CN106501519A - Human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent and preparation method thereof - Google Patents
Human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent and preparation method thereof, human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent includes：HIV (human immunodeficiency virus) monoclonal antibody and the chemiluminescent labels of the coated carboxylated magnetic particle of human immunodeficiency virus antigen and HIV (human immunodeficiency virus) monoclonal antibody and human immunodeficiency virus antigen labelling.This human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent can with Full-automatic chemiluminescence immunoassay analysis meter as detect instrument, complete human immunodeficiency virus antigen and（Or）The detection of antibody.This human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent, through experiment, its antigen detection sensitivity reaches 1.25IU/mL, relative to the detection method of traditional human immunodeficiency virus antigen antibody, sensitivity at least improves 5 times, and the accuracy of detection of this human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent is higher.
Description
Technical field
The present invention relates to vitro detection field, more particularly to a kind of human immunodeficiency virus antigen antibody chemical luminescence is exempted from
Epidemic disease detection kit and preparation method thereof.
Background technology
HIV (human immunodeficiency virus)（Human Immunodeficiency Virus, HIV）It is a kind of infection human immunity
The slow viruss of system cells, belong to one kind of retrovirus retrovirus.It is believed that the infection of HIV (human immunodeficiency virus) causes acquired immune deficiency syndrome (AIDS)
（AIDS, Acquired Immune Deficiency Syndrome, acquired immunity lack syndrome）, acquired immune deficiency syndrome (AIDS) is posteriority
There is defect and causes serious opportunistic infections and/or secondary tumor a kind of fatal disease in cellular immune function.Acquired immune deficiency syndrome (AIDS) is certainly
It is identified and develops into the whole world in the U.S. within 1981 and be very popular, to the end of the year 2003, adds up to cause two over thousands of ten thousand people dead.
Inhibition of HIV is spherical in shape or oval granule, diameter 100-140nm, be with tunicary RNA retrovirus,
Belong to the lentiviridae in Retroviridae in classification.Inhibition of HIV single copy gene group RNA is about 9.2-9.7Kb.At present
It was found that there is HIV-1 and HIV-2 amphitypys, both types are much like on biological characteristicses, and their genome has 40%-50%
Homology, the core protein of two of which virus has the cross reaction of height.HIV has three main structural genes：
Env, gag, pol, they be separately encoded produce difference in functionality protein i.e. antigen, antigen can cause immune response so as to
Produce corresponding antibody.
According to HIV gene differences, it is divided into HIV-1 and HIV-2, the homology of aminoacid sequence is 40% ~ 60% between amphitypy.
HIV-1 can be further divided into different hypotypes, including M hypotype groups, O hypotypes group and N hypotype groups.At present Global prevalence mainly
HIV-1, the popular HIV-1 hypotypes in different regions have differences, in the mainly subtype B that the U.S. and West Europe are popular, China with
HIV-1 is main epidemic strain, it has been found that have A, B, B ', C, D, E, F and G totally 8 hypotypes, also different prevalence recombinant types.
HIV-2 is mainly popular in West Africa number state.In recent years, minority HIV-2 case was also found successively in Europe and minority Asian countries.
Find in China some areas from 1999 and confirm minority HIV-2 type the infected.
When HIV enters human body to be infected, the immune system of human body can be produced accordingly to multiple virus proteins of HIV
Antibody.In 5-12 weeks generally after infected by HIV, the antibody of HIV can be found in serum.Due to resisting for immune epitope
Former decision basic sequence is different, particularly HIV-1/M types, and HIV-1/O types are different with the memebrane protein of HIV-2, it is therefore desirable to specificity
Antigen can successfully detect HIV to guarantee immunoassay.By determining recent infection patient（High virus quantity）Blood specimen
Middle HIV-1 p24 antigens, antibody testing method that can be more traditional find HIV in more than 6 days ahead of time.HIV immunology detection is passed through
Gone through the development course of several generations, at present antigen and antibody test kit of state-of-the-art 4th generation HIV can detect simultaneously anti-HIV-1/
2 antibody and p24 antigens, shorten the diagnostic window phase.
The common methods of Clinical detection human immunodeficiency virus antigen antibody have enzyme linked immunosorbent assay, immune-gold labeled
Method, enzyme-catalyzed chemical luminescence method, but in place of these methods all have some shortcomings.
First, enzyme linked immunosorbent assay
Enzyme linked immunosorbent assay（ELISA）It is widely used, but the method there is also following weak points：
（1）12 × 8 types, 6 × 8 types, 8 × 12 types or 96 hole Special micro porous plate of complete plate is used to use as antigen or antibody coating
Tool and reaction vessel, using when can only be divided into 12 batches, 6 batches, 8 batches or imposite first use, it is impossible to carry out independent,
The detection of single part；
（2）Detection reagent type used is more, and each detectable will be contained with reagent bottle, and often using one
It is required for changing imbibition nozzle to be filled in the micropore of microwell plate respectively when planting reagent, not only reagent bottle species is more, fills reagent
Operation also extremely loaded down with trivial details；
（3）Lack the corresponding mark to detection information, can only just will appreciate that by checking the mark of test kit external packing box or know
Product batch number and the effect duration information of detectable is known, and the information that is known is uncontrolled in detection process, with very big
Randomness；
（4）Detectable in open space, easily causes the cross-contamination between various reagents and shadow in detection process
Ring the accuracy of testing result；
（5）Dosage more than detection process using manual operations, reagent or sample is not bery accurate, and operating process is extremely loaded down with trivial details and multiple
Miscellaneous, bust is susceptible to, the accuracy and precision of testing result is poor；
（6）Item number × 48/96 person-portion is in the quantity configuration of detection project reagent set and using on, if necessary to examine
10 projects are surveyed, then the configuration of reagent and the use of number must be 10 × 48/96 person-portions, if only a sample needs to detect 10
Different project, it is also desirable to configure the reagent of 10 × 48/96 person-portions, haves the shortcomings that inadequate economical rationality.
2nd, immune-gold labeled method
Gold colloidal be by gold chloride (HAuCl4) reducing agent such as white phosphorus, ascorbic acid, sodium citrate, tannic acid etc. effect under, can
Aggregate into a certain size gold grain, and a kind of stable colloidal state is become due to electrostatic interaction.Gold colloidal is in mild alkaline conditions
Under negatively charged, firm combination can be formed with the positive charge group of antigen, antibody.Immuno-gold labeling technology (Immunogold
Labelling techique) mainly make use of gold grain that there is the characteristic of high electron density, at gold mark protein binding,
Visible dark brown coloured particles under microscope, when these labels are assembled at corresponding part in a large number, naked eyes red color visible or powder
Punctation；The advantage of the method is easy to operate quick, but sensitivity is relatively low.
3rd, chemoluminescence method
Chemoluminescence method can be divided into direct chemiluminescence and enzyme-catalyzed chemical luminescence by principle of luminosity.
Enzyme-catalyzed chemical luminescence mainly has horseradish peroxidase（HRP）With two kinds of alkali phosphatase, but there is certain office
Sex-limited, horseradish peroxidase major defect is：Luminol, also can be by H2O2 in the case of existing without horseradish peroxidase
Oxidation itself lights, and background is of a relatively high, affects signal to noise ratio, and kinetics is complicated, and influence factor is more, is as a result not sufficiently stable,
The substrate for obtaining sensitivity height and plateau length is not easy.Alkali phosphatase major defect is：Substrate reach plateau when
Between long, substrate high cost, cause testing cost high, patient burden's weight.
Acridinium ester compares enzyme-catalyzed chemical luminescence with detailed advantage, main performance as the direct chemiluminescence of label
?：Reaction does not need catalyst, as long as alkaline environment can be carried out, is swift in response, and background luminescence is low, and signal to noise ratio is high, disturb because
Element is few, and reagent stability is good, can be simple with two-point calibration, system, exciting liquid low cost, acridinium ester easily and protein bind, and
After connection, photon yield is not reduced.
Content of the invention
It is based on this, it is necessary to provide a kind of detection sensitivity higher human immunodeficiency virus antigen antibody chemical luminescence
Immunity detection reagent and preparation method thereof.
A kind of human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent, including：Human immune deficiency
Viral monoclonal antibodies and the coated carboxylated magnetic particle of human immunodeficiency virus antigen and HIV (human immunodeficiency virus) list
Clonal antibody and the chemiluminescent labels of human immunodeficiency virus antigen labelling.
In one embodiment, the HIV (human immunodeficiency virus) monoclonal antibody and human immunodeficiency virus antigen bag
In the carboxylated magnetic particle of quilt, the HIV (human immunodeficiency virus) monoclonal antibody and human immunodeficiency virus antigen and institute
The ratio for stating carboxylated magnetic particle is 1：25~100.
In one embodiment, the HIV (human immunodeficiency virus) monoclonal antibody and human immunodeficiency virus antigen mark
In the chemiluminescent labels of note, the HIV (human immunodeficiency virus) monoclonal antibody and human immunodeficiency virus antigen and institute
The ratio for stating chemiluminescent labels is 1:10~100.
In one embodiment, the particle diameter of the carboxylated magnetic particle is 0.05 μm ~ 1 μm.
In one embodiment, the chemiluminescent labels are luminol, different luminol, tris (bipyridine) ruthenium or acridine
Ester.
In one embodiment, also include Chemoluminescent substrate, the Chemoluminescent substrate includes A liquid and B liquid.
In one embodiment, the A liquid is H2O2Solution, the B liquid are NaOH solution.
In one embodiment, HIV (human immunodeficiency virus) chemiluminescence immune detection reagent kit calibration product are also included.
In one embodiment, human immunodeficiency virus antigen antibody calibration product be COI be respectively 0 ~ 0.5,1 ~
20 serum or blood plasma.
A kind of preparation method of above-mentioned human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent, bag
Include following steps：
The suspension of carboxylated magnetic particle is taken, Magneto separate is used MES buffer resuspended after removing supernatant, is subsequently added into EDC aqueous solutions,
The surface carboxyl groups of the magnetic particle of activated carboxyl, are subsequently added into HIV (human immunodeficiency virus) monoclonal antibody or human immune deficiency
Virus antigen, suspension 2h ~ 10h under room temperature, Magneto separate use Tris buffer resuspended after removing supernatant, obtain human immunodeficiency
Malicious monoclonal antibody or the coated carboxylated magnetic particle of human immunodeficiency virus antigen；And
HIV (human immunodeficiency virus) monoclonal antibody or human immunodeficiency virus antigen is taken, is mixed after adding carbonate buffer solution
Even, mix after being subsequently adding chemiluminescent labels, under room temperature, remove impurity after lucifuge reaction 1h ~ 2h, obtains human immunodeficiency
Malicious monoclonal antibody or the chemiluminescent labels of human immunodeficiency virus antigen labelling.
This human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent can be sent out with full-automatic chemical
Light immunity analysis instrument be detection instrument, complete human immunodeficiency virus antigen and（Or）The detection of antibody.This human immunity
Defective viruss antigen-antibody chemiluminescence immune detection reagent kit, through experiment, its antigen detection sensitivity reaches 1.25IU/
ML, the detection method sensitivity relative to traditional human immunodeficiency virus antigen antibody at least improve 5 times, this mankind
The accuracy of detection of immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent is higher.
Description of the drawings
Fig. 1 is the preparation of the human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent of an embodiment
The flow chart of method；
Specific embodiment
Understandable for enabling the above objects, features and advantages of the present invention to become apparent from, below in conjunction with the accompanying drawings and concrete real
Apply example to be described in detail the specific embodiment of the present invention.Elaborate in the following description a lot of details in order to
Fully understand the present invention.But the present invention can be implemented with being much different from alternate manner described here, art technology
Personnel can do similar improvement without prejudice to intension of the present invention in the case of, and therefore the present invention is not embodied as by following public
Restriction.
The human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent of one embodiment, including：The mankind
Immunodeficiency viruss monoclonal antibody and the coated carboxylated magnetic particle of human immunodeficiency virus antigen and human immunity lack
Sunken viral monoclonal antibodies and the chemiluminescent labels of human immunodeficiency virus antigen labelling.
Preferably, HIV (human immunodeficiency virus) monoclonal antibody and human immunodeficiency virus antigen are coated carboxylated
In magnetic particle, the ratio of HIV (human immunodeficiency virus) monoclonal antibody and human immunodeficiency virus antigen and carboxylated magnetic particle
Example is 1：25~100.
Preferably, the chemiluminescence of HIV (human immunodeficiency virus) monoclonal antibody and human immunodeficiency virus antigen labelling
In label, the ratio of HIV (human immunodeficiency virus) monoclonal antibody and human immunodeficiency virus antigen and chemiluminescent labels
Example is 1：10~100.
Preferably, the particle diameter of carboxylated magnetic particle is 0.05 μm ~ 1 μm.
Chemiluminescent labels can be luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.Wherein, chemiluminescence
Label is preferably acridinium ester.
In other examples, above-mentioned human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent is also
Including Chemoluminescent substrate.
Chemoluminescent substrate includes A liquid and B liquid.A liquid can be H2O2Solution, B liquid can be NaOH solution.
In the present embodiment, A liquid is the H that concentration is 0.1mol/L2O2Solution, B liquid are that concentration is molten for the NaOH of 0.25mol/L
Liquid.
In other examples, above-mentioned human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent is also
Product are calibrated including human immunodeficiency virus antigen antibody.
Human immunodeficiency virus antigen antibody calibration product are serum or the blood plasma that COI is respectively 0 ~ 0.5,1 ~ 20.
Specifically, human immunodeficiency virus antigen antibody calibration product can adopt human immunodeficiency virus antigen antibody
Negative serum or blood plasma, human immunodeficiency virus antibody positive material is thrown human immunodeficiency virus antigen negative antibody
Serum or blood plasma be configured to COI be respectively 0 ~ 0.5,1 ~ 20 serum or blood plasma.
This human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent is used for human immunodeficiency
During malicious antigen and antibody, product are calibrated to human immunodeficiency virus antigen antibody using Full-automatic chemiluminescence immunoassay analysis meter
Detected, determine Cutoff values, be built in computer software；Then actual sample is tested, sample is calculated according to sample luminous value
COI；Performance is carried out to human immunodeficiency virus antigen antibody automatic chemiluminescence immunoassay system finally（Sensitivity,
Precision, interference）Evaluation.
This human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent can be sent out with full-automatic chemical
Light immunity analysis instrument is detection instrument, completes the detection of human immunodeficiency virus antigen antibody.This human immunodeficiency
Malicious antigen-antibody chemiluminescence immune detection reagent kit, through experiment, its antigen detection sensitivity reaches 1.25IU/mL, relative
Detection method sensitivity in traditional human immunodeficiency virus antigen antibody at least improves 5 times, and this human immunity lacks
The accuracy of detection of sunken virus antigen-antibody chemiluminescence immune detection reagent kit is higher.
Additionally, this human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent is also with following excellent
Point：
1st, acridinium ester being selected as marker material, and being applied to chemiluminescence immunoassay system, the luminescence system is directly change
Learn and light, compared with traditional enzyme-catalyzed chemical luminescence, the reaction does not need the participation of enzyme, more cost-effective；
2nd, from the chemiluminescence immunoassay system RLU wide ranges of acridinium ester, RLU can reach 200 ~ 5000000, and traditional
Human immunodeficiency virus antigen antibody detection method range of absorbency be 0 ~ 4；
3rd, acridinium ester chemiluminescent immunoassay system repeatability is high, and within 5%, this is other chemistry to batch interior and difference between batch
Luminescence immunoassay system is unapproachable；
4th, chemiluminescence immunoassay system has realized the qualitative of sample, by built-in Cutoff to test software, only needs to test
Sample can directly obtain the COI of sample；
5th, chemiluminescence immunoassay system can realize that the interpolation of full-automation, reagent and sample has instrument to complete entirely, operation
Easier, reduce artificial error.
The preparation side of above-mentioned human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent as shown in Figure 1
Method, comprises the steps：
The suspension of carboxylated magnetic particle is taken, Magneto separate is used MES buffer resuspended after removing supernatant, is subsequently added into EDC aqueous solutions,
The surface carboxyl groups of the magnetic particle of activated carboxyl, are subsequently added into HIV (human immunodeficiency virus) monoclonal antibody or human immune deficiency
Virus antigen, suspension 2h ~ 10h under room temperature, Magneto separate use Tris buffer resuspended after removing supernatant, obtain human immunodeficiency
Malicious monoclonal antibody or the coated carboxylated magnetic particle of human immunodeficiency virus antigen.
MES（2- (N- morpholines) ethyl sulfonic acid）The concentration of buffer is 0.02M, and pH is 5.5.
The concentration of Tris buffer is 0.1M and contains 2%BSA, and pH is 8.0.
EDC（1- ethyl -3- (3- dimethyl aminopropyls)-carbodiimides）The concentration of aqueous solution is 10mg/mL ~ 20mg/
The ratio of mL, EDC and carboxylated magnetic particle is 0.05：0.1~1.
Preferably, HIV (human immunodeficiency virus) monoclonal antibody or human immunodeficiency virus antigen are coated carboxylated
In magnetic particle, the ratio of HIV (human immunodeficiency virus) monoclonal antibody or human immunodeficiency virus antigen and carboxylated magnetic particle
Example is 1：25~100.
Preferably, the particle diameter of carboxylated magnetic particle is 0.05 μm ~ 1 μm.
HIV (human immunodeficiency virus) monoclonal antibody or human immunodeficiency virus antigen is taken, after adding carbonate buffer solution
Mix, mix after being subsequently adding chemiluminescent labels, remove impurity after lucifuge reaction 1h ~ 2h, obtains human immune deficiency under room temperature
Viral monoclonal antibodies or the chemiluminescent labels of human immunodeficiency virus antigen labelling.
Carbonate buffer solution concentration is 0.1M, and pH is 9.0 ~ 9.5,
The operation of remove impurity is that centrifugation desalting column desalination, concrete operations are：First respectively with pure water and TBS buffer（40 mM
Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0）Centrifugation desalting column is processed, the human immunodeficiency for obtaining is eventually adding
Malicious monoclonal antibody or the chemiluminescent labels of human immunodeficiency virus antigen labelling, finally collect the liquid in centrifuge tube
Body.
Preferably, the chemiluminescence of HIV (human immunodeficiency virus) monoclonal antibody or human immunodeficiency virus antigen labelling
In label, the ratio of HIV (human immunodeficiency virus) monoclonal antibody or human immunodeficiency virus antigen and chemiluminescent labels
Example is 1：10~100.
Chemiluminescent labels can be luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.Wherein, chemiluminescence
Label is preferably acridinium ester.
The HIV (human immunodeficiency virus) monoclonal antibody for obtaining or human immunodeficiency virus antigen are coated carboxylated
Magnetic particle and the chemiluminescent labels of HIV (human immunodeficiency virus) monoclonal antibody or human immunodeficiency virus antigen labelling
Combination is obtained above-mentioned human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent.
This human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent is when using, in addition it is also necessary to chemistry
Luminous substrate liquid and human immunodeficiency virus antigen antibody calibration product.
Chemoluminescent substrate and human immunodeficiency virus antigen antibody calibration product can voluntarily be prepared and be obtained.
Chemoluminescent substrate includes A liquid and B liquid.A liquid can be H2O2Solution, B liquid can be NaOH solution.
In the present embodiment, A liquid is the H that concentration is 0.1mol/L2O2Solution, B liquid are that concentration is molten for the NaOH of 0.25mol/L
Liquid.
Specifically, human immunodeficiency virus antigen antibody calibration product can adopt human immunodeficiency virus antigen antibody
Negative serum or blood plasma, human immunodeficiency virus antibody positive material is thrown human immunodeficiency virus antigen negative antibody
Serum or blood plasma be configured to COI be respectively 0 ~ 0.5,1 ~ 20 serum or blood plasma.
The preparation method of this human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent is simple and convenient,
The detection sensitivity of obtained human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent is higher, with good
Application prospect.
It is below specific embodiment.
Embodiment 1：The preparation of human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent
（1）HIV (human immunodeficiency virus) monoclonal antibody or the coated carboxylated magnetic particle of human immunodeficiency virus antigen
Prepare：
Take containing the carboxylated magnetic particle that 50mg particle diameters are 0.05 μm ~ 1 μm（MagnaBind, article No. 21353）Suspension,
Magneto separate removes supernatant, and it is that 5.5 MES buffer are resuspended to use 0.02 M, pH, and the EDC of the 10mg/mL for adding 1mL newly to configure is water-soluble
Liquid, activated magnetic beads surface carboxyl groups add 1mg HIV (human immunodeficiency virus) monoclonal antibodies（Biorbyt, article No. orb48780）Or
Human immunodeficiency virus antigen（Producer？, article No.？）, suspension 6h under room temperature, Magneto separate remove supernatant, use containing 2%BSA
0.1M, pH are that 8.0 Tris buffer is resuspended to 1mg/mL, obtain HIV (human immunodeficiency virus) monoclonal antibody or human immunity
The carboxylated magnetic particle of defective viruss antigen coat, every bottle of 5mL subpackage be stored in 4 DEG C standby.
（2）The system of the acridinium ester of HIV (human immunodeficiency virus) monoclonal antibody or human immunodeficiency virus antigen labelling
Standby：
HIV (human immunodeficiency virus) monoclonal antibody or human immunodeficiency virus antigen that 50 μ L concentration are 25mg/mL is taken, plus
It is 9.0 ~ 9.5 carbonate buffer solution to enter 150 μ L concentration for 0.1M, pH, mixes, and is subsequently adding 1.5 μ L concentration for 5mg/mL's
Acridine ester solution is mixed, lucifuge reaction under room temperature, is taken out after 1.5h, with the zeba centrifugation desalting column desalting processing of 2mL, desalination
Processed with pure water and TBS buffer respectively in journey first, be eventually adding the HIV (human immunodeficiency virus) monoclonal for obtaining
The acridine ester solution of antibody or human immunodeficiency virus antigen labelling, the liquid that collects in centrifuge tube are in control the mankind to preservation
Immunodeficiency viruss monoclonal antibody or the acridinium ester of human immunodeficiency virus antigen labelling, every bottle of 5mL subpackage are stored in 4 DEG C
Standby.
（3）Human immunodeficiency virus antigen antibody calibrates the preparation of product：
With the negative serum of HIV (human immunodeficiency virus) antigen-antibody or blood plasma, by human immunodeficiency virus antibody positive material
Throw human immunodeficiency virus antigen negative antibody serum or blood plasma be configured to COI be respectively 0 ~ 0.5,1 ~ 20 serum or
Blood plasma, per bottle of 1.0 mL subpackages, 4 DEG C save backup.
Embodiment 2：Human immunodeficiency virus antigen antibody chemical luminescence immunologic detection method
With Full-automatic chemiluminescence immunoassay analysis meter（YHLO, article No. iFlash3000）For detecting instrument, methodology pattern is double
Antibody/dual-antigen sandwich method, i.e. instrument sequentially add the sample of 100 μ L, 50 μ L containing HIV (human immunodeficiency virus) Dan Ke
Grand antibody and the sample treatment solution of the coated carboxylated magnetic particle of human immunodeficiency virus antigen and 50 μ L, reaction 20
After min, then plus 50 μ L a word used for translation containing HIV (human immunodeficiency virus) monoclonal antibody and human immunodeficiency virus antigen labelling
Pyridine ester and the test diluent of 50 μ L, after 20 min of reaction, carry out Magneto separate, and reactant mixture is sent into darkroom by instrument, according to
Secondary addition luminous substrate A liquid（H2O2）And B liquid（NaOH）Luminescence-producing reaction is carried out, luminous value is finally recorded.
Embodiment 3：Human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent performance evaluation
Human immunodeficiency virus antigen antibody calibration product are detected using the method in embodiment 2, obtain Cutoff values.
Then actual sample is tested, sample COI is calculated according to sample luminous value.
The detection of sensitivity：
With reference to CLSI EP17-A file recommendation experimental programs, the immunity of human immunodeficiency virus antigen antibody chemical luminescence is calculated
The sensitivity of detection kit, the antigen sensitivity that tries to achieve are 1.25IU/ mL.
Precision is determined：
Two human immunodeficiency virus antibody positive samples and antigen positive sample that COI be 10 are taken respectively, and each sample is each
Do 3 parallel, detected with three batches of test kits, calculated in test kit batch and difference between batch, as a result show in the test kit batch and
Difference between batch is respectively less than 5%.
Interference is tested：
Taking pooled serum and adding chaff interference respectively includes：Conjugated bilirubin, unconjugated bilirubin, hemoglobin, ascorbic acid, glycerol
Ester, adding proportion is according to 1：20 are carried out, and are determined pooled serum respectively and be with the addition of the measured value of pooled serum after various chaff interferences,
Deviation therebetween is calculated, with ± 10% as tolerance interval.As a result show, interference reaches the files-designated of NCCLS
Standard, can be used for the accurate evaluation of clinical laboratory's human immunodeficiency virus antigen antibody situation.
Embodiment 4, the contrast experiment of human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent
It is 20IU/mL, 10IU/mL, 5IU/ with chemical luminescence detection method and traditional enzyme linked immunosorbent assay to concentration respectively
ML, 2.5IU/mL, 1.25IU/mL, 0.625IU/mL, 0.312IU/mL, 0.156IU/mL, 0.078IU/mL, 0IU/mL, people
Para-immunity defective viruss antigen positive sample detects that two methods detection sensitivity is compared, and data are as shown in the table：
| Concentration | Chemiluminescence detection（COI） | Enzyme linked immunosorbent assay is detected（COI） |
| 20IU/mL | 11.92 | 3.31 |
| 10IU/mL | 6.65 | 1.62 |
| 5IU/mL | 3.71 | 0.76 |
| 2.5IU/mL | 1.93 | 0.42 |
| 1.25IU/mL | 1.17 | 0.30 |
| 0.625IU/mL | 0.81 | 0.22 |
| 0.312IU/mL | 0.72 | 0.16 |
| 0.156IU/mL | 0.60 | 0.141 |
| 0.078IU/mL | 0.41 | 0.14 |
| 0IU/mL | 0.14 | 0.12 |
| Sensitivity（IU/mL） | 1.25 | 10 |
As can be seen from the above table, the sensitivity of chemical luminescence detection method improves about 5 times compared with enzyme linked immunosorbent assay.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more concrete and detailed, but can not
Therefore the restriction to the scope of the claims of the present invention is interpreted as.It should be pointed out that for the person of ordinary skill of the art,
Without departing from the inventive concept of the premise, some deformations and improvement can also be made, these belong to the protection model of the present invention
Enclose.Therefore, the protection domain of patent of the present invention should be defined by claims.
Claims (10)
1. a kind of human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent, it is characterised in that include：The mankind
Immunodeficiency viruss monoclonal antibody and the coated carboxylated magnetic particle of human immunodeficiency virus antigen and human immunity lack
Sunken viral monoclonal antibodies and the chemiluminescent labels of human immunodeficiency virus antigen labelling.
2. human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent according to claim 1, its are special
Levy and be, the HIV (human immunodeficiency virus) monoclonal antibody and the coated carboxylated magnetic of human immunodeficiency virus antigen are micro-
In grain, the HIV (human immunodeficiency virus) monoclonal antibody, human immunodeficiency virus antigen and the carboxylated magnetic particle
Ratio be 1：25~100.
3. human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent according to claim 1, its are special
Levy and be, the chemiluminescent labeling of the HIV (human immunodeficiency virus) monoclonal antibody and human immunodeficiency virus antigen labelling
In thing, the HIV (human immunodeficiency virus) monoclonal antibody and human immunodeficiency virus antigen and the chemiluminescent labels
Ratio be 1：10~100.
4. human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent according to claim 1, its are special
Levy and be, the particle diameter of the carboxylated magnetic particle is 0.05 μm ~ 1 μm.
5. human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent according to claim 1, its are special
Levy and be, the chemiluminescent labels are luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.
6. human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent according to claim 1, its are special
Levy and be, also include Chemoluminescent substrate, the Chemoluminescent substrate includes A liquid and B liquid.
7. human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent according to claim 6, its are special
Levy and be, the A liquid is H2O2Solution, the B liquid are NaOH solution.
8. human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent according to claim 1, its are special
Levy and be, also include human immunodeficiency virus antigen antibody calibration product.
9. human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent according to claim 8, its are special
Levy and be, the human immunodeficiency virus antigen antibody calibration product are serum or the blood plasma that COI is respectively 0 ~ 0.5,1 ~ 20.
10. human immunodeficiency virus antigen antibody chemical luminescence immunity of the one kind according to any one of claim 1 ~ 9
The preparation method of detection kit, it is characterised in that comprise the steps：
The suspension of carboxylated magnetic particle is taken, Magneto separate is used MES buffer resuspended after removing supernatant, is subsequently added into EDC aqueous solutions,
The surface carboxyl groups of the magnetic particle of activated carboxyl, are subsequently added into HIV (human immunodeficiency virus) monoclonal antibody or human immune deficiency
Virus antigen, suspension 2h ~ 10h under room temperature, Magneto separate use Tris buffer resuspended after removing supernatant, obtain human immunodeficiency
Malicious monoclonal antibody or the carboxylated magnetic particle of mankind's cotton dress defective viruss antigen coat；And take HIV (human immunodeficiency virus)
Monoclonal antibody or human immunodeficiency virus antigen, mix after adding carbonate buffer solution, are subsequently adding chemiluminescent labeling
Mix after thing, remove impurity after lucifuge reaction 1h ~ 2h under room temperature obtains HIV (human immunodeficiency virus) monoclonal antibody or human immunity lacks
The chemiluminescent labels of sunken virus antigen labelling.
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