CN101470117A - Chemiluminescent ligand analysis method for quantitative detection of human auto-antibody - Google Patents
Chemiluminescent ligand analysis method for quantitative detection of human auto-antibody Download PDFInfo
- Publication number
- CN101470117A CN101470117A CNA2007100602148A CN200710060214A CN101470117A CN 101470117 A CN101470117 A CN 101470117A CN A2007100602148 A CNA2007100602148 A CN A2007100602148A CN 200710060214 A CN200710060214 A CN 200710060214A CN 101470117 A CN101470117 A CN 101470117A
- Authority
- CN
- China
- Prior art keywords
- tpo
- autoantibody
- chemiluminescence
- quantitative
- magnetic particle
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
A chemical luminous ligand analysis method for quantitatively checking human antibodies belongs to the biomedical technical field, which comprises: using the generality that staphylococcal protein A (SPA) can react with Fc point of IgG in mice; using the monoclonal antibody of high affinity and specificity to prepare a standard product to establish a standard curve; respectively reacting the monoclonal antibody and the antibody in the sample with enzyme-labeled antigens; using the nanometer magnetic particles coated by SPA as ligand to separate solid and liquid; and using enzymatic chemical illumination reaction catalyzed by horseradish peroxidase (HRP) to process illumination check; thereby establishing a chemical luminous ligand quantitative analysis on human antibodies. The chemical luminous ligand analysis method is suitable for quantitatively checking all human antibodies, for resolving the problem of prior art while most human antibodies can not be accurately and quantitatively checked, avoiding cross reaction and avoiding false negative result or false positive result.
Description
Technical field:
The present invention relates to field of biomedicine technology, is a kind of chemiluminescence part (nanometer magnetic particle) quantitative analysis method that is used for autoantibody in the external detection by quantitative human body.
Background technology:
Autoimmune disease (Autoimmune diseases) is meant that body causes autologous tissue, organ damage and corresponding function obstacle caused one big class disease to autoantigen generation immune response.Putative at present autoimmune disease has kind more than 30 at least.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 ", thereby autoimmune response takes place, and produces autoantibody.Therefore people's autoantibody is meant the antibody that human body produces autologous tissue, organ, cell and cell component generation immune response.
The autoantibody of low titre can be arranged in normal person's body fluid, 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 all has crucial meaning for diagnosis, prevention, treatment and the therapeutic evaluation of numerous disease.As thyroid peroxidase autoantibody (TPO-Ab), thyroglobulin autoantibody (TG-Ab), thyroid microsomal autoantibody (TM-Ab) and glutamate decarboxylase
65Autoantibody (GAD
65-Ab), islet cells autoantibody (ICA), insulin autoantibody (IAA) etc., be that example specifies with thyroid gland peroxidase thing autoantibody (TPO-Ab) below.
Thyroid peroxidase (TPO) is the membrane bound enzyme in the thyroid cell, and molecular weight is about 100KDa, and it is one of three kinds of thyroid antigens, plays an important role to the thyroid gland hormone is synthetic.The TPO autoantibody makes the thyroid hormone secretion deficiency cause the relevant hypothyroidism of autoimmunity by the cell-mediated cytotoxicity that relies on antibody, and characteristics of autoimmune thyroid disease are exactly the autoantibody that has thyroid antigen.Thereby the detection of TPO autoantibody helps to determine chronic autoimmune thyroiditis and to the hypothyroidism somatotype.The TPO autoantibody detects also has important value to hyperthyroidism, especially with suspicion the and TSH-REZAK of clinical symptoms
Detect negative case.The thyroid gland autoimmune disease of in addition the normal goitre of thyroid function being got rid of and being given birth to is helpful.
Domestic autoantibody detection kit adopts elisa technique mostly at present, adopts elisa technique as the TPO of Wuhan Boster Biological Technology Co., Ltd. autoantibody detection kit.The ELISA method mainly adopts the antigen coated microwell plate of TPO, detects by adding enzyme-labeled secondary antibody and chromogenic substrate with TPO autoantibody reaction back, belongs to indirect method and carries out qualitative detection.This method can't be carried out accurately quantitatively autoantibody, therefore can not provide reliable basis for the diagnosis and the therapeutic evaluation of autoimmune disease.Beijing Bei'aikang Biotechnology Co., Ltd. provides a kind of human thyroid peroxidase antibody enzyme-linked immunity detection method: use the antigen coated immunomagnetic beads of recombined human thyroid peroxidase as solid phase, make it with autoantibody reaction after carry out Separation of Solid and Liquid, the anti-human IgG antibody of goat (second antibody) and the substrate that add alkali phosphatase enzyme mark then carry out color developing detection, and this method belongs to the ELISA indirect method and measures.
The present invention adopts the enzymatic chemical luminous immunoassay technology to reach from the staphylococcal protein A bag of creating by nanometer magnetic particle ligand technique, the denominator of utilizing staphylococcal protein A to react with the Fc binding site of Immunoglobulin IgG in people and the mouse body, with the antigen of horseradish peroxidase-labeled respectively with standard items in monoclonal antibody and the human serum autoantibody in the sample combine and form the Ag-Ab immune complex.Under the albumin A effect of nanometer magnetic particle surface, formed the sandwich style compound system at bag then, set up typical curve with the monoclonal antibody standard items, set up the quantitative analysis method of part forensic chemistry electrochemiluminescent immunoassay, be used for the diagnosis of autoimmune disease and the observation of result of treatment.This method belongs to the direct method detection by quantitative, accurately the concentration of detection by quantitative autoantibody.Monoclonal antibody and the autoantibody in the blood serum sample as standard items in this method do not enter same reaction system, just having utilized them to have the general character that combines with the albumin A specificity 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.
Summary of the invention:
The objective of the invention is to set up the platform of quantitative measurement technology fast and accurately of autoantibody in a kind of human serum, can be used for the detection by quantitative of multiple autoantibody.
A kind of chemiluminescence ligand analysis method of detection by quantitative people's autoantibody, chemiluminescence immunoassay detection by quantitative CLIA technology is combined with nanometer magnetic particle ligand technique, it is a kind of direct assay method that detects principle based on part, the denominator that its core is to utilize dexterously staphylococcal protein A (SPA) to react with the FC binding site of Immunoglobulin IgG in people and the mouse body is with being set up the quantitative immune analysis method of chemiluminescence part of autoantibody by nanometer magnetic particle ligand technique from creating the SPA bag.(Thyroid peroxidase, TPO) detection by quantitative of autoantibody (TPO-Ab) is that example specifies with thyroid gland peroxidase thing below.
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 (highly purified TPO-HRP) respectively with standard items in TPO monoclonal antibody and the TPO autoantibody in the blood serum sample combine and form immunocomplex.This complex under the effect of the albumin A on nano magnetic particle surface, forms the sandwich style compound system at the bag quilt, because albumin A can be by the Fc part combination of (TPO-Ab)-TPO-HRP immunocomplex.Wherein albumin A partly wraps by on the nano magnetic particle surface, thereby forms a solid phase, carries out Solid-Liquid Separation with magnetic sheet.
Suction adds enhanced chemical luminous substrate working fluid after removing to contain the supernatant of binding label not and washing pearl, and the luminous substrate luminol in the working fluid is at the catalysis and the luminous startup reagent (NaOH+H of horseradish peroxidase (HRP)
2O
2) effect is down luminous.The emission maximum optical wavelength of luminol is 425nm and since luminol system luminous be flash type and signal a little less than, so we use tetraphenylboron sodium and cinnamic acid as the associating reinforcing agent with the enhancing luminous signal.Luminous intensity depends on the concentration of horseradish peroxidase in the immune response.Measure its relative light unit RLU behind the reaction 20min, set up typical curve, can find the concentration value of patient TPO autoantibody from typical curve with the standard items of TPO monoclonal antibody preparation.
The high more then label of TPO-Ab concentration specificity is in conjunction with many more in sample or the standard items, and therefore detected luminous intensity is also strong more, and promptly its relative light unit RLU is directly proportional with TPO-Ab concentration.Do not have in sample that no immune system forms under the situation of TPO-Ab, because the enzyme labeling thing is only in conjunction with TPO-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 has successfully solved the accurately difficult problem of detection by quantitative of autoantibody, is the new direction of future labels immuno analytical method development.
The chemiluminescence ligand analysis method of detection by quantitative people's autoantibody is an example with the TPO autoantibody, and its method of production is as follows:
1) kit constituent:
The key component of the quantitative immune analysis diagnosis reagent kit of TPO-Ab chemiluminescence part is: label---TPO-HRP, standard items---TPO monoclonal antibody, ligand reagent---SPA-nanometer magnetic particle, enhanced chemical luminous substrate working fluid---A liquid and B liquid, cleansing solution;
2) preparation of each component of kit:
(1) preparation of horseradish peroxidase HRP labelled antigen TPO: under the effect of sodium periodate, form TPO-HRP: adopt dialysis and HPLC to carry out secondarily purified with HRP mark TPO;
(2) preparation of monoclonal antibody specific: prepare TPO monoclonal antibody (TPO-Ab) by the Fusion of Cells hybridoma technology, with it as the standard items of drawing with reference to curve;
(3) staphylococcal protein A (SPA)-nanometer magnetic preparation of granules: with SPA by the carbodiimide bag by in organic-silylation magnetic nano particle sub-surface;
(4) optimization of enhanced chemiluminescence substrate working fluid preparation: with the luminol is luminous substrate, adopts tetraphenylboron sodium, cinnamic acid and luminol to prepare by a certain percentage as enhanced chemical luminous substrate working fluid to improve luminous intensity; 3) TPO-Ab chemiluminescence part (nanometer magnetic particle) quantitative analysis method and clinical detection establishment of standard
(1) sets up chemiluminescence part (the nanometer magnetic particle) quantitative analysis method of TPO-Ab in the human serum, and formulate the experimental implementation method and the quality control standard of this kit;
(2) lab scale of TPO-Ab chemiluminescence part quantitative test diagnostic kit and part clinical trial:
Gather clinical samples, with the accurate content of TPO-Ab in this kit measurement person under inspection serum, a large amount of clinical test results show its clinical coincidence rate 〉=95%; Therefore, the TPO-Ab clinical assays value of this kit can be autoimmune thyroid disease diagnosis, early intervention treatment, correctly taking drugs and therapeutic evaluation reliable basis is provided; 4) set up a maturation, sensitivity, autoantibody chemiluminescence part (nanometer magnetic particle) quantitative measurement technology platform efficiently, can utilize a large amount of people's autoantibody series chemical luminescent ligand body (nanometer magnetic particle) the quantitative test diagnostic kit of this method exploitation, form the research and development and the production system of a brand-new autoantibody chemiluminescence part (nanometer magnetic particle) detection by quantitative kit.
The chemiluminescence ligand analysis method of detection by quantitative people's autoantibody is an example with the TPO autoantibody, and the experimental implementation program of this kit 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) get a microwell plate and be numbered, all experiments are all done diplopore and are repeated;
(3) standard items, quality controlled serum and sample 25～200 μ l that get each concentration add in the hole of corresponding numbering;
(4) every hole all adds 50～200 μ l TPO-HRP;
(5) be inverted abundant mixing SPA-nanometer magnetic particle, every hole all adds 25～150 μ lSPA-nanometer magnetic particles;
(6) abundant mixing, room temperature vibration 1～4 hour;
(7) place it in absorption magnetic particle on the magnetic sheet, inhale and remove supernatant;
(8) remove magnetic sheet, every hole all adds 200～1000 μ l and washes plate liquid;
(9) vibration placed it in absorption magnetic particle on the magnetic sheet after 4～15 seconds, inhaled and removed supernatant;
(10) come again step 8,9;
(11) every hole adds luminous enhancing A liquid 100～300 μ l;
(12) every hole adds into luminous enhancing B liquid 100～300 μ l;
(13) measure the luminous intensity in every hole with Chemiluminescence Apparatus;
(14) read the concentration of sample and quality controlled serum from typical curve.
Description of drawings
Fig. 1 SPA combination rate figure
Fig. 2 TPO-Ab chemiluminescence part (nanometer magnetic particle) quantitative test principle schematic
Embodiment:
The content of this method specifies by following examples:
The foundation of TPO autoantibody chemiluminescence part (nanometer magnetic particle) quantitative analysis method
(1) preparation of each component of kit:
TPO-Ab chemiluminescence part quantitative test diagnostic kit key component is: label (TPO-HRP), standard items (TPO monoclonal antibody), ligand reagent (SPA-nanometer magnetic particle), the luminous working fluid of enhanced chemical, cleansing solution.
1, the preparation and the detection of labelled antigen (TPO-HRP):
A, preparation: adopt sodium periodate enzyme process labelling technique to prepare TPO-HRP:
(1) take by weighing TPO 2.0mg and put into the 5ml vial, (4 ℃ store for future use for 0.05mol/L, pH7.5) carbonate buffer solution dissolving to add 1.0ml;
(2) HRP that takes by weighing 10.0mg puts into 16 * 100mm glass test tube, adds the 1.0ml deionized water, vibration, treat that it dissolves fully after, take out 0.74ml and put into another 16 * 100mm glass test tube, add the NaIO of 0.1mol/L
4Solution 0.2ml (NaIO
4The necessary fresh preparation of solution), with glass test tube mouth jam-pack, the lucifuge reaction is 2 hours under the room temperature with plug;
(3) take out and to have dissolved good TPO antigen (step 1) is added in the mixed liquor that derives from step (2), and the lucifuge reaction is 3 hours under the room temperature;
(4) add the NaBH that concentration is 5mg/ml with 0.1mmol NaOH dissolving
4Solution (NaBH
4Necessary fresh preparation), lucifuge was reacted 0.5 hour under the room temperature;
(5) above-mentioned reactant liquor is packed in the bag filter, dialyse with 0.01mol/L PBS pH7.5 damping fluid, 4 ℃ of placements are spent the night;
(6) continue dialysis again behind the replacing dislysate, 4 ℃ of placements are spent the night;
(7) collect reactant liquor and add isopyknic 0.01mol/L PBS pH7.5 damping fluid that contains 10%BSA;
(8) carry out secondarily purifiedly with high pressure lipuid chromatography (HPLC), collect protein peak 1.47ml and in-20 ℃ of freezing preservations down.
The purity detecting of b, HRP labelled antigen: recording HRP concentration with ultraviolet spectrophotometry is 0.78mg/ml, and TPO concentration is 1.35mg/ml.
Therefore the ratio of its volumetric molar concentration is: HRP:TPO=(0.78 ÷ 40000): (1.35 ÷ 100000)=1.44:1.00.
Enzyme total amount=0.78mg/ml * 1.47ml=1.15mg in the bond
Enzyme combination rate (%)=(1.15mg/7.4mg) * 100%=15.54%
The antigenic mark rate: (1.35mg/ml * 1.47ml)/2.0mg * 100%=99.23%
The activity of c, enzyme-labelled antigen detects:
Make enzyme-labelled antigen and TPO monoclonal antibody form precipitation line with agar diffusion method, through PBS rinsing 1 day, again with distilled water immersion 1 hour, be dipped in the enzyme substrate solution agar gel sheet painted, stronger color reaction appears in the result, soaks with physiological saline again, and color does not still take off, the existing enzymatic activity of expression bond also has antibody activity.Bond is after colour developing, and fine jade expands titre and reaches 1:26, shows that enzyme-labelled antigen is functional.
Determining of d, enzyme-labelled antigen working concentration:
Enzyme-labelled antigen is pressed the multiple dilution of 1:500,1:750,1:1000,1:1500 and 1:2000,1:2500,1:3000,1:4000 and 1:5000 respectively with 0.01mol/LPBS damping fluid (pH=7.5), carry out the square formation titration with the CLIA method, the result shows that reaction pattern is best when dilutability is 1:3000, illustrates that also enzyme-labelled antigen has good enzymatic activity and antigen active.Therefore the working concentration of determining enzyme-labelled antigen is: 1.35mg/ml ÷ 3000=0.45 μ g/ml.
2, TPO MONOCLONAL ANTIBODIES SPECIFIC FOR and Performance Detection:
(1) antigen is purified and animal immune:
Thyroid peroxidase (TPO) adds complete freund adjuvant and through fully emulsified, selects 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, draws culture supernatant behind limiting dilution, detects antibody content with ELISA.Filter out high antibody-secreting hole, cell in the hole is carried out cloning again, 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.
(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 staphylococcal protein 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 eluent 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:
TPO monoclonal antibody the key technical indexes
| The ELISA titre | 1:(1.21×10 5) |
| Detection sensitivity | <4.93pg/mL |
| Cross reacting rate | <0.1％ |
| Affinity costant | 5.16×10 10M -1 |
Titre detects: be to detect antigen with TPO, measure antibody titer, its titre (in 1.0mg/ml)=1:(1.12 * 10 by indirect elisa method
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: the specificity of anti-TPO 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, wrap successively by microwell plate with 4 concentration TPO of 5,2.5,1.25 and 0.625 μ g/ml dilution, get its maximum OD value half (be OD 50%) by graphing method and locate 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=5.16 * 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 5mg organic-silylation magnetic particle, wash 1-2 time with redistilled water;
2. the PBS with 0.02mol/L pH5.6-6.2 washes once;
3. the PBS adjustment cumulative volume with 0.02mol/L pH5.6-6.2 is about 10ml;
4. add a certain amount of SPA and be placed on and carry out homogenize 10-20 minutes 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 5.0mg of carbodiimide (EDC);
6. room temperature reaction is placed on and carries out homogenize 10 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/LpH7.4PBS;
10. wash 3 times with 0.05mol/L Tris+0.004mol/L EDTA pH7.5 damping fluid;
(2) mensuration of SPA binding capacity: the SPA binding capacity shows with the scale of every milliliter or every gram organic-silylation nanometer magnetic particle coupling SPA that generally its numerical value can be used as the parameter of decision affinity adsorbent actual amount, adopts direct 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 of magnetic particle and SPA during to 1:50, 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 | 1:100 |
| SPA combination rate (%) | 25.64 | 38.13 | 47.18 | 62.71 | 63.76 | 64.97 |
4, the preparation of enhanced chemical luminous substrate working fluid:
(1) the Tris-HCl damping fluid of preparation 0.05mol/L pH9.0
(2) A liquid: in above-mentioned Tris-HCl damping fluid, add the 4.58mmol/L luminol.
(3) B liquid: in above-mentioned Tris-HCl damping fluid, add following material:
1. 0.81mmol/L tetraphenylboron sodium;
2. 0.38mmol/L cinnamic acid;
3. 7.56mmol/L hydrogen peroxide.
5, the preparation of cleansing solution:
①TrisBase 6.05g/L；
②NaCl 8.55/L；
③Tween-20 0.5ml/L；
4. transfer pH to 7.8 ± 0.1 with HCl.
(3) kit experimental implementation method and step:
Set up the quantitative analysis method of TPO-Ab chemiluminescence part, 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) get a microwell plate and be numbered, all experiments are all done diplopore and are repeated;
(3) standard items, quality controlled serum and sample 25～200 μ l that get each concentration add in the hole of corresponding numbering;
(4) every hole all adds 50～200 μ l TPO-HRP;
(5) be inverted abundant mixing SPA-nanometer magnetic particle, every hole all adds 25～150 μ lSPA-nanometer magnetic particles;
(6) abundant mixing, room temperature vibration 1～4 hour;
(7) place it in absorption magnetic particle on the magnetic sheet, inhale and remove supernatant;
(8) remove magnetic sheet, every hole all adds 200～1000 μ l and washes plate liquid;
(9) vibration placed it in absorption magnetic particle on the magnetic sheet after 4～15 seconds, inhaled and removed supernatant;
(10) come again step 8,9;
(11) every hole adds luminous enhancing A liquid 100～300 μ l;
(12) every hole adds into luminous enhancing B liquid 100～300 μ l;
(13) measure the luminous intensity in every hole with Chemiluminescence Apparatus;
(14) read the concentration of sample and quality controlled serum from typical curve.
The sensitivity of this kit can reach 0.1U/mL, and does not have obvious cross reaction, and accuracy reaches 95-105%, and it is 0.1-2000U/mL that typical curve satisfies the required measurement range of clinical samples, clinical coincidence rate 〉=95%.
1, autoimmune thyroid disease and control group TPO-Ab testing result are relatively
| Group | The example number | Detect routine number | Recall rate |
| Hyperthyroid group | 143 | 138 | 96.50％ |
| The low group of first | 132 | 126 | 95.45％ |
| Normal person's group | 117 | 0 | 0 |
2, clinical effectiveness analytic statistics
| Hyperthyroidism | First is low | Remarks | |
| Sensitivity (True Positive Rate) | 96.50％ | 95.45％ | |
| Specificity (True Positive Rate) | 100％ | 100％ | |
| False |
0％ | 0％ | |
| False negative rate | 3.50％ | 4.55％ | |
| Crude agreement (Crude agreement) | 98.08％ | 97.59％ | |
| Adjust consistance (adjusted agreement) | 98.10％ | 97.64％ | |
| Youden index (youden ' s index) | 0.9650 | 0.9545 | |
| The predictive value of |
100％ | 100％ | |
| The predictive value of negative test | 95.90％ | 95.12％ |
With the content of TPO-Ab in this kit measurement person under inspection serum, the TPO-Ab recall rate is 0 in the normal human serum; Hyperthyroid patient group TPO-Ab recall rate is 96.50%; It is 95.45% that the low patient of first organizes TPO antibody recall rate.Therefore, the TPO-Ab clinical assays result of this kit, have sensitivity and accuracy height, high specificity, testing result waits advantage accurately and reliably, the disease early diagnosis, early intervention treatment, correctly taking drugs and the therapeutic evaluation that can be autoimmunity thyroid gland disease provide reliable basis, and the treatment of autoimmunity thyroid gland disease is had crucial meaning.
Claims (6)
1, a kind of chemiluminescence ligand analysis method of detection by quantitative people's autoantibody, it is characterized in that: chemiluminescence immunoassay detection by quantitative CLIA technology is combined 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 staphylococcal protein A (SPA) can with the F of Immunoglobulin IgG in people and the mouse body
CThe denominator that the site reacts is with the chemiluminescence part quantitative analysis method of being set up autoantibody by nanometer magnetic particle ligand technique from the SPA of wound bag.
2, the chemiluminescence ligand analysis method of a kind of detection by quantitative people's autoantibody according to claim 1 is an example with the TPO autoantibody, it is characterized in that: at first with the staphylococcal protein A bag by in nanometer magnetic particle surface; Utilize the high-affinity that oneself prepares, the TPO monoclonal antibody of high specific to prepare standard items to set up typical curve, allow the TPO monoclonal antibody in the standard items form (TPO-Ab)-TPO-HRP immunocomplex with relative excessive enzyme-labelled antigen TPO-HRP reaction respectively with autoantibody in the blood serum sample, nanometer magnetic particle with albumin A bag quilt carries out Separation of Solid and Liquid as part, because albumin A can be by the Fc part combination of (TPO-Ab)-TPO-HRP immunocomplex, thereby form a solid phase, separate with the magnetic sheet method; Luminous detection is carried out in enzyme-catalyzed chemical luminescence reaction by horseradish peroxidase (HRP) catalysis; Luminous value according to standard items is set up typical curve, can find the concentration value of TPO autoantibody patients serum's sample from typical curve.
3, the chemiluminescence ligand analysis method of a kind of detection by quantitative people's autoantibody according to claim 2, it is characterized in that: monoclonal antibody and the autoantibody in the blood serum sample as standard items do not enter same system, the general character of just having utilized them to have with the albumin A specific bond contrasts, therefore cross reaction can not take place, testing result accurately and reliably, measurement result false negative phenomenon on the low side can not occur, having solved the autoantibody detection can't an accurately quantitative difficult problem.
4, according to the chemiluminescence ligand analysis method of claim 2 or 3 described a kind of detection by quantitative people's autoantibodies, be example, it is characterized in that method of production is with the TPO autoantibody:
1) kit constituent
The quantitative immune analysis diagnosis reagent kit key component of TPO-Ab chemiluminescence part is: label---TPO-HRP, standard items---TPO monoclonal antibody, ligand reagent---SPA-nanometer magnetic particle, enhanced chemical luminous substrate working fluid---A liquid and B liquid, cleansing solution;
2) preparation of each component of kit
(1) preparation of horseradish peroxidase HRP mark TPO antigen: under the effect of sodium periodate, TPO forms TPO-HRP with the HRP mark, adopts dialysis and HPLC to carry out secondarily purified;
(2) preparation of monoclonal antibody specific: prepare TPO monoclonal antibody (TPO-Ab) by the Fusion of Cells hybridoma technology, with it as the standard items of drawing with reference to curve;
(3) staphylococcal protein A (SPA)-nanometer magnetic preparation of granules: with SPA by the carbodiimide bag by in organic-silylation magnetic nano particle sub-surface;
(4) optimization of enhanced chemiluminescence substrate working fluid preparation: with the luminol is luminous substrate, adopts tetraphenylboron sodium, cinnamic acid and luminol to prepare by a certain percentage as enhanced chemical luminous substrate working fluid to improve luminous intensity;
3) TPO-Ab chemiluminescence part (nanometer magnetic particle) quantitative analysis method and clinical detection establishment of standard
(1) sets up chemiluminescence part (the nanometer magnetic particle) quantitative analysis method of TPO-Ab in the human serum, and formulate the experimental implementation method and the quality control standard of this kit;
(2) lab scale of TPO-Ab chemiluminescence part quantitative test diagnostic kit and part clinical trial:
Gather clinical samples, with the accurate content of TPO-Ab in this kit measurement person under inspection serum, a large amount of clinical test results show its clinical coincidence rate 〉=95%; Therefore, the TPO-Ab clinical assays value of this kit can be autoimmune thyroid disease diagnosis, early intervention treatment, correctly taking drugs and therapeutic evaluation reliable basis is provided;
4) set up a maturation, sensitivity, autoantibody chemiluminescence part (nanometer magnetic particle) quantitative measurement technology platform efficiently, can utilize a large amount of serial people's autoantibody chemiluminescence part (nanometer magnetic particle) the quantitative test diagnostic kit of this method exploitation, form one completely newly, the research and development and the production system of people's autoantibody chemiluminescence part (nanometer magnetic particle) detection by quantitative kit efficiently.
5, according to the chemiluminescence ligand analysis method of claim 2 or 3 described a kind of detection by quantitative people's autoantibodies, be example with the TPO autoantibody, it is characterized in that: the experimental implementation program of this kit is:
(1) will test required reagent and sample and place room temperature, balance just can be operated more than 30 minutes at least;
(2) get a microwell plate and be numbered, all experiments are all done diplopore and are repeated;
(3) standard items, quality controlled serum and sample 25～200 μ l that get each concentration add in the hole of corresponding numbering;
(4) every hole all adds 50～200 μ l TPO-HRP;
5) be inverted abundant mixing SPA-nanometer magnetic particle, every hole all adds 25～150 μ lSPA-nanometer magnetic particles;
(6) abundant mixing, room temperature vibration 1～4 hour;
(7) place it in absorption magnetic particle on the magnetic sheet, inhale and remove supernatant;
(8) remove magnetic sheet, every hole all adds 200～1000 μ l and washes plate liquid;
(9) vibration placed it in absorption magnetic particle on the magnetic sheet after 4～15 seconds, inhaled and removed supernatant;
(10) come again step 8,9;
(11) every hole adds luminous enhancing A liquid 100～300 μ l;
(12) every hole adds into luminous enhancing B liquid 100～300 μ l;
(13) measure the luminous intensity in every hole with Chemiluminescence Apparatus;
(14) read the concentration of sample and quality controlled serum from typical curve.
6, the chemiluminescence ligand analysis method of a kind of detection by quantitative people's autoantibody according to claim 4 is an example with the TPO autoantibody, it is characterized in that: the experimental implementation program of this kit is:
(1) will test required reagent and sample and place room temperature, balance just can be operated more than 30 minutes at least;
(2) get a microwell plate and be numbered, all experiments are all done diplopore and are repeated;
(3) standard items, quality controlled serum and sample 25～200 μ l that get each concentration add in the hole of corresponding numbering;
(4) every hole all adds 50～200 μ l TPO-HRP;
(5) be inverted abundant mixing SPA-nanometer magnetic particle, every hole all adds 25～150 μ lSPA-nanometer magnetic particles;
(6) abundant mixing, room temperature vibration 1～4 hour;
(7) place it in absorption magnetic particle on the magnetic sheet, inhale and remove supernatant;
(8) remove magnetic sheet, every hole all adds 200～1000 μ l and washes plate liquid;
(9) vibration placed it in absorption magnetic particle on the magnetic sheet after 4～15 seconds, inhaled and removed supernatant;
(10) come again step 8,9;
(11) every hole adds luminous enhancing A liquid 100～300 μ l;
(12) every hole adds into luminous enhancing B liquid 100～300 μ l;
(13) measure the luminous intensity in every hole with Chemiluminescence Apparatus;
(14) read the concentration of sample and quality controlled serum from typical curve.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100602148A CN101470117A (en) | 2007-12-26 | 2007-12-26 | Chemiluminescent ligand analysis method for quantitative detection of human auto-antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100602148A CN101470117A (en) | 2007-12-26 | 2007-12-26 | Chemiluminescent ligand analysis method for quantitative detection of human auto-antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101470117A true CN101470117A (en) | 2009-07-01 |
Family
ID=40827781
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007100602148A Pending CN101470117A (en) | 2007-12-26 | 2007-12-26 | Chemiluminescent ligand analysis method for quantitative detection of human auto-antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101470117A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102520195A (en) * | 2011-12-30 | 2012-06-27 | 天津市协和医药科技集团有限公司 | Chromogranin A chemiluminescence immunoassay reagent kit and preparation method thereof |
| CN102565032A (en) * | 2010-12-15 | 2012-07-11 | 北京勤邦生物技术有限公司 | Magnetic granule chemiluminescence kit for detecting nitrofurantoin metabolite and application of magnetic granule chemiluminescence kit |
| CN103645318A (en) * | 2013-12-09 | 2014-03-19 | 重庆原伦生物科技有限公司 | Detection method and detection kit of antigen-specific IgG (immunoglobulin G) antibody of staphylococcus aureus SpA5 mutant |
| CN104849475A (en) * | 2015-05-02 | 2015-08-19 | 王贤俊 | Quantified detection method for luteinizing hormone (LH) |
| WO2017039967A1 (en) * | 2015-09-01 | 2017-03-09 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Method and device for detecting antigen-specific antibodies in a biological fluid sample by using neodymium magnets |
| CN112312921A (en) * | 2018-07-02 | 2021-02-02 | 美国西门子医学诊断股份有限公司 | Novel immunoassay for thyroid peroxidase autoantibodies |
| CN112710839A (en) * | 2020-12-14 | 2021-04-27 | 三诺生物传感股份有限公司 | GAD65 autoantibody detection kit and preparation method thereof |
| CN113092392A (en) * | 2021-03-05 | 2021-07-09 | 苏州西山中科药物研究开发有限公司 | Universal method for detecting total concentration of monoclonal antibody drug targeting soluble protein in monkey serum |
| JP2021529948A (en) * | 2018-07-02 | 2021-11-04 | シーメンス・ヘルスケア・ダイアグノスティックス・インコーポレイテッド | Direct immunoassay measurement of autoantibodies |
-
2007
- 2007-12-26 CN CNA2007100602148A patent/CN101470117A/en active Pending
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102565032A (en) * | 2010-12-15 | 2012-07-11 | 北京勤邦生物技术有限公司 | Magnetic granule chemiluminescence kit for detecting nitrofurantoin metabolite and application of magnetic granule chemiluminescence kit |
| CN102520195A (en) * | 2011-12-30 | 2012-06-27 | 天津市协和医药科技集团有限公司 | Chromogranin A chemiluminescence immunoassay reagent kit and preparation method thereof |
| CN102520195B (en) * | 2011-12-30 | 2014-07-23 | 天津市协和医药科技集团有限公司 | Chromogranin A chemiluminescence immunoassay reagent kit and preparation method thereof |
| CN103645318A (en) * | 2013-12-09 | 2014-03-19 | 重庆原伦生物科技有限公司 | Detection method and detection kit of antigen-specific IgG (immunoglobulin G) antibody of staphylococcus aureus SpA5 mutant |
| CN103645318B (en) * | 2013-12-09 | 2016-11-02 | 成都欧林生物科技股份有限公司 | Staphylococcus aureus SpA5 mutant antigen specific IgG antibodies detection method and detection kit |
| CN104849475A (en) * | 2015-05-02 | 2015-08-19 | 王贤俊 | Quantified detection method for luteinizing hormone (LH) |
| WO2017039967A1 (en) * | 2015-09-01 | 2017-03-09 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Method and device for detecting antigen-specific antibodies in a biological fluid sample by using neodymium magnets |
| US10564152B2 (en) | 2015-09-01 | 2020-02-18 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Method and device for detecting antigen-specific antibodies in a biological fluid sample by using neodymium magnets |
| CN112312921A (en) * | 2018-07-02 | 2021-02-02 | 美国西门子医学诊断股份有限公司 | Novel immunoassay for thyroid peroxidase autoantibodies |
| JP2021529948A (en) * | 2018-07-02 | 2021-11-04 | シーメンス・ヘルスケア・ダイアグノスティックス・インコーポレイテッド | Direct immunoassay measurement of autoantibodies |
| US11327074B2 (en) | 2018-07-02 | 2022-05-10 | Siemens Healthcare Diagnostics Inc. | Thyroid peroxidase autoantibody immunoassay |
| US11835518B2 (en) | 2018-07-02 | 2023-12-05 | Siemens Healthcare Diagnostics Inc. | Thyroid peroxidase autoantibody immunoassay |
| CN112710839A (en) * | 2020-12-14 | 2021-04-27 | 三诺生物传感股份有限公司 | GAD65 autoantibody detection kit and preparation method thereof |
| CN113092392A (en) * | 2021-03-05 | 2021-07-09 | 苏州西山中科药物研究开发有限公司 | Universal method for detecting total concentration of monoclonal antibody drug targeting soluble protein in monkey serum |
| CN113092392B (en) * | 2021-03-05 | 2022-11-08 | 苏州西山中科药物研究开发有限公司 | Universal method for detecting total concentration of monoclonal antibody drug targeting soluble protein in monkey serum |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101470117A (en) | Chemiluminescent ligand analysis method for quantitative detection of human auto-antibody | |
| JP3108103B2 (en) | Monoclonal antibodies against human cardiac myoglobin | |
| AU2010274320B2 (en) | Insulin measurement method | |
| CN101738473B (en) | Treponema pallidum antibody diagnostic kit and preparation method thereof | |
| CN101360997A (en) | Modified cardiolipin and uses therefor | |
| CN105745542B (en) | The assay method of influenza A virus | |
| CN1924579A (en) | Cross-linked composite used as standard diagnosing reagent replacing positive serum and method for use as standard reagent | |
| CN104849251A (en) | Time resolution fluorescence immunoassay method and kit for fast detecting gutter oil | |
| CA3033035C (en) | Method for the diagnosis of acute pancreatitis (ap) by detection of glycoprotein 2 isoform alpha (gp2a) | |
| CN103045541A (en) | Hybridoma cell strain and monoclonal antibody of anti-heart-type fatty acid-binding protein generated by same | |
| CN102876634B (en) | PMSG (pregnant mare serum gonadotropin) double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) kit | |
| CN101957365B (en) | Kit for detecting cyclic citrullinated peptide (CCP) and immunoglobulin G (IgG) resistant bispecific antibody | |
| CA2785678C (en) | Human insulin assay and assay reagent | |
| JP5798679B2 (en) | Monoclonal antibody specifically recognizing human liver-carboxyl esterase 1, hybridoma cell line producing said antibody and use thereof | |
| US20140186861A1 (en) | Soluble treponema pallidum protein tp0453, tp0453-tp0326 fusion protein, and use in syphilis diagnosis | |
| CN101256189A (en) | Human-outer autoantibody radio-immunity quantitative determination method | |
| CN105403706A (en) | Heat shock protein 90 (HSP90) chemiluminescence detection kit | |
| CN104764884A (en) | Preparation method and application of anti-alpha-enolase antibody chromatography test strip | |
| Miyazawa et al. | A reverse-sandwich ELISA for IgG antibody to a pollen allergen | |
| CN105987998B (en) | Human tissue kallikrein 1ELISA immue quantitative detection reagent boxes | |
| CN105988004B (en) | The collaurum quantitative testing test paper card of human tissue kallikrein 1 | |
| CN104297489A (en) | Anti-PR3-ANC antibody detection test paper preparation method and purpose thereof | |
| Jeon et al. | ELISA-on-a-chip for on-site, rapid determination of anti-rabies virus antibodies in canine serum | |
| CN111217910A (en) | Monoclonal antibody pair and application thereof in detecting myeloperoxidase protein | |
| JP2878317B2 (en) | Laminin measurement reagent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20090701 |