CN109100516A - A kind of detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit and preparation method thereof - Google Patents
A kind of detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit and preparation method thereof Download PDFInfo
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- CN109100516A CN109100516A CN201811092187.7A CN201811092187A CN109100516A CN 109100516 A CN109100516 A CN 109100516A CN 201811092187 A CN201811092187 A CN 201811092187A CN 109100516 A CN109100516 A CN 109100516A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
Abstract
The present invention relates to a kind of detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kits and preparation method thereof, belong to technical field of immunoassay.Kit of the invention includes calibration object；R1 reagent；R2 reagent；R3 reagent；R1 reagent is that magnetic bead is coated with CMV antigen；R2 reagent is the anti-human IgG antibodies of acridinium ester label；R3 reagent is project dilution.The present invention also provides the preparation methods of kit.Chemical luminescence immune assay determination reagent kit provided by the invention has the advantages that high sensitivity, high specificity, reproducible, detection range is wide, advantage of lower cost, can be widely used for clinical detection for detecting cytomegalovirus IgG antibody, detection method.Solve existing common cytomegalovirus IgG antibody detection method such as RIA there are radioactive pollution, marker half-life short, have the shortcomings that radioactive damage to operator, cumbersome, detection time is long；ELISA has the shortcomings that sensitivity is low, detection range is narrow, detection time is long, poor repeatability.
Description
Technical field
The invention belongs to technical field of immunoassay, and in particular to a kind of detection cytomegalovirus IgG antibody chemiluminescence
Immune analytic reagent kit and preparation method thereof.
Background technique
Human cytomegalovirus (hCMV) belongs to herpetoviridae, is that a kind of couple of people has pathogenic herpesviral.It is extensively
In the presence of and tool kind of a group specificity, the close contact for passing through crowd propagate.Viral capsid is positive 20 face bodies, by 162 shell particle groups
At including a DNA core, one or more outer membranes containing lipid are looped around ambient envelope.Cytomegalovirus infection is divided into primary
Sexuality dye and secondary infection.Primary infection can be obtained by the different routes of transmission physiology phase different with people (for example, dividing
For congenital infection and acquired infection).After primary infection, cytomegalovirus enters incubation period, during this period, bone-marrow-derived lymphocyte
In can find virus.When the immunoloregulation function of host changes, such as pregnancy, major disease, immunosuppressive therapy, spirit are pressed
Power etc., the duplication of latent virus are just activated (secondary infection).Congenital infection to fetus or is gone out by pregnant woman through placental infection
Fetus is transmitted to when raw.Even if existing anti-hCMV antibody (being re-infected by exogenous virus), the infection of fetus in pregnant woman's body
It can also happen that.If seronegative women contacts giant cell cause of disease between period of pregnancy, giant cell has thus been infected
Virus can cause miscarriage, stillbirth or newborn teratogenesis.During pregnancy infect hCMV virus, produce normal infant a possibility that be
50%.The clinical manifestation of congenital cytomegalovirus infection is usually very serious, shows as dementia, deaf, retina choroid
Inflammation, head deformity, hydrocephalus, heart disease, hepatitis, hepatosplenomegaly, the symptoms such as decrease of platelet, the death rate are very high.
Most people (40%-90%) infects hCMV virus for the first time during childhood or adult, infects for posteriority.The day after tomorrow
Sexuality dye is by contacting infected body fluid (such as urine, saliva, milk, sperm, cervical secretions, excrement) and contaminated
Blood product infects, and infects occasionally through organ transplant.Posteriority infects the clinical manifestation in the normal the infected of immune function
It is usually to compare mitigation or non-evident sympton.Common manifestation increases and for transaminase level in fever, uncomfortable, blood without Huang
Subcutaneous ulcer.And the patient's (organ transplant patients, AIDS patient, lymphoid malignant hyperplasia patient or cancer patient) for thering is immune class to inhibit,
HCMV infection can be because virus diffusion or intrusion internal organ generate serious symptom, comprising: splenomegaly, pneumonia, haemolytical anaemia,
Myocarditis and encephalitis.HCMV infection may be fatal to this kind of patient.IgM can be produced into vivo after infected patient's several weeks
Antibody, with it is latter week in can generate IgG antibody.
The detection of specific IgG helps to distinguish whether infect hCMV virus.This takes suitable prevention to suspected patient
Measure is even more important.The immune system situation of detection hCMV is of great significance: after (a) can inhibiting patient's infection with epidemic prevention
Cause fatal harm；(b) Women of Childbearing Age or pregnant woman can be prevented viral infection to fetus；It (c) can be with prevention of organ transplant
Caused by infect；(d) infection caused by transfusion procedure can be prevented.Leucocyte can carry hCMV, can be moved by blood or organ
Plant infection blood donor or organ transplant person.Detection hCMV specific IgM antibodies help adequately to treat patient.It can be with
Prevent hCMV infection by the viral specific immunoglobulin of height.In addition, there is the patient of manifest symptom that should use spy
Anisotropic antiviral drug is treated.
The method of currently used detection cytomegalovirus IgG antibody has radioimmunoassay technique (RIA), ELISA
Technology (ELISA), but there are many deficiencies for both methods, such as RIA there are radioactive pollution, marker half-life short, to behaviour
Author has the shortcomings that radioactive damage, cumbersome, detection time is long；ELISA is with sensitivity is low, detection range is narrow, inspection
The disadvantages of surveying long time, poor repeatability.
Summary of the invention
The invention solves in the prior art detect cytomegalovirus IgG antibody method validity period it is short, it is cumbersome,
The technical problem that sensitivity is low, testing cost is high provides a kind of detection cytomegalovirus IgG antibody chemiluminescence immune assay survey
Determine kit and preparation method thereof.
To achieve the goals above, the invention provides the following technical scheme:
A kind of detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit, comprising: 1) calibration object；2)
R1 reagent；3) R2 reagent；4) R3 reagent；
The main component of the R1 reagent is that magnetic bead is coated with CMV antigen；
The main component of the R2 reagent is the anti-human IgG antibodies of acridinium ester label；
The R3 reagent is project dilution.
In the above-mentioned technical solutions, the R1 reagent is stored in the phosphate containing protein stabiliser that pH is 6.0-8.0
In buffer, concentration 0.03%-0.5%；The R2 reagent is stored in the citric acid solution that pH is 6.0-8.0,
Its mass concentration is 0.1-0.5 μ g/mL；The R3 reagent is that the citric acid that the pH containing 0.5%-3% albumen is 6.0-8.0 is slow
Fliud flushing.
In the above-mentioned technical solutions, the R1 reagent buffer be containing 0.05% tween and 0.05%Proclin300,
The 100mM phosphate buffer of pH6.0-8.0；The R2 reagent buffer is to contain 0.05% tween and 0.05%
The 100mM citrate buffer solution of Proclin300, pH6.0-8.0；The R3 reagent buffer be containing 0.5%-3% albumen,
0.05% tween and 0.05%Proclin300, the 100mM citrate buffer solution of pH6.0-8.0.
In the above-mentioned technical solutions, the coating quality of CMV antigen and magnetic bead ratio is in the magnetic bead coating CMV antigen
0.5%-3%.
In the above-mentioned technical solutions, in the anti-human IgG antibodies of acridinium ester label anti-human IgG antibodies and acridinium ester molar ratio
1:3-1:15。
In the above-mentioned technical solutions, the detection cytomegalovirus IgG antibody chemiluminscence immunoassay reagent
Box further includes two o'clock enterprise calibration object, and the concentration containing cytomegalovirus IgG antibody is respectively 30U/mL and 100U/ in calibration object
mL。
In the above-mentioned technical solutions, the buffer of the calibration object is the 100mM PBS buffering containing 20% calf serum
Liquid.
A kind of preparation method detecting cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit, including with
Lower step:
S1, preparation R1 reagent:
It after mixing well magnetic bead, is placed in centrifuge tube, collects magnetic, remove supernatant, sealer is added, collects again after mixing well
Magnetic removes supernatant, repeats closing 3 times, sealer is added again, mixes well, and coating CMV antigen is added, is rotated using 360 °
After blending instrument room temperature rotates blending incubation, collect magnetic, remove supernatant, cleaning buffer solution is added, mix, collects magnetic, go supernatant, repeated washing
3 times, obtain magnetic bead coating CMV antigen；With the 100mM phosphorus containing 0.05% tween and 0.05%Proclin300, pH6.0-8.0
Phthalate buffer is configured to the solid phase R1 reagent that magnetic bead concentration is 0.03%-0.5%；
S2, preparation R2 reagent:
50mM-200mM PBS buffer solution is added in anti-human IgG antibodies, anti-human IgG antibodies are mixed with acridinium ester, are filled
Divide and mixes；After being placed at room temperature for 2-4 hours, lysine confining liquid is added, mixes, closing；It is molten to acridinium ester label antibody after closing
Liquid is purified, and purified solution is collected, and obtains the anti-human IgG antibodies of acridinium ester label；With containing 0.05% tween and
The 100mM citrate buffer solution of 0.05%Proclin300, pH6.0-8.0 are diluted to the R2 examination of final concentration of 0.1-0.5 μ g/mL
Agent；
S3, preparation R3 reagent:
Contained with the citrate buffer solution preparation of 0.05% tween and 0.05%Proclin300, the 100mM of pH6.0-8.0
The R3 reagent of 0.5%-3% albumen；
S4, calibration object is prepared
Cytomegalovirus IgG antibody is diluted to various concentration with the 100mM PBS buffer solution containing 20% calf serum
Calibration object；
S5, packing calibration object, R1 reagent, R2 reagent and R3 reagent, are assembled into finished product.
In the above-mentioned technical solutions, in step S2 purification step using AKTA protein purification instrument.
In the above-mentioned technical solutions, the detection cytomegalovirus IgG antibody chemiluminscence immunoassay reagent
The detecting step of box are as follows: sample to be tested and R1 reagent and R3 reagent are incubated for 18min, washing is added R2 reagent and is incubated for 5min again,
Washing is added exciting liquid and records relative luminous intensity；In a certain range, in sample the content of cytomegalovirus IgG antibody with
Relative luminous intensity is directly proportional.
The present invention uses the reaction pattern of indirect method, the content of cytomegalovirus IgG antibody in quantitatively determining human serum,
Ensure the sensitivity detected.Every Testing index of the invention is superior to the average level of similar import reagent box.
The beneficial effects of the present invention are:
Detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit provided by the invention uses acridinium ester
Chemical luminous system, stability is good, and high specificity, high sensitivity, the range of linearity is wide, and detection time is short, provides for clinical diagnosis
More special, stable, quick, reliable detection means.
Detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit provided by the invention is huge for detecting
Cell virus IgG antibody, detection method have high sensitivity, high specificity, reproducible, detection range is wide, cost is opposite
Lower advantage, can be widely used for clinical detection.Solves existing common cytomegalovirus IgG antibody detection method such as RIA
There are radioactive pollution, marker half-life short, operator is lacked with radioactive damage, cumbersome, detection time length etc.
Point；ELISA has the shortcomings that sensitivity is low, detection range is narrow, detection time is long, poor repeatability.
Detailed description of the invention
Invention is further described in detail with reference to the accompanying drawings and detailed description.
Fig. 1 is the preparation side of detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit of the invention
And preparation flow design drawing.
Fig. 2 is the survey using detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit of the invention
The standard curve of calibration object detection relative light unit is tried, abscissa is concentration, and unit U/mL, ordinate is relative light intensity.
Specific embodiment
The present invention is illustrated in conjunction with Fig. 1, and a kind of detection cytomegalovirus IgG antibody chemiluminescence immune assay survey is provided
Determine kit, mainly include following reagent:
R1 reagent: the liquid containing magnetic bead coating CMV antigen；
R2 reagent: the liquid of the anti-human IgG antibodies containing acridinium ester label；
R3 reagent: project dilution.
Wherein, CMV antigen and the coated mass ratio of magnetic bead are 0.5%-3% in R1 reagent, and magnetic bead is coated with the dense of CMV antigen
Degree is 0.03%-0.5%；
The molar ratio of anti-human IgG antibodies and acridinium ester is 1:3-1:15 in R2 reagent；The anti-human IgG antibodies of acridinium ester label
Concentration be 0.1-0.5 μ g/mL；
R3 reagent is the project dilution containing 0.5%-3% albumen.
The pH value of R1, R2 and R3 buffer is 6.0-8.0.
Further, above-mentioned detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit further includes
Two o'clock enterprise calibration object, the concentration containing cytomegalovirus IgG antibody is respectively 30U/mL and 100U/mL in calibration object.
Further, calibration object buffer is the 100mM PBS buffer solution containing 20% calf serum.
Further, R1 reagent the preparation method comprises the following steps: mixing well magnetic bead after, take 1mL (i.e. 10mg magnetic bead) in centrifuge tube
In, collect magnetic, remove supernatant, 1mL sealer is added, collects magnetic after mixing well again, remove supernatant, repeats closing 3 times, be added again
1mL sealer, mixes well, and 100 μ g CMV envelope antigens are added, and rotates blending incubation 3h using 360 ° of rotation blending instrument room temperatures
Afterwards, collect magnetic, remove supernatant, 1mL cleaning buffer solution is added, mix, collect magnetic, remove supernatant, repeated washing 3 times, obtain magnetic bead coating CMV
Antigen.With the 100mM phosphate buffered saline containing 0.05% tween and 0.05%Proclin300, pH6.0-8.0 at magnetic
Pearl concentration is the solid phase R1 reagent of 0.03%-0.5%.
Further, R2's the preparation method comprises the following steps: by anti-human IgG antibodies be added 50mM-200mM PBS buffer solution, will resist
Human IgG antibody is mixed with acridinium ester by a mole volume ratio 1:3-1:15, is mixed well；After being placed at room temperature for 2-4 hours, 1mL is added
20% lysine confining liquid mixes, off-period 1h；Acridinium ester label antibody-solutions are purified after closing, are collected pure
Solution after change, with 0.05% tween and 0.05%Proclin300 is contained, the 100mM citrate buffer solution of pH6.0-8.0 dilutes
To the R2 reagent of final concentration of 0.1-0.5 μ g/mL.
Further, R3's the preparation method comprises the following steps: with 0.05% tween and 0.05%Proclin300, pH6.0-8.0's
The citrate buffer solution of 100mM prepares the R3 reagent containing 0.5%-3% albumen.
Further, above-mentioned purification step is using AKTA protein purification instrument.
The present invention also provides the preparations of above-mentioned cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit
Method, comprising the following steps:
S1, preparation R1 reagent, R1 reagent the preparation method comprises the following steps: mixing well magnetic bead after, take 1mL (i.e. 10mg magnetic bead) in from
In heart pipe, collect magnetic, remove supernatant, 1mL sealer is added, collects magnetic after mixing well again, remove supernatant, repeat closing 3 times, again plus
Enter 1mL sealer, mix well, 100 μ g CMV envelope antigens are added, rotates blending incubation using 360 ° of rotation blending instrument room temperatures
After 3h, collect magnetic, remove supernatant, 1mL cleaning buffer solution is added, mix, collect magnetic, removes supernatant, repeated washing 3 times, obtain magnetic bead coating
CMV antigen.With the 100mM phosphate buffered saline containing 0.05% tween and 0.05%Proclin300, pH6.0-8.0 at
Magnetic bead concentration is the solid phase R1 reagent of 0.03%-0.5%.
S2, preparation R2 reagent, R2 reagent buffer the preparation method comprises the following steps: 50mM-200mM PBS is added in anti-human IgG antibodies
Anti-human IgG antibodies are mixed with acridinium ester by a mole volume ratio 1:3-1:15, are mixed well by solution；It is placed at room temperature for 2-4 hours
Afterwards, 20% lysine confining liquid of 1mL is added, mixes, off-period 1h；Acridinium ester label antibody-solutions are carried out after closing
Purifying, collect purified solution, with contain 0.05% tween and 0.05%Proclin300, the 100mM citric acid of pH6.0-8.0
Buffer is diluted to the R2 reagent of final concentration of 0.1-0.5 μ g/mL.
S3, preparation R3 reagent, R3 reagent the preparation method comprises the following steps: with 0.05% tween and 0.05%Proclin300,
The citrate buffer solution of the 100mM of pH6.0-8.0 prepares the R3 reagent containing 0.5%-3% albumen.
S4, calibration object is prepared
S5, packing calibration object, R1 reagent, R2 reagent and R3 reagent, are assembled into finished product.
Detecting step of the invention: sample to be tested and R1 reagent and R3 reagent are incubated for 18min, R2 reagent is added in washing
It is incubated for 5min again, washs, exciting liquid record relative luminous intensity (RLU) is added.In a certain range, cytomegalovirus in sample
The content of IgG antibody is directly proportional to relative luminous intensity.
The present invention uses the reaction pattern of indirect method, the content of cytomegalovirus IgG antibody in quantitatively determining human serum,
Ensure the sensitivity detected.Every Testing index of the invention is superior to the average level of similar import reagent box.
In order to make those skilled in the art more fully understand technical solution of the present invention, below in conjunction with attached drawing and implementation
Example is further detailed the present invention.
The preparation of the main calibration object of embodiment 1
Cytomegalovirus IgG antibody is diluted to calibration object with the 100mM PBS buffer solution containing 20% calf serum, point
Dress up 6 bottles of main calibration objects of 0U/mL, 12U/mL, 25U/mL, 50U/mL, 100U/mL, 180U/mL.It is quasi- based on main calibration object
Conjunction curve carries out back value and obtains two o'clock calibration value, and wherein the concentration of enterprise's two o'clock calibration is respectively 30U/mL and 100U/mL.
It is shown in Figure 2, CMV IgG chemical luminescence immune assay determination reagent kit dose response calibration curve, linear phase
Relationship number r >=0.9900, calibration is qualified, and design parameter is shown in Table 1.
1 calibration object of table detects opposite light value
The preparation of the detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit of embodiment 2
The preparation of R1 reagent: after mixing well magnetic bead, taking 1mL (i.e. 10mg magnetic bead) in centrifuge tube, collects magnetic, removes supernatant,
1mL sealer is added, collects magnetic after mixing well again, removes supernatant, repeats closing 3 times, 1mL sealer is added again, it is sufficiently mixed
It is even, 100 μ g CMV envelope antigens are added, after rotating blending incubation 3h using 360 ° of rotation blending instrument room temperatures, collects magnetic, removes supernatant,
1mL cleaning buffer solution is added, mixes, collects magnetic, removes supernatant, repeated washing 3 times, obtains magnetic bead coating CMV antigen.With containing
The 100mM phosphate buffered saline of 0.05% tween and 0.05%Proclin300, pH6.0 are 0.03% at magnetic bead concentration
Solid phase R1 reagent, 2~8 DEG C of preservations.
The preparation of R2 reagent: by anti-human IgG antibodies be added 50mM-200mM PBS buffer solution, by anti-human IgG antibodies with
Acridinium ester is mixed well by a mole volume ratio 1:3 mixing；After being placed at room temperature for 2-4 hours, the closing of 20% lysine of 1mL is added
Liquid mixes, off-period 1h；Acridinium ester label antibody-solutions are purified after closing, collect purified solution, with containing
0.05% tween and 0.05%Proclin300, the 100mM citrate buffer solution of pH6.0 are diluted to final concentration of 0.1 μ g/mL's
R2 reagent, 2~8 DEG C of preservations.
The preparation of R3: matched with the citrate buffer solution of 0.05% tween and 0.05%Proclin300, the 100mM of pH6.0
Make the R3 reagent containing 0.5% albumen.
The preparation of 3 cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit of embodiment
The preparation of R1 reagent: after mixing well magnetic bead, taking 1mL (i.e. 10mg magnetic bead) in centrifuge tube, collects magnetic, removes supernatant,
1mL sealer is added, collects magnetic after mixing well again, removes supernatant, repeats closing 3 times, 1mL sealer is added again, it is sufficiently mixed
It is even, 100 μ g CMV envelope antigens are added, after rotating blending incubation 3h using 360 ° of rotation blending instrument room temperatures, collects magnetic, removes supernatant,
1mL cleaning buffer solution is added, mixes, collects magnetic, removes supernatant, repeated washing 3 times, obtains magnetic bead coating CMV antigen.With containing
The 100mM phosphate buffered saline of 0.05% tween and 0.05%Proclin300, pH7.0 are 0.1% at magnetic bead concentration
Solid phase R1 reagent, 2~8 DEG C of preservations.
The preparation of R2 reagent: by anti-human IgG antibodies be added 50mM-200mM PBS buffer solution, by anti-human IgG antibodies with
Acridinium ester is mixed well by a mole volume ratio 1:10 mixing；After being placed at room temperature for 2-4 hours, the closing of 20% lysine of 1mL is added
Liquid mixes, off-period 1h；Acridinium ester label antibody-solutions are purified after closing, collect purified solution, with containing
0.05% tween and 0.05%Proclin300, the 100mM citrate buffer solution of pH7.0 are diluted to final concentration of 0.3 μ g/mL's
R2 reagent, 2~8 DEG C of preservations.
The preparation of R3: matched with the citrate buffer solution of 0.05% tween and 0.05%Proclin300, the 100mM of pH7.0
Make the R3 reagent containing 1% albumen.
The preparation of 4 cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit of embodiment
The preparation of R1 reagent: after mixing well magnetic bead, taking 1mL (i.e. 10mg magnetic bead) in centrifuge tube, collects magnetic, removes supernatant,
1mL sealer is added, collects magnetic after mixing well again, removes supernatant, repeats closing 3 times, 1mL sealer is added again, it is sufficiently mixed
It is even, 100 μ g CMV envelope antigens are added, after rotating blending incubation 3h using 360 ° of rotation blending instrument room temperatures, collects magnetic, removes supernatant,
1mL cleaning buffer solution is added, mixes, collects magnetic, removes supernatant, repeated washing 3 times, obtains magnetic bead coating CMV antigen.With containing
The 100mM phosphate buffered saline of 0.05% tween and 0.05%Proclin300, pH8.0 are 0.5% at magnetic bead concentration
Solid phase R1 reagent, 2~8 DEG C of preservations.
The preparation of R2 reagent: by anti-human IgG antibodies be added 50mM-200mM PBS buffer solution, by anti-human IgG antibodies with
Acridinium ester is mixed well by a mole volume ratio 1:15 mixing；After being placed at room temperature for 2-4 hours, the closing of 20% lysine of 1mL is added
Liquid mixes, off-period 1h；Acridinium ester label antibody-solutions are purified after closing, collect purified solution, with containing
0.05% tween and 0.05%Proclin300, the 100mM citrate buffer solution of pH8.0 are diluted to final concentration of 0.5 μ g/mL's
R2 reagent, 2~8 DEG C of preservations.
The preparation of R3 reagent: with 0.05% tween and 0.05%Proclin300, the lemon acid buffering of the 100mM of pH8.0
Liquid prepares the R3 reagent containing 3% albumen.
The Performance Evaluation of the detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit of embodiment 5
2 reagent preparation box of embodiment is examined and determine according to the manufacture and vertification regulation of this field routine, the results are shown in Table 2.
2 kit performance evaluation result of table
To sum up, in research process of the invention, the present invention has carried out screening and quality control to raw material used first
It makes, the research of concentration, purity, affinity including antibody, antibody activity after magnetic bead coating and acridinium ester label.Then to magnetic bead
Coating and acridinium ester label system are studied, and are tested using not isolabeling ratio and label buffer, by repeatedly
Experiment and comparative experiments have eventually found method simplicity, high, at low cost, reliable in quality the labeling method of yield.
In addition, the present invention also explores the reaction pattern of kit and condition.By different reaction pattern and
Reaction time comparative experiments has finally determined that this kit uses indirect two steps, two incubation reaction mode, and examination has finally been determined
Agent incubation time is 18min+5min, shortens detection time, improves detection efficiency.
The method of the detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit of the invention of embodiment 6
Learn calibrating
It is examined and determine according to the kit that professional standard in this field and vertification regulation prepare embodiment 2, as a result as follows:
1. the kit range of linearity measures
By the kit prepared in embodiment 2 wherein 1 batch, measurement range of linearity sample 0U/mL, 12U/mL, 25U/mL,
50U/mL, 100U/mL, 150U/mL show that the range of linearity is 5U/mL~150U/mL, linearly dependent coefficient r >=0.9900.
The measurement of the 3 kit range of linearity of table
| It measures linear sample concentration (U/mL) | Relative light intensity (RLU) |
| 0 | 775 |
| 12 | 60646 |
| 25 | 111590 |
| 50 | 209544 |
| 100 | 384284 |
| 180 | 671306 |
2. the measurement of kit sensitivity
Kit prepared by embodiment 2 wherein 1 batch, 20 zero-dose samples are measured, calculate average value (M) and standard deviation
(SD), it obtains M+2SD, two o'clock regression fit is carried out according to the concentration-RLU between zero-dose calibration object and adjacent calibration object and is obtained
Linear function brings the RLU of M+2SD into equation, show that respective concentration value is kit sensitivity 0.0067U/mL, as a result sees
Table 4 and table 5.
4 first luminous values of table
First point of mean value M that shines1=782, SD1=16.92, M1+2SD1=815.84
5 second point luminous value of table
Second point shines mean value M=61322
First point (0,782) and second point (12,61322) line matched curve, sensitivity=0.0067U/mL of calculating.
3. kit precision is tested
(1) withinrun precision
Kit prepared by embodiment 2 wherein 1 batch, the sample of two various concentrations of high level and low value is repeated to survey respectively
It is 10 times fixed, obtain variation within batch coefficient < 8%.
6 withinrun precision measurement result of table
| It measures standard substance concentration (U/mL) | Measure number | Coefficient of variation CV% |
| 12 | 10 | 5.82 |
| 150 | 10 | 2.36 |
(2) betweenrun precision
Kit prepared by embodiment 2 wherein 1 batch, the sample of two various concentrations of high level and low value is repeated to survey respectively
It is 10 times fixed, obtain interassay coefficient of variation less than 15%.
7 betweenrun precision measurement result of table
| It measures standard substance concentration (ng/mL) | Measure number | Coefficient of variation CV% |
| 12 | 30 | 7.28 |
| 150 | 30 | 3.52 |
4. kit accuracy determination
Kit prepared by embodiment 2 wherein 1 batch, the standard substance that measurement concentration is 12U/mL and 150U/mL, relatively
Deviation < 10%, test result is as follows:
8 accuracy determination result of table
| It measures standard substance concentration (ng/mL) | Measurement result | Relative deviation % |
| 12 | 12.3 | 2.5% |
| 150 | 150.9 | 0.6% |
5. kit specificity experiments
Cytomegalovirus IgG antibody is negative, and Epstein-Barr virus (IgG) is positive, cytomegalovirus (IgG) is positive, toxoplasma (IgG)
Positive, rubella virus (IgG) positive, HSV-2 (IgG) is positive, varicella-zoster (IgG) is positive, human papilloma virus sun
Property, the microspironema pallidum positive clinical sample tested, test result is as follows:
9 specific assay result of table
| Project | Sample yin and yang attribute | Testing result |
| Epstein-Barr virus (IgG) | It is positive | It is negative |
| Cytomegalovirus (IgG) | It is positive | It is negative |
| Toxoplasma (IgG) | It is positive | It is negative |
| Rubella virus (IgG) | It is positive | It is negative |
| HSV-2(IgG) | It is positive | It is negative |
| Varicella-zoster (IgG) | It is positive | It is negative |
| Human papilloma virus | It is positive | It is negative |
| Microspironema pallidum | It is positive | It is negative |
6. stabilization of kit is tested
Kit prepared by embodiment 2 carries out 4 DEG C and 37 DEG C of 14 days stability experiments respectively, the results showed that kit mark
In the normal range, kit validity period was up to 12 months for the indexs such as variation, precision, the accuracy of quasi- product luminous intensity.
By a large amount of repeated experiments, kit index of the invention is as follows:
Detection range: 3-180U/mL；
Sensitivity: minimum detection limit is not higher than 0.0067U/mL；
Precision: less than 10%；
Accuracy: measured value standard substance deviation is less than 10%.
The detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit of preparation of the embodiment of the present invention uses
Acridinium ester chemiluminescent system, for detecting cytomegalovirus IgG antibody, detection method have high sensitivity, high specificity,
The advantages of reproducible, detection range is wide, advantage of lower cost, can be widely used for clinical detection.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (10)
1. a kind of detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit characterized by comprising 1)
Calibration object；2) R1 reagent；3) R2 reagent；4) R3 reagent；
The main component of the R1 reagent is that magnetic bead is coated with CMV antigen；
The main component of the R2 reagent is the anti-human IgG antibodies of acridinium ester label；
The R3 reagent is project dilution.
2. detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit according to claim 1, special
Sign is that the R1 reagent is stored in the phosphate buffer containing protein stabiliser that pH is 6.0-8.0, and concentration is
0.03%-0.5%；The R2 reagent is stored in the citric acid solution that pH is 6.0-8.0, mass concentration 0.1-
0.5μg/mL；The R3 reagent is the citrate buffer solution that the pH containing 0.5%-3% albumen is 6.0-8.0.
3. detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit according to claim 2, special
Sign is that the R1 reagent buffer is the 100mM phosphorus containing 0.05% tween and 0.05%Proclin300, pH6.0-8.0
Phthalate buffer；The R2 reagent buffer be containing 0.05% tween and 0.05%Proclin300, pH6.0-8.0's
100mM citrate buffer solution；The R3 reagent buffer is to contain 0.5%-3% albumen, 0.05% tween and 0.05%
The 100mM citrate buffer solution of Proclin300, pH6.0-8.0.
4. detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit according to claim 1, special
Sign is that the coating quality of CMV antigen and magnetic bead ratio is 0.5%-3% in the magnetic bead coating CMV antigen.
5. detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit according to claim 1, special
Sign is, the molar ratio 1:3-1:15 of anti-human IgG antibodies and acridinium ester in the anti-human IgG antibodies of acridinium ester label.
6. detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit according to claim 1, special
Sign is, further includes two o'clock enterprise calibration object, in calibration object the concentration containing cytomegalovirus IgG antibody be respectively 30U/mL and
100U/mL。
7. detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit according to claim 6, special
Sign is that the buffer of the calibration object is the 100mM PBS buffer solution containing 20% calf serum.
8. a kind of system of detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit as claimed in claim 3
Preparation Method, which comprises the following steps:
S1, preparation R1 reagent:
It after mixing well magnetic bead, is placed in centrifuge tube, collects magnetic, remove supernatant, sealer is added, collects magnetic after mixing well again, goes
Supernatant repeats closing 3 times, sealer is added again, mixes well, and coating CMV antigen is added, uses 360 ° of rotation blending instruments
After room temperature rotates blending incubation, collect magnetic, remove supernatant, cleaning buffer solution is added, mix, collects magnetic, remove supernatant, repeated washing 3 times, obtain
CMV antigen is coated with to magnetic bead；It is slow with the 100mM phosphate containing 0.05% tween and 0.05%Proclin300, pH6.0-8.0
Fliud flushing is configured to the solid phase R1 reagent that magnetic bead concentration is 0.03%-0.5%；
S2, preparation R2 reagent:
50mM-200mM PBS buffer solution is added in anti-human IgG antibodies, anti-human IgG antibodies are mixed with acridinium ester, it is sufficiently mixed
It is even；After being placed at room temperature for 2-4 hours, lysine confining liquid is added, mixes, closing；After closing to acridinium ester label antibody-solutions into
Row purifying, collects purified solution, obtains the anti-human IgG antibodies of acridinium ester label；With contain 0.05% tween and 0.05%
The 100mM citrate buffer solution of Proclin300, pH6.0-8.0 are diluted to the R2 reagent of final concentration of 0.1-0.5 μ g/mL；
S3, preparation R3 reagent:
Contained with the citrate buffer solution preparation of 0.05% tween and 0.05%Proclin300, the 100mM of pH6.0-8.0
The R3 reagent of 0.5%-3% albumen；
S4, calibration object is prepared
Cytomegalovirus IgG antibody is diluted to the school of various concentration with the 100mM PBS buffer solution containing 20% calf serum
Quasi- product；
S5, packing calibration object, R1 reagent, R2 reagent and R3 reagent, are assembled into finished product.
9. the system of detection cytomegalovirus IgG antibody chemical luminescence immune assay determination reagent kit according to claim 8
Preparation Method, which is characterized in that purification step is using AKTA protein purification instrument in step S2.
10. detection cytomegalovirus IgG antibody chemiluminscence immunoassay described in -7 any one according to claim 1
Kit, which is characterized in that the detecting step of the kit are as follows: sample to be tested and R1 reagent and R3 reagent are incubated for 18min,
Washing is added R2 reagent and is incubated for 5min again, washs, and exciting liquid is added and records relative luminous intensity；In a certain range, in sample
The content of cytomegalovirus IgG antibody is directly proportional to relative luminous intensity.
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Cited By (7)
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| CN111007249A (en) * | 2019-11-27 | 2020-04-14 | 迪瑞医疗科技股份有限公司 | Herpes simplex virus antibody IgG chemiluminescence immunoassay kit and preparation method thereof |
| CN111089975A (en) * | 2019-12-26 | 2020-05-01 | 迪瑞医疗科技股份有限公司 | Chemiluminescence immunoassay determination kit for detecting Toxoplasma gondii IgG antibody and preparation method thereof |
| CN111856004A (en) * | 2020-03-25 | 2020-10-30 | 潍坊市康华生物技术有限公司 | Reagent for detecting COVID-19 by chemiluminescence immunoassay and detection method thereof |
| CN113295873A (en) * | 2020-02-21 | 2021-08-24 | 深圳麦科田生物医疗技术股份有限公司 | Kit and method for detecting novel coronavirus SARS-CoV-2 antibody |
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