CN104714028B - Immunodetection method for detecting IgM antibody - Google Patents
Immunodetection method for detecting IgM antibody Download PDFInfo
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Abstract
The invention discloses an immunodetection method for detecting an IgM antibody, and relates to an immunodetection technology of the IgM antibody. The immunodetection method comprises the following steps: 1, curing processing of anti-IgM; 2, combination of IgM to be detected with the anti-IgM; 3, combination of an antigen and a conjugated monoclonal antibody with the IgM to be detected on a solid phase carrier; and 4, signal detection. The immunodetection method has the advantages of greatly reducing the interference from the IgM to the detection result, reducing the background and false positive rate of the detection, improving the detection specificity, enhancing the detection signal, reducing the false negative rate and improving the detection sensitivity.
Description
Technical field
The present invention relates to technical field of biomedical detection, more particularly, to igm antibody mediated immunity detection technique field.
Background technology
In existing immunologic detection method, mainly test substance in applied immunology detection technique detection sample；In clinic
In inspection, the material such as antibody or antigen in the main detection body fluid by antigen-antibody reaction.According to the difference of Cleaning Principle, immunity
Detection can be divided into indirect method, sandwich method, competition law and prize law.
In normal serum, igg is the main component of serum immune globulin, accounts for Immunoglobulin in Serum total content
75%, and igm then constitutes about the 6% of Immunoglobulin in Serum total amount, for certain antigen specific igm often with special
Different in nature igg exists simultaneously, and specific igg often disturbs the mensure of igm antibody.In existing four kinds of detection methods, specifically
Property igg is mainly manifested in as follows to the interference of igm TPPA:
When indirect method surveys igm antibody, by envelope antigen, examined with the pattern that enzyme or other instruction substance markers two resist
Survey, it is not strong that specificity easily in this kind of method, is easily disturbed by igg in sample to be tested；
When sandwich method surveys igm antibody, it is not strong specificity equally, is easily disturbed by igg, detection sensitivity relatively captures
Method is not high, and the antigen simultaneously marking can be vied each other with coated antigen and is combined with igm to be measured, lead to detection signal strong or
Person does not have；
When competition law surveys igm antibody, because igm antibody molecule amount is larger, it is not to be especially suitable for using the method, and and
Connection is the same, and its specificity is not strong, is easily disturbed by igg in sample to be tested, marks igm antibody used to be difficult to obtain simultaneously.
When measuring igm antibody at present, mainly use prize law, capture rule is by being coated anti-igm, with enzyme or other instructions
The pattern of substance markers antigen is detected, i.e. first (immunoglobulin (Ig) igm all in serum are included specific igm and non-specific
Property igm) be all fixed in solid phase, remove igg interference after, then detect to be measured specificity igm, but this in detection process,
Detection sensitivity easily occurs not high, be easily caused false negative phenomenon.
Content of the invention
Present in immunologic detection method for existing detection igm antibody, specificity is not strong, is easily subject in sample to be tested
Igg interference and detection sensitivity not high, false-negative problem easily occurs, the invention aims to solve foregoing problems and
A kind of immunologic detection method of detection igm antibody is proposed.
For reaching above-mentioned technical purpose, present invention employs following technical scheme:
The present invention is a kind of immunologic detection method of detection igm antibody: comprises the steps of
Step one, the curing process of anti-igm, anti-igm is placed on solid phase carrier, under conditions of 2 DEG C～8 DEG C, incubation
12 hours and more than, be washed out 3～5 times；
Step 2, igm to be measured are combined with anti-igm, and the igm addition to be measured as sample has been had cured consolidating of anti-igm
In phase carrier, it is incubated 0.5～1.0 hour, wash 3～5 times；
Step 3, antigen, mark monoclonal antibody are combined with the igm to be measured on solid phase carrier, can be first by antigen and mark monoclonal antibody
After mixing, add in the solid phase carrier washing in step 2, in insulating box, 37 DEG C of constant temperature incubations 0.5～1
Hour, incubation terminates, and washs 3～5 times；Or successively antigen, mark monoclonal antibody are added the solid phase carrier washing in step 2
In, first antigen is added, and then add mark monoclonal antibody, in insulating box, 37 DEG C of condition constant-temperature incubations 0.5～1 hour, incubate
Educate end, wash 3～5 times, remain to detect；
Step 4, signal detection, after the completion for the treatment of step 3, according to the difference of the indicant on mark monoclonal antibody, corresponding
Its reaction signal detection is carried out on instrument and equipment.
More preferably, during the incubation of described antigen, mark monoclonal antibody and igm to be measured, antigen share can be tied simultaneously
The mark monoclonal antibody of conjunction 2 and above different binding sites, to increase reaction signal.
More preferably, described solid phase carrier is microwell plate, glass plate or nc film.
More preferably, described washing process adopts the cleaning solution containing surfactant.
More preferably, described anti-igm is the goat-anti igm or anti-igm of mouse.
More preferably, the indicant in described mark monoclonal antibody is enzyme, collaurum, collargol, electroselenium, latex, radioactive material
Matter, chemiluminescent substance and fluorescent material.
Beneficial effects of the present invention: the present invention eliminates the competitive knot with antigen of igg and igm to be measured in sample first
Closing, greatly reduce the interference to testing result for the igg, thus reducing background and the false positive rate of detection, improving detection special
Property, adopt the mark monoclonal antibody of 2 and different binding sites above simultaneously, strengthen detection signal, reduce false negative rate, improve detection
Sensitivity.
Brief description
Fig. 1 is a kind of reaction principle schematic diagram of the immunologic detection method of present invention detection igm antibody.
Wherein, 1- solid phase carrier；The anti-igm of 2-；Igm to be measured in 3- sample；4- antigen；5- marks monoclonal antibody.
Specific embodiment
In order to the present invention is better described, it is described further with accompanying drawing in conjunction with embodiment.
The present invention is a kind of immunologic detection method of detection igm antibody, reaction principle as shown in Figure 1, the step being related to
For:
Step one, the curing process of anti-igm2.Anti- igm2 is placed in microwell plate or glass plate or the nc of detecting system
On film, these microwell plates or glass plate or nc film are the solid phase carrier 1 of experiment, under conditions of 2 DEG C～8 DEG C, are incubated 12
Hour and more than, then wash 3～5 times with the cleaning solution containing surfactant, the purpose of washing is mainly removal and not yet combines
The anti-igm2 of upper solid phase carrier 1.
Step 2, igm3 to be measured are combined with anti-igm2.Have cured consolidating of anti-igm2 by adding containing igm3 sample to be measured
In phase carrier 1, it is incubated 0.5～1.0 hour, after the anti-igm2 on igm3 to be measured and solid phase carrier 1 combines, with containing surface-active
The cleaning solution of agent washs 3～5 times, to remove the specific igg in sample, it is to avoid the interference to detection for the specific igg.
Step 3, antigen, mark monoclonal antibody are combined with the igm3 to be measured on solid phase carrier.Can be by antigen and at least 2 marks
After note monoclonal antibody mixes, add and be attached in anti-igm2 and the solid phase carrier of igm3 to be measured, in insulating box, constant temperature temperature
37 DEG C of degree, is incubated 0.5～1 hour, incubation terminates, and is washed 3～5 times using the cleaning solution containing surfactant；Or can be first
Antigen is added the solid phase load being attached to anti-igm2 knot and sample zhogn igm3 to be measured in the insulating box being placed on 37 DEG C
In body, and then add at least 2 mark monoclonal antibodies, be equally incubated 0.5～1 hour, incubation terminates, using containing surfactant
Cleaning solution washs 3～5 times, obtains being incubated thing, to be detected；
Step 4, signal detection.After the completion for the treatment of step 3, according to the difference of indicant on mark monoclonal antibody, in corresponding instrument
Its reaction signal detection is carried out on device equipment.
In particular it relates to mark monoclonal antibody be 2 and the monoclonal antibody above with different binding sites.
Specifically, anti-igm used in the present invention can be the goat-anti igm or anti-igm of mouse.
Specifically, present invention could apply to ELISA, fast detection method, radio-immunoassay, fluorescence immunoassay
Detection method.
Specifically, the indicant of the present invention can be enzyme, collaurum, collargol, electroselenium, latex, radioactive substance, change
Learn luminescent substance and fluorescent material.
Specific embodiment:
Rubella virus igm antibody assay kit (ELISA) is adopted in the present embodiment.
The preparation of 1.1 microwell plates
Be coated: by anti-igm (specially goat-anti people igm, be purchased from sigma company) be coated liquid (cbs (1l, ph=9.6):
na2co31.59g、nahco32.93g) prepare according to 1:1000, then add microwell plate (the one of solid phase carrier 1 using pipettor
Kind) in, every hole 100ul, then at 2 DEG C～8 DEG C, incubation time is more than or equal to 12 hours, then washs 3 with cleaning solution
Secondary；
Closing: the microwell plate having washed is patted dry residual liquid on gauze, then at 37 DEG C, every hole adds 200 μ l
Confining liquid (pbs+0.5%bsa), close 2 hours；Directly confining liquid is outwelled, after patting dry Liquid Residue after end；If no
Can use immediately, then dry microwell plate under conditions of room epidemic disaster is for less than 50%, then sealing preserve；
The preparation of 1.2 antigens: using Sample dilution (pbs), rubella virus antigen being diluted to concentration is 0.5～1mg/
ml；
1.3 hrp- monoclonal antibodies 1 and the preparation of hrp- monoclonal antibody 2: monoclonal antibody 1 and monoclonal antibody 2 (making by oneself) adopt horseradish peroxidase
(abbreviation hrp is purchased from sigma company) is marked according to the glutaraldehyde method of improvement, first uses Sample dilution according to 1 using front:
20000 are diluted, and reuse；
1.4 substrate solutions and the preparation of terminate liquid:
Substrate a liquid: tmb200mg, absolute ethyl alcohol (or dmso) 100ml, plus distilled water is to 1000ml；
Substrate b liquid: na2hpo414.60g, citric acid 9.33g, 0.75% hydrogen peroxide urea 6.4ml, plus tri-distilled water is extremely
1000ml, adjusts ph to 5.0～5.4；
Terminate liquid: measure the concentrated sulfuric acid of 13.6ml98%, be then slowly added in distilled water, and ceaselessly stir, finally
It is settled to 500ml；
The detection method of 1.5 kits:
Band detection sample is used sample diluting liquid (pbs) to dilute according to 1:100, is subsequently adding the microwell plate preparing
In, every hole adds 100ul, does 2 critical comparisons simultaneously, at 37 DEG C, is incubated 1 hour, then washs 3 with cleaning solution (pbst)
Secondary；
Antigen, hrp- monoclonal antibody 1 and hrp- monoclonal antibody 2 are mixed and made into every hole after compound according to 1:1:1 and add 100ul,
At 37 DEG C, it is incubated 0.5～1 hour, then washs 3 times with cleaning solution (pbst)；
Every hole adds substrate a liquid and each mixing of b liquid, and lucifuge stands 15～30 minutes at room temperature；
Every hole adds terminate liquid 1, reads its od value with ELIASA in 30 minutes at 450nm wavelength；
Specific result judgement standard is as follows:
Negative: the mean value of od value ＜ 0.9 × critical control wells；
Positive: the mean value of od value ＜ 1.1 × critical control wells；
Grey area: od value is in the range of the 1 ± 10% of critical control wells mean value.
1.6 testing result
1.6.1 sensitivity for analysis and specificity
Measured through clinical 100 negative sample and 30 confirming with the rubella virus igm antibody assay kit preparing
Example positive sample, the od numerical value of reading is as shown in table 1.
The testing result of table 1 kit
Sensitivity for analysis: 30/ (30+0) × 100%=100%
Analysis specificity: 100/ (100+0) × 100%=100%
1.6.2 experiment detection
By the antigen being manufactured separately using rubella virus+hrp- monoclonal antibody 1, antigen+hrp- monoclonal antibody 2, antigen+hrp- monoclonal antibody 1+
28 parts of Blood serum plate (who) are detected by hrp- monoclonal antibody 2, the kit (buying other companies) of direct labelled antigen, detection knot
Fruit is as shown in table 2.
The testing result of table 2 Blood serum plate (who)
It can be seen from the results above that after the present invention adopts 2 mark monoclonal antibodies, detection signal is substantially strengthened, especially in inspection
When surveying weakly positive sample, performance is especially prominent, and when detecting negative sample, then basically identical with additive method；Simultaneously from table
In as can be seen that market on using direct labelled antigen pattern prize law, false positive phenomenon also occurs in detection process.
Described in synthesis, technical scheme sufficiently effective can complete foregoing invention purpose, and the present invention
Structural principle and the principle of work and power are all sufficiently verified in an embodiment, and can reach expected effect and purpose, and
Embodiments of the invention can also be applied equally to other classes according to these principles in the different enterprising line translations of solid phase carrier
The antigen of type, therefore, the present invention include all be previously mentioned in claim in the range of all replacement contents.Any
The equivalence changes made in scope of the present invention patent, all belong within the scope of the claims of this case application.
Claims (5)
1. a kind of immunologic detection method of detection igm antibody it is characterised in that: comprise the steps of
Step one, the curing process of anti-igm, anti-igm is placed on solid phase carrier, under conditions of 2 DEG C～8 DEG C, is incubated 12
Hour and more than, be washed out 3～5 times；
Step 2, igm to be measured are combined with anti-igm, and the igm addition to be measured as sample has been had cured consolidating of anti-igm
In phase carrier, it is incubated 0.5～1.0 hour, wash 3～5 times；
Step 3, antigen, mark monoclonal antibody are combined with the igm to be measured on solid phase carrier, first mix antigen with mark monoclonal antibody
Afterwards, add in the solid phase carrier washing in step 2, in insulating box, 37 DEG C of constant temperatures are incubated 0.5～1 hour,
Incubation terminates, and washs 3～5 times；Or successively antigen, mark monoclonal antibody are added in the solid phase carrier washing in step 2,
First antigen is added, and then add mark monoclonal antibody, in insulating box, 37 DEG C of condition constant-temperature incubations 0.5～1 hour, incubate
Educate end, wash 3～5 times, remain to detect；
Step 4, signal detection, after the completion for the treatment of step 3, according to the difference of the indicant on mark monoclonal antibody, in corresponding instrument
Its reaction signal detection is carried out on equipment；
During described antigen, the incubation of mark monoclonal antibody and igm to be measured, antigen molecule can in combination with 2 and more than
The mark monoclonal antibody of different binding sites, to increase reaction signal.
2. a kind of detection igm antibody as described in claim 1 immunologic detection method it is characterised in that: described solid phase carries
Body is microwell plate, glass plate or nc film.
3. a kind of detection igm antibody as described in claim 1 immunologic detection method it is characterised in that: described washed
Cheng Caiyong contains the cleaning solution of surfactant.
4. a kind of detection igm antibody as described in claim 1 immunologic detection method it is characterised in that: described anti-igm
For the goat-anti igm or anti-igm of mouse.
5. a kind of detection igm antibody as described in claim 1 immunologic detection method it is characterised in that: described mark is single
Indicant in anti-is enzyme, collaurum, collargol, electroselenium, latex, radioactive substance, chemiluminescent substance and fluorescent material.
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| CN105785045A (en) * | 2016-04-13 | 2016-07-20 | 柏荣诊断产品（上海）有限公司 | High-performance detection reagent kit for human blood immunoglobulin M |
| CN108169475A (en) * | 2017-12-18 | 2018-06-15 | 郑州安图生物工程股份有限公司 | A kind of Respiratory Syncytial Virus(RSV) IgM antibody detection kit |
| CN116143931B (en) * | 2021-11-20 | 2023-10-31 | 东莞市朋志生物科技有限公司 | Anti-human IgM antibody and preparation method and application thereof |
| CN116148462A (en) * | 2021-11-22 | 2023-05-23 | 菲鹏生物股份有限公司 | Method for detecting antibody by capture method and application |
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| EP0189389B1 (en) * | 1985-01-24 | 1989-04-05 | Institut International De Pathologie Cellulaire Et Moleculaire | Immunoassay method for antibodies of different classes in a liquid sample |
| WO1999026067A1 (en) * | 1997-11-18 | 1999-05-27 | Bio-Rad Laboratories, Inc. | Multiplex flow immunoassays with magnetic particles as solid phase |
| CN1902496A (en) * | 2003-11-07 | 2007-01-24 | 赫普金尼克斯股份有限公司 | Binding assay components |
| CN101013133B (en) * | 2007-02-12 | 2011-01-26 | 北京英诺特生物技术有限公司 | Colloidal gold chromatography strip for detecting specific IgM antibody and method for making same |
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Effective date of registration: 20190422 Address after: 518000 6 301A, No. 14, Hangzi Zhongxing Road, Hangzi Street, Pingshan District, Shenzhen City, Guangdong Province Patentee after: Shenzhen Kerunda Bioengineering Co., Ltd. Address before: 518172 Room B1301, 13 floors, Building B, No. 2, Tian'an Digital Innovation Park, Longgang District, Shenzhen, Guangdong Province, at the junction of Qinglin West Road and Huangge North Road, Longgang District, Longcheng Street Center Patentee before: Kai Ruide Bioisystech Co., Ltd of Shenzhen |
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