disease patient CIC
Note: ± ,+, ++, +++ represent that positive degree increases progressively ,-represent negative findings.* after CIC not being separated, carry out anti-dsDNA detection, therefore the anti-dsDNA positive detecting cannot judge that whether this antibody is from the antibody in CIC or free antibodies.
2. the immune complex described in employing embodiment 3 cushions the agent of dissociating and detects the Antibody Results in CIC in different body fluid
15 parts of the type ii diabetes patients serums of the medium-term and long-term use of exogenous insulin of collection routine work, 7 parts of rheumatoid arthritis patients arthral fluids, adopt embodiment 3 to dissociate to the antibody in above-mentioned humoral specimen CIC, then adopt ELISA, chemoluminescence method, western blot test to detect corresponding antibody, the results are shown in Table 3.
Table 3 embodiment 3 is to the antibody test result in CIC in different body fluid
| Sick kind | Sample type | Number of cases | Test item | Implementation method | Positive rate (%) |
| Type ii diabetes | Serum | 15 | Insulin antibody | Embodiment 3 | 46.7 |
| Rheumatoid arthritis | Arthral fluid | 7 | Anti-CCP, anti-RA33 | Embodiment 3 | 100 |
In a word, the foregoing is only preferred embodiment of the present invention, all equalizations of doing 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 (4)
1. for the immune complex buffering CIC antibody damping fluid of the agent neutralizing agent that dissociates that dissociates, it is characterized in that: the CIC antibody damping fluid of the every 1L neutralizing agent that dissociates comprises that the tromethamine of following component: 6.8g-10.0g, the sodium chloride of 6.5g-9.6g, liquid bio antiseptic, the surplus of 100 μ l-500 μ l are distilled water.
2. claimed in claim 1 a kind of for the immune complex buffering CIC antibody damping fluid of the agent neutralizing agent that dissociates that dissociates, it is characterized in that: the CIC antibody damping fluid of the every 1L neutralizing agent that dissociates comprises that the tromethamine of following component: 7.8g-9.5g, the sodium chloride of 6.8g-9.0g, liquid bio antiseptic, the surplus of 120 μ l-490 μ l are distilled water.
3. claimed in claim 1 a kind of for the immune complex buffering CIC antibody damping fluid of the agent neutralizing agent that dissociates that dissociates, it is characterized in that: the CIC antibody damping fluid of the every 1L neutralizing agent that dissociates comprises that the tromethamine of following component: 8.4g-9.5g, the sodium chloride of 8.0g-9.0g, liquid bio antiseptic, the surplus of 200 μ l-490 μ l are distilled water.
4. claimed in claim 1 a kind of for the immune complex buffering CIC antibody damping fluid of the agent neutralizing agent that dissociates that dissociates, it is characterized in that: the CIC antibody damping fluid of the every 1L neutralizing agent that dissociates comprises that tromethamine, the sodium chloride of 8.0g, the liquid bio antiseptic of 200 μ l, the surplus of following component: 8.4g are distilled water.
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