CN101256189A - Human-outer autoantibody radio-immunity quantitative determination method - Google Patents

Abstract

The invention relates to a quantificationally detecting method for autoantibody in vitro, belonging to biomedicine technical field. The general character that the staphylococcal bacteria protein A (SPA) can react with Fc site of IgG in human and mouse is skillfully used and monoclonal antibodies of high affinity and specificity are prepared into standard substance to establish standard curve, therefore the monoclonal antibodied and autoantibody in sample respectively react with the labelled antigens, and the SPA enveloped nm magnetic grains are used as ligands to perform solid-liquid separation, after radioactivity measurement of <SUP>125</SUP>I, finally the radioactive ligand immunity quantitative analysis method of autoantibody is established. The said method is suitable for quantificationally detecting the autoantibody, so as to solve the problem that most of the antoantibody can not be accurately and quantificationally detected and at the same time prevent cross reaction without false negative or false positive phenomena.

Description

A kind of human-outer autoantibody radio-immunity quantitative determination method

Technical field:

The present invention relates to the field of biomedicine technology detection method, is that a kind of radioligand (nanometer magnetic particle) quantitative analysis method that is used for the outer autoantibody detection by quantitative of human body is a kind of human-outer autoantibody radio-immunity quantitative determination method

Background technology:

Autoantibody is meant the antibody of anti-autologous tissue, organ, cell and cell component.The growth of human body, growth and existence have keeping of complete autoimmunity tolerance mechanism, and normal immune response has the protectiveness defense reaction, promptly immune response is not taken place for autologous tissue, composition.In case the integrality of self tolerance is destroyed, then body is looked autologous tissue, composition is " foreign matter ", and autoimmune response takes place, and produces autoantibody.The autoantibody of low titre can be arranged in normal human's blood, but disease can not take place.If but the titre of autoantibody surpasses certain level, just may produce damage to health, induce an illness.The generation of autoantibody may be pathogenic antigens (bacterium, virus etc.) with self component between have some identical molecular structure: the immune response with autoantigen generation cross reaction has appearred; Also may be that some infective agent makes the autoantigen sex change, immune system produces autoantibody to the neoantigen of these exposures.The detection by quantitative of some specific autoantibodies has crucial meaning for diagnosis, prevention, treatment and the therapeutic evaluation of numerous disease.

Be that example is done and specified below with the Glutamic Acid Decarboxylase autoantibody, (Glutamic aciddecarboxylase is the synzyme that suppresses neurotransmitter γ-An Jidingsuan (GABA) in people and the animal body GAD) to glutamate decarboxylase, and γ-An Jidingsuan has GAD ₆₅With GAD ₆₇The isodynamic enzyme of two kinds of forms.Diabetes are second killers that harm is only second to cancer in the modern disease, and type 1 diabetes is the autoimmune disease of inheritance susceptible individuality by beta Cell of islet that immune response the causes destruction of autoantigen mediation, GAD ₆₅Be the initiating target antigen of this immune response key, so GAD ₆₅-Ab is the more special immune indexes of prediabetes individuality.Because most of glycosuria patients' the cause of the death comes from the long-term complications and the sequelae of disease, and before diabetes are clarified a diagnosis, patient's complication begins in fact already, 80%～90% beta Cell of islet afunction, various immunologic intervention measures this moment all are difficult to prove effective, so the key of preventing and controlling is early diagnosis.So using suitable diagnostic criteria carries out early diagnosis and has very significant meaning to the prevention of diabetes and control and to the treatment of its complication.In a word, detection by quantitative GAD ₆₅Autoantibody has crucial value to the observation of early diagnosis, somatotype and the medication effect of clinical diabetes.

Summary of the invention:

The object of the present invention is to provide a kind of autoantibody radio-immunity quantitative determination method: set up the platform of quantitative measurement technology fast and accurately of autoantibody in the human serum, can be used for the detection by quantitative of multiple autoantibody.With glutamate decarboxylase Glutamic acid decarboxylase, GAD ₆₅Autoantibody radio-immunity quantitative determination method is set up and is illustrated,

This method combines radiommunoassay (Radioimmunoassay RIA) quantitative measurement technology with nanometer magnetic particle ligand technique, be a kind ofly to detect the direct detecting method of principle based on part, its core be to utilize dexterously SPA can with the F of Immunoglobulin IgG in people and the mouse body _CThe denominator that binding site reacts is with being set up the part immune quantitative analytical approach of autoantibody by nanometer magnetic particle ligand technique from creating the SPA bag.Below with GAD ₆₅Autoantibody (GAD ₆₅-Ab) detection by quantitative is that example specifies.

At first with the albumin A bag by in nanometer magnetic particle surface, utilize the monoclonal antibody preparation standard items (in order to set up typical curve) of high-affinity, high specific, with excessive relatively labelled antigen * GAD ₆₅(highly purified ¹²⁵I-GAD ₆₅) respectively with standard items in GAD ₆₅GAD in monoclonal antibody and the blood serum sample ₆₅Autoantibody is in conjunction with forming immunocomplex (GAD ₆₅-Ab)-* GAD ₆₅Under the participation effect of the albumin A on nano magnetic particle surface, form the sandwich style compound system at the bag quilt, reason is that albumin A can be by (GAD ₆₅-Ab)-* GAD ₆₅The Fc part combination of immunocomplex.Wherein albumin A partly wraps by on the nano magnetic particle surface, thereby form a solid phase, carry out Solid-Liquid Separation with magnetic sheet, after suction is removed to comprise the supernatant of binding label not and is washed pearl, directly detect activity (CPM) with γ-counter, CPM value according to standard items is set up typical curve, can find patient GAD from typical curve ₆₅The concentration value of autoantibody.

GAD in sample or the standard items ₆₅The high more then label of-Ab concentration specificity is in conjunction with many more, and therefore detected radioactive intensity is also strong more, and promptly activity (CPM) is and GAD ₆₅-Ab concentration is directly proportional.In sample, there is not GAD ₆₅No immune system forms under-Ab the situation, because label is only in conjunction with GAD ₆₅-Ab, and not in conjunction with albumin A.

Monoclonal antibody and the autoantibody in the blood serum sample as standard items in this method do not enter same reaction system, the general character of just having utilized them to have with the SPA specific bond contrasts, therefore cross reaction can not take place, have high specificity, sensitivity and accuracy advantages of higher so detect effect, and false negative or false positive phenomenon can not occur.

This method may further comprise the steps:

(1) kit constituent:

GAD ₆₅-Ab radio-immunity part quantitative test diagnostic kit key component is: label ( ¹²⁵I-GAD ₆₅), standard items (GAD ₆₅Monoclonal antibody), ligand reagent (SPA-nanometer magnetic particle), cleansing solution.

(2) preparation of each component of kit:

1, the preparation of labelled antigen: adopt the preparation of lactoperoxidase enzyme process labelling technique ¹²⁵I-GAD ₆₅, its radiochemicsl purity is reached more than 95%.

2, the preparation of monoclonal antibody specific: use GAD ₆₅The antigen immune mouse prepares GAD by the Fusion of Cells hybridoma technology ₆₅Monoclonal antibody (GAD ₆₅-Ab), as the standard items of drawing with reference to curve.

3, the preparation of staphylococcal protein A (SPA)-nanometer magnetic particle: with SPA by the carbodiimide bag by in organic-silylation magnetic nano particle sub-surface, as the ligand reagent of carrying out Separation of Solid and Liquid.

(2) GAD ₆₅-Ab radioligand (nanometer magnetic particle) quantitative analysis method and clinical detection establishment of standard.

1, sets up GAD in the human serum ₆₅The radioligand of-Ab (nanometer magnetic particle) quantitative analysis method, and the experimental implementation method and the quality control standard of formulation kit.

2, show GAD in the kit measurement person under inspection serum of finishing with this project through a large amount of clinical test results ₆₅The content of-Ab is in order to judge diagnosis of diabetes and somatotype, its clinical coincidence rate 〉=95%.Therefore, the GAD of this kit ₆₅-Ab clinical assays value can be diabetes early diagnosis and correct somatotype thereof, and early intervention treatment, correctly taking drugs and therapeutic evaluation provide reliable basis.

Description of drawings

Fig. 1 is radiochemicsl purity figure

Fig. 2 is combination rate figure

Embodiment:

The content of this method specifies by following examples:

GAD ₆₅The foundation of autoantibody chemiluminescence part (nanometer magnetic particle) quantitative analysis method

(1) preparation of each component of kit:

1, labelled antigen ( ¹²⁵I-GAD ₆₅) preparation

(1) adopts improved lactoperoxidase enzyme process labelling technique preparation ¹²⁵I-GAD ₆₅:

Claim GAD ₆₅10～50 μ g are dissolved among PH6.5 phosphate buffer 50～200 μ l, add Na ¹²⁵I 1～8mCi, lactoperoxidase enzyme solutions 40～150ng (10～50 μ l), hydrogen peroxide (H ₂O ₂) 400～800ng (50～100 μ l), in 37 ℃ of incubations, drip H ₂O ₂400～800ng (50～100 μ l), finish in 5～10 minutes, continue reaction again after 2～5 minutes, add 0.5～2.0ml 20mmol/L mercaptoethanol 30～60 seconds with cessation reaction, reactant liquor is transferred to chromatography on the Sephadex G50 that uses PH6.5～7.5 phosphate buffer balances in advance or the Sephadex G25 post rapidly, with the equilibrium liquid wash-out, collect automatically, detect activity with γ-counter.Collect first peak.Identify with paper chromatography ¹²⁵I-glutamate decarboxylase (GAD ₆₅) radiochemicsl purity, identify with polyacrylamide gel electrophoresis ¹²⁵I-glutamate decarboxylase (GAD ₆₅) chemical purity.

(2) mensuration of radiochemicsl purity

Radiochemicsl purity refers to be combined in the number percent that radioactivity on the antigen accounts for this label gross activity activity in label.We adopt two kinds of methods to identify:

A, paper chromatography are identified

1) 1cm * 12cm filter paper is dipped in spend the night in the 1%KI solution standby.

2) filter paper that will soak before the use dries up, and marks with pencil by every 1cm distance.

3) with label to be identified 5 μ L～10 μ L point samples in distance end points 1cm place, dry.

4) the filter paper bar of point sample is put in the chromatography cylinder, adds developping agent then.Developping agent is a normal butyl alcohol: ethanol: ammoniacal liquor: water=20: 1: 0.5: 1.The chromatography time is 2 hours.

5) after the expansion filter paper is taken out and dries up, cut off by every 1cm distance then, and segmentation is counted.With every section cpm is ordinate, and the sequence number of segmentation paper slip is the horizontal ordinate mapping.The first peak of nearly initial point is the labelled antigen peak.

Referring to Fig. 1

6) calculate radiochemicsl purity by following formula:

$=\frac{1285740\left(\mathrm{cpm}\right)}{1306471\left(\mathrm{cpm}\right)}\&times;100\%=98.41\%\left(\mathrm{cpm}\right)$

B, free-iodine inspection technique

Inspection method is as follows: gets 0.1mL and collects liquid, add 1% carrier protein solution and 5% trichloroacetic acid (TCA) 4.8mL, occur white precipitate immediately, leave standstill a few hours, centrifugal 10min under the 4000rpm, survey supernatant and precipitation radioactive intensity partly respectively:

$=\frac{751\left(\mathrm{cpm}\right)}{63760+751\left(\mathrm{cpm}\right)}\&times;100\%=1.16\%\left(\mathrm{cpm}\right)$

2, GAD ₆₅MONOCLONAL ANTIBODIES SPECIFIC FOR and Performance Detection:

(1) antigen is purified and animal immune:

Glutamate decarboxylase (GAD ₆₅) add complete freund adjuvant and through fully emulsified, select to carry out animal immune with the BALB/c healthy mice of used myeloma cell's homology.After the last immunity 3～4 days, separate the splenocyte after merging.

(2) preparation of myeloma cell and feeder cells

Adopting the Sp2/0 myeloma cell strain to cultivate, do to adapt to the nutrient culture media that contains the 8-azaguanine earlier before fusion and cultivate, is 2 * 10 in the previous day of Fusion of Cells with fresh cultured keynote cell concentration ⁵/ ml generally is the exponential phase cell next day.Adopting the mouse peritoneal cell is feeder cells (feedercell).

(3) Fusion of Cells

The myeloma cell is mixed back adding PEG with splenocyte merge cell each other.With nutrient solution dilute PEG, eliminate the effect of PEG thereafter.Cell after merging is suitably diluted, split to cultivate in the plate hole and cultivate.

(4) limiting dilution assay screening positive cell strain

The myeloma cell of the positive usefulness of selecting good strains in the field for seed of screening is the strain of HAT sensitive cells.Fused cell is clonal growth, behind limiting dilution, draws culture supernatant and detects antibody content with ELISA.Filter out high antibody-secreting hole, with the capable again cloning of cell in the hole, the ELISA that carries out antigen-specific then measures, and selects the strain of high secretion specific cell to carry out enlarged culture or frozen.Be the ELISA The selection result that one of them hybridoma merges plate below, positive colony blue italic font representation, the positive contrast of H12.

A typical hybridoma merges the ELISA The selection result of plate

	1	2	3	4	5	6	7	8	9	10	11	12
													A	0.368	1.53	1.24	1.806	2.537	1.634	2.604	2.404	0.668	2.563	0.092	2.382
B	1.918	1.733	0.082	2.186	2.361	0.071	1.446	0.77	2.413	0.915	1.603	0.732
													C	2.326	0.077	2.209	2.105	0.082	0.784	0.989	1.97	0.784	1.399	0.906	1.771
D	1.101	1.911	0.455	0.288	1.26	2.111	0.6	1.59	1.381	0.785	1.334	1.523
													E	2.327	0.779	1.679	0.605	0.075	1.439	0.547	0.7	1.664	1.687	1.508	2.217
F	2.432	1.848	2.457	2.675	0.694	1.358	0.871	0.741	2.524	2.906	0.803	1.660
													G	0.245	2.759	0.864	1.406	0.074	2.442	1.64	1.917	1186	2.181	0.083	2.227
H	1.028	2.964	1.033	2.829	1.205	2.193	0.08	0.587	2.45	1.617	2.818	3.043

(5) MONOCLONAL ANTIBODIES SPECIFIC FOR and frozen

Antibody Preparation adopts mice celiac inoculation, selects for use BALB/c mouse or its parental generation mouse to carry out lumbar injection.Usually after one week of inoculation, promptly there is tangible ascites to produce.Select positive cell strain and frozen early.

(6) Purification of Monoclonal Antibodies

With staphylococcus A affinity column monoclonal antibody purification, the recovery reaches more than 90%.The UV Absorption method bigness scale of antibody concentration in the eluent.Behind low pH wash-out, in collection tube, preset neutralizer or speed adds neutralizer, to keep the activity of antibody purification.

(7) Performance Detection of monoclonal antibody

We have carried out comprehensive evaluation to titre, purity and the specificity of monoclonal antibody and affinity etc., and its key technical indexes is as follows:

GAD ₆₅The key technical indexes of monoclonal antibody

ELISA titre (in 1.0mg/ml)	1∶10 5
		Detection sensitivity	＜5.0pg/ml
Cross reacting rate	＜0.1％
		Affinity costant	4.97×10 10M -1

Titre detects: with GAD ₆₅For detecting antigen, measure antibody titer, its titre (in 1.0mg/ml)=1: 10 by indirect ELISA ⁵

Purity detecting: detect monoclonal antibody purity with non-reduced SDS-PAGE method after extracting purifying, the result has bar bands of a spectrum clearly about 160kD, reach more than 95% through its purity of albumin A affinity chromatography.

Specificity: anti-GAD ₆₅The specificity of monoclonal antibody is good, and is very little from the interference of ANA, DNA and the rheumatism factor not with the human serum albumins association reaction, cross reacting rate＜0.1%.

Affinity detects: according to methods that the people set up such as Beatty, with 4 concentration GAD of 5,2.5,1.25 and 0.625 μ g/ml ₆₅Dilution wraps successively by microwell plate, gets its maximum OD value half (be OD 50%) by graphing method and locates corresponding antibody concentration.Substitution formula K=(n-1)/2 (nAb '-Ab) calculating affinity costant, Ab and Ab ' represent respectively when antigen concentration is Ag and Ag ' in the formula, produce the antibody concentration (mol/L) of half light absorption value, n=Ag/Ag '.Ask its average to get affine normal K=4.97 * 10 ¹⁰M ^-1

3, the preparation of SPA-nanometer magnetic particle:

(1) preparation of SPA-nanometer magnetic particle: we have carried out a series of discussion tests to the reaction ratio of nanometer magnetic particle and SPA.Concrete preparation method is as follows:

1. get 5～10mg organic-silylation magnetic particle, wash 1-2 time with redistilled water;

2. the PBS with 0.01～0.5mol/L pH5.6-6.2 washes once;

3. the PBS adjustment cumulative volume with 0.01～0.5mol/L pH5.6-6.2 is about 10ml;

4. add a certain amount of SPA and be placed on and carry out homogenize 10-60 minute on the shaking table, the concrete quality of SPA according to the reaction mass of nanometer magnetic particle and SPA than deciding;

5. add the about 5.0～10mg of carbodiimide (EDAC);

6. room temperature reaction is placed on and carries out homogenize 10～48 hours on the shaking table;

7. with 0.1mol/L pH7.4PBS+0.1%BSA washing three times;

8. wash 3 times with redistilled water;

9. wash 3 times with 0.1mol/L pH7.4PBS;

10. wash 3 times with 0.05mol/L Tris+0.004mol/L EDTA pH 7.5 damping fluids;

With 0.05mol/L Tris+0.004mol/L EDTA pH 7.5 damping fluid constant volumes is 1.0mg/ml, and 2-4 ℃ of preservation is stand-by.

(2) mensuration of SPA binding capacity: the SPA binding capacity generally shows with the scale of every milliliter or every gram organic-silylation nanometer magnetic particle coupling SPA, its numerical value can be used as the parameter of decision affinity adsorbent actual amount, adopts direct determination method or indirect determination method to measure binding capacity.

Nanometer magnetic particle and SPA wrapped by different proportion reacted, measure the combination rate of SPA.Measurement result shows, when the ratio to 1 of magnetic particle and SPA: in the time of 50, the binding capacity of SPA is intimate saturated, and therefore, magnetic particle that we adopted and the ratio of SPA are 1: 50.

SPA combination rate measurement result

Nanometer magnetic particle/SPA	1∶5	1∶10	1∶20	1∶50	1∶75	100
							SPA combination rate (%)	25.64	38.13	47.18	62.71	63.76	64.97

Referring to Fig. 2

(2) kit experimental implementation method and step:

Set up GAD ₆₅The quantitative analysis method of-Ab radioligand, the experimental implementation step of taking is as follows:

1, will test required reagent and sample and place room temperature, balance just can be operated more than 30 minutes at least;

2, go that required test tube is some to be numbered, two-tube repetition is all done in all experiments, and is arranged in it on test tube rack in order;

3, standard items, quality controlled serum and sample 25～200 μ l that get each concentration add in the test tube of corresponding numbering;

4, every pipe all adds 50～200 μ l ¹²⁵I-GAD ₆₅Solution;

5, be inverted abundant mixing SPA-nanometer magnetic particle, every pipe all adds 25～100 μ l SPA-nanometer magnetic particles;

6, abundant mixing, room temperature vibration 1～6 hour;

7, test tube rack is placed on absorption magnetic particle on the magnetic sheet, inhales and remove supernatant;

8, remove magnetic sheet, every pipe all adds 500～1000 μ l cleansing solutions;

9, vibration placed absorption magnetic particle on the magnetic sheet with test tube rack after 5 seconds, inhaled and removed supernatant;

10, come again step 8,9;

11, detect the radioactive intensity of every pipe with γ-counter;

12, read the concentration of sample and quality controlled serum from typical curve.

(3), clinical detection applicable cases statistical study:

1, diabetes and control group GAD ₆₅-Ab testing result relatively

2, data statistics

3, clinical effectiveness analysis

With GAD in this kit measurement person under inspection serum ₆₅The content of-Ab, GAD in the normal human serum ₆₅-Ab recall rate is 0; The teenager sends out patient GAD among the type 1 diabetes patient ₆₅-Ab recall rate is 96.08%, apparently higher than 10.38% of diabetes B group; Tardy autoimmune type diabetes group GAD is grown up ₆₅The antibody recall rate is 97.69%, also apparently higher than the recall rate of diabetes B group, coincide with bibliographical information.

Therefore, the GAD of this kit ₆₅-Ab clinical assays value; have sensitivity and accuracy height; high specificity; testing result waits advantage accurately and reliably; can be the diabetes early diagnosis and correct somatotype, early intervention treatment, correctly taking drugs and therapeutic evaluation provide reliable basis; strive at prediabetes even more early give immunologic intervention or use exogenous insulin to protect remaining beta Cell of islet function that the treatment of diabetes and complication thereof is had crucial meaning.

Claims (2)

1. a human-outer autoantibody radio-immunity quantitative determination method is used glutamate decarboxylase Glutamic aciddecarboxylase, GAD ₆₅Autoantibody radio-immunity quantitative determination method is set up and is illustrated, and it is characterized in that:

This method is with radiommunoassay---Radioimmunoassay RIA---, and quantitative measurement technology combines with nanometer magnetic particle ligand technique, be a kind ofly to detect the direct detecting method of principle based on part, its core be to utilize dexterously SPA can with the F of Immunoglobulin IgG in people and the mouse body _CThe denominator that binding site reacts is with being set up the part immune quantitative analytical approach of autoantibody by nanometer magnetic particle ligand technique from creating the SPA bag.

2. a kind of autoantibody radio-immunity quantitative determination method according to claim 1, it is characterized in that: at first with the albumin A bag by in nanometer magnetic particle surface, the monoclonal antibody preparation standard items----that utilize high-affinity, high specific are in order to set up typical curve, with excessive relatively labelled antigen ^*GAD ₆₅, adopt highly purified ¹²⁵I-GAD ₆₅, respectively with standard items in GAD ₆₅GAD in monoclonal antibody and the blood serum sample ₆₅Autoantibody is in conjunction with forming immunocomplex (GAD ₆₅-Ab)- ^*GAD ₆₅Under the participation effect of the albumin A on nano magnetic particle surface, form the sandwich style compound system at the bag quilt, reason is that albumin A can be by (GAD ₆₅-Ab)- ^*GAD ₆₅The Fc part combination of immunocomplex; Wherein albumin A partly wraps by on the nano magnetic particle surface, thereby form a solid phase, carry out Solid-Liquid Separation with magnetic sheet, after suction is removed to comprise the supernatant of binding label not and is washed pearl, directly detect activity CPM with γ-counter, CPM value according to standard items is set up typical curve, can find patient GAD from typical curve ₆₅The concentration value of autoantibody;

This method may further comprise the steps:

(1) kit constituent:

GAD ₆₅-Ab radio-immunity part quantitative test diagnostic kit key component is: label--- ¹²⁵I-GAD ₆₅, standard items---GAD ₆₅Monoclonal antibody, ligand reagent---SPA-nanometer magnetic particle, cleansing solution;

(2) preparation of each component of kit:

1), the preparation of labelled antigen: adopt the preparation of lactoperoxidase enzyme process labelling technique ¹²⁵I-GAD ₆₅, its radiochemicsl purity is reached more than 95%;

2), the preparation of monoclonal antibody specific: personnel selection GAD ₆₅The antigen immune mouse prepares GAD by the Fusion of Cells hybridoma technology ₆₅Monoclonal antibody---GAD ₆₅-Ab is as the primary raw material of preparation standard items;

3), the preparation of staphylococcal protein A (SPA)-nanometer magnetic particle: with SPA by the carbodiimide bag by in organic-silylation magnetic nano particle sub-surface, as the ligand reagent of carrying out Separation of Solid and Liquid;

GAD ₆₅-Ab radioligand---nanometer magnetic particle---quantitative analysis method and clinical detection establishment of standard;

1), sets up GAD in the human serum ₆₅The radioligand of-Ab---nanometer magnetic particle---quantitative analysis method, and the experimental implementation method and the quality control standard of formulation kit;

2), show, GAD in the kit measurement person under inspection serum of finishing with this project through a large amount of clinical test results ₆₅The content of-Ab is in order to judge diagnosis of diabetes and somatotype, its clinical coincidence rate 〉=95%; Therefore, the GAD of this kit ₆₅-Ab clinical assays value can be diabetes early diagnosis and correct somatotype thereof, and early intervention treatment, correctly taking drugs and therapeutic evaluation provide reliable basis.

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