CN109142753A - Squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit and preparation method thereof - Google Patents
Squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit and preparation method thereof Download PDFInfo
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- CN109142753A CN109142753A CN201811118543.8A CN201811118543A CN109142753A CN 109142753 A CN109142753 A CN 109142753A CN 201811118543 A CN201811118543 A CN 201811118543A CN 109142753 A CN109142753 A CN 109142753A
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- cell carcinoma
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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
Abstract
The present invention provides a kind of squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit and preparation method thereof, belongs to vitro detection technical field.The kit includes: the squamous cell carcinoma-related antigen antibody of Streptavidin magnetic particle suspension liquid, the squamous cell carcinoma-related antigen monoclonal antibody of chemiluminescent labels label and coupling label.The present invention also provides a kind of preparation methods of squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit.Kit of the invention is full automatic measurement sample, and directly gives numerical value, reduces manual operation error, and realizes unattended, and time needed for shortening clinical detection, detection accuracy are higher, and reagent and instrument form closed system, and systematic error is small.
Description
Technical field
The invention belongs to vitro detection technical fields, and in particular to a kind of squamous cell carcinoma-related antigen chemiluminescence immunoassay
Detection kit and preparation method thereof.
Background technique
Squamous cell carcinoma-related antigen (SCC) is a kind of glycoprotein extracted from uterine tissue, abbreviation SCC.SCC is one
Group belongs to serine/cysteine proteinase mortifier family glycoprotein, and by very high homology, biological function is completely different
SCC1 and SCC2 composition, contain 390 amino acid, molecular weight is about 45KDa.SCC is widely present in normal tissue (content
It is atomic) and the epithelial cell of malignant change in.SCC concentration in serum is related with the differentiation degree of squamous cell carcinoma, normal person
SCC concentration < 1.5ng/ml in serum.A small number of benign diseases can also be shown in that SCC is increased, such as pulmonary infection, dermatitis, renal failure and liver
Disease.It has higher diagnostic value to cervix cancer, and sensibility and specificity are preferable, while the squamas such as auxiliary diagnosis lung, esophagus
Cancer, it is horizontal related to the development degree of tumour, accuracy and sensitivity can be improved with CYFRA21-1 joint-detection.
At present measurement squamous cell carcinoma-related antigen method have radiommunoassay (RIA), EIA enzyme immunoassay (EIA),
Chemiluminescence immunoassay (CLEIA) etc..However, ELISA hair especially limitation, poor specificity, sensitivity is low, accuracy is not high
Deng.Although radio-immune method is considered as accurately and reliably method, but radioisotopic radiation effect, at waste
The drawbacks of special laboratory of reason needs, at high cost and half-life short, this method is made gradually to suffer exit.
SCC chemiluminescence immunoassay test agent box compares traditional detection method, and the combination of antigen, antibody is in similar liquid
It is carried out under concrete conditions in the establishment of a specific crime, reaction process is more succinct, it is sensitive to be swift in response, the range of linearity is wider and specific more preferable；On the other hand,
SCC chemiluminescence immunoassay test agent box and chemiluminescence immunoassay detecting instrument form closed system, the addition and inspection of reagent, sample
Survey task is automatically performed by instrument, reduces the error of manual operation, improves the sensitivity and accuracy of whole system.
Summary of the invention
The purpose of the present invention is to solve existing SCC assay kit, there are operating time length, poor accuracy, linear
The problem of narrow range, and a kind of squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit is provided and preparation method thereof.
Present invention firstly provides a kind of squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit, the kit packets
It includes:
Streptavidin magnetic particle suspension liquid, the squamous cell carcinoma-related antigen monoclonal of chemiluminescent labels label are anti-
The squamous cell carcinoma-related antigen antibody of body and coupling label.
Preferably, in the kit, the mass fraction of Streptavidin magnetic particle is 0.01%~1%.
Preferably, in the Streptavidin magnetic particle suspension liquid, the partial size of Streptavidin magnetic particle is 0.05
~3 μm.
Preferably, in the squamous cell carcinoma-related antigen monoclonal antibody of the chemiluminescent labels label, squamous
The molar ratio of cell carcinoma antigen monoclonal antibody and chemiluminescent labels is 1:3~20.
Preferably, the chemiluminescent labels are acridinium ester, luminol, different luminol and tris (bipyridine) ruthenium.
Preferably, in the squamous cell carcinoma-related antigen antibody of the coupling label, squamous cell carcinoma-related antigen is anti-
The molar ratio of body and coupling marker is 1:5~20.
Preferably, the coupling marker is biotin or antigen.
Preferably, the kit further includes Chemoluminescent substrate, the Chemoluminescent substrate include A liquid and
B liquid, the A liquid are nitric acid solution, and B liquid is sodium hydroxide solution.
Preferably, the kit further includes squamous cell carcinoma-related antigen calibration object, the squamous cell
The concentration of cancer antigen calibration object is respectively 0.00ng/mL, 0.5ng/mL, 1.00ng/mL, 10ng/mL, 50ng/mL, 250ng/mL
With the squamous cell carcinoma-related antigen solution of 500ng/mL.
The present invention also provides a kind of preparation method of squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit, packets
Include following steps:
Step 1: the preparation of Streptavidin magnetic particle suspension liquid
After Streptavidin magnetic particle solution and TBST solution are mixed, be placed on magnetic separator, until supernatant without
Muddiness abandons supernatant, leaves and takes magnetic particle, be made into solid-phase reagent after cleaning in buffer, obtains the suspension of Streptavidin magnetic particle
Liquid；
Step 2: the preparation of the squamous cell carcinoma-related antigen monoclonal antibody of chemiluminescent labels label
SCC antibody is put into centrifuge tube and is centrifuged, phosphate buffer is then added, chemiluminescent labels are added after mixing
Solution centrifugation is protected from light being put into after the centrifugation seal of tube in magazine, then magazine is put into gas bath constant temperature oscillator and is mixed, is added
Confining liquid is put into gas bath constant temperature oscillator and mixes, and by the antibody closed by purifying, collection, is then placed in buffer
Dilution saves；
Step 3: it is coupled the preparation of the squamous cell carcinoma-related antigen antibody of label
SCC antibody is put into centrifuge tube and is centrifuged, phosphate buffer is then added, coupling marker solution is added after mixing
Centrifugation is added confining liquid, is put into gas bath constant temperature oscillator and mixes, and by the antibody closed by purifying, collection, is then placed in
It dilutes, saves in buffer.
Beneficial effects of the present invention
1, the present invention selects acridinium ester etc. for the marker material of chemiluminescence immunoassay system, which has excitation state to return
The energy jump generated when to ground state is direct chemiluminescence, does not need the participation of enzyme, saves time and cost.
2, the present invention can be firmly combined together using the analog of Streptavidin MagneSphere and biotin labeling, be subtracted
Few non-specific adsorption, improves the accuracy of test sample, strong antijamming capability.
3, squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit of the invention is full automatic measurement sample,
And numerical value is directly given, manual operation error is reduced, and realize unattended.
4, chemical luminescence immune analysis reagent box of the invention can be detection with Full-automatic chemiluminescence immunoassay analysis meter
Tool completes the detection to squamous cell carcinoma-related antigen, and this chemical luminescence immune analysis reagent box and instrument are mating, shortens
Time needed for clinical detection, detection accuracy are higher, and reagent and instrument form closed system, and systematic error is small.
5, chemiluminescence immune detection reagent kit of the invention can substitute market conventional reagents box in terms of accuracy, be
Client provides more accurate measurement data and cheap testing cost.
Detailed description of the invention
Fig. 1 is the preparation stream of the preparation method of the squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit of invention
Journey design drawing；
Fig. 2 is the canonical plotting of kit of the invention and available reagent box.
Specific embodiment
It is right in the following with reference to the drawings and specific embodiments in order to keep advantages of the present invention, purpose and method more full and accurate understandable
A specific embodiment of the invention is described in detail.Many details are explained in the following description in order to understand, but this
Invention can be implemented with different with other modes that are describing.
Present invention firstly provides a kind of squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit, the kit packets
It includes:
Streptavidin magnetic particle suspension liquid, the squamous cell carcinoma-related antigen monoclonal of chemiluminescent labels label are anti-
The squamous cell carcinoma-related antigen antibody of body and coupling label.
According to the present invention, in the kit, the mass fraction of Streptavidin magnetic particle is preferably 0.01%~
1%, more preferably 0.072%.
According to the present invention, in the Streptavidin magnetic particle suspension liquid, the partial size of Streptavidin magnetic particle is preferred
It is 0.05~3 μm, more preferably 3 μm, when the partial size of Streptavidin magnetic particle is lower than 0.05 μm, antigen or antibody and magnetic
Pearl Percentage bound is low, and it is relatively low to may cause whole light quantity subnumber；It is non-specific when the partial size of Streptavidin magnetic particle is higher than 1 μm
Property combination effect is obvious, may cause kit poor sensitivity etc..
According to the present invention, in the squamous cell carcinoma-related antigen monoclonal antibody of the chemiluminescent labels label, squama
Columnar epithelium cell carcinoma antigen monoclonal antibody and the molar ratio of chemiluminescent labels are 1:3~20, more preferably 1:3.It is described
Chemiluminescent labels be preferably acridinium ester, luminol, different luminol and tris (bipyridine) ruthenium, more preferably acridinium ester.
According to the present invention, in the squamous cell carcinoma-related antigen antibody of the coupling label, squamous cell carcinoma-related antigen
The molar ratio of antibody and coupling marker is preferably 1:5~20, more preferably 1:5；The coupling marker is preferably biotin
Or antigen, more preferably biotin.
According to the present invention, the kit further includes Chemoluminescent substrate, and the Chemoluminescent substrate includes A liquid
With B liquid, the A liquid is preferably nitric acid solution, and B liquid is preferably sodium hydroxide solution.
According to the present invention, the kit further includes squamous cell carcinoma-related antigen calibration object, and the scaly epithelium is thin
The concentration of born of the same parents' cancer antigen calibration object preferably be respectively 0.00ng/mL, 0.5ng/mL, 1.00ng/mL, 10ng/mL, 50ng/mL,
The squamous cell carcinoma-related antigen solution of 250ng/mL and 500ng/mL.
The present invention also provides a kind of preparation method of squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit, systems
Standby process is as shown in Figure 1, include the following steps:
Step 1: the preparation of Streptavidin magnetic particle suspension liquid
After Streptavidin magnetic particle solution and TBST solution are mixed, be placed on magnetic separator, until supernatant without
Muddiness abandons supernatant, leaves and takes magnetic particle, be made into solid-phase reagent after cleaning in buffer, obtains the suspension of Streptavidin magnetic particle
Liquid；
The concentration of the Streptavidin magnetic particle solution is preferably 50~100mg/ml；Streptavidin magnetic particle is molten
The volume ratio of liquid and TBST solution is preferably (0.5~1): (5~10)；The mixing time is preferably 10-15 minutes, described
Buffer be 50mM MES, 0.05% Tween-20,0.05%Proclin300, pH6.5；The concentration of the solid-phase reagent
Preferably 0.01%~1%；The source of the Streptavidin magnetic particle solution is commercially available.
Step 2: the preparation of the squamous cell carcinoma-related antigen monoclonal antibody of chemiluminescent labels label
SCC antibody is put into centrifuge tube and is centrifuged, is preferably centrifuged 10s~30s at room temperature, guarantees that antibody is located at centrifuge tube
Then phosphate buffer is added in bottom position, the centrifugation of chemiluminescent labels solution is added after mixing, and the centrifuging temperature is excellent
It is selected as room temperature, centrifugation time is preferably 0.5min~3min；It is protected from light being put into after the centrifugation seal of tube in magazine, then puts magazine
Enter in gas bath constant temperature oscillator (25 DEG C) and mix, the mixing time is preferably 2-4h, and confining liquid is added, is put into gas bath constant temperature
Closing is mixed in oscillator, the off-period is preferably 1-2h, and 250 column of sephadex G on the antibody closed is pure
Change, eluted with buffer, collect, be then placed in buffer and dilute, saves；
The quality (μ g) of the SCC antibody: the volume (μ l) of chemiluminescent labels solution is preferably 500:5；It is described
The concentration of chemiluminescent labels solution be preferably 2mg/mL；The confining liquid is preferably lysine, and mass fraction is preferred
It is 20%；The buffer is 100mM Bis-tris propane, 0.05% tween, 0.05%Proclin300, pH6.0；
Step 3: it is coupled the preparation of the squamous cell carcinoma-related antigen antibody of label
SCC antibody is put into centrifuge tube and is centrifuged, is preferably centrifuged 10s~30s at room temperature, guarantees that antibody is located at centrifuge tube
Then phosphate buffer is added in bottom position, coupling marker solution centrifugation is added after mixing, and the centrifuging temperature is preferably
Room temperature, centrifugation time are preferably 0.5min~3min；Then it mixes, the mixing temperature is preferably 2-8 DEG C, and mixing time is excellent
It is selected as 2-4h, confining liquid is added, is put into gas bath constant temperature oscillator and mixes closing, the off-period is preferably 1-2h, will
250 column purification of sephadex G on the antibody closed is eluted with buffer, is collected, is then placed in buffer and dilutes, and is protected
It deposits；
The quality (μ g) of the SCC antibody: the volume (μ l) of coupling marker solution is preferably 500:2；The idol
The concentration for joining marker solution is preferably 2mg/mL；The confining liquid is preferably lysine, and mass fraction is preferably 20%；Institute
The buffer stated is 100mM Bis-tris propane, 0.05% tween, 0.05%Proclin300, pH6.0；
Squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit of the invention is for detecting squamous cell
When cancer antigen, squamous cell carcinoma-related antigen calibration object is examined using Full-automatic chemiluminescence immunoassay analysis meter (CM180)
It surveys, draws standard curve, be built in computer software；Then clinical sample is tested according to demand, according to the light quantity subnumber meter of sample
Calculate the concentration of squamous cell carcinoma-related antigen；Finally to squamous cell carcinoma-related antigen automatic chemiluminescence immunoassay system
Carry out the evaluation of performance (sensitivity, linear, anti-interference/specificity).
Further detailed description is done to the present invention combined with specific embodiments below.
Embodiment 1: the preparation of squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit:
(1) preparation of Streptavidin magnetic particle suspension liquid
Taking concentration is 0.72 milliliter of the Streptavidin magnetic particle solution (72mg) of 100mg/ml, and the TBST that 10mL is added is molten
Liquid mixes well after ten minutes, is placed on magnetic separator, until supernatant abandons supernatant, leave and take magnetic particle without muddiness.Repeated washing
Being made into magnetic bead concentration in 50mM MES, 0.05% tween, 0.05%Proclin300, the buffer of pH7.4 after 3 times is
0.072% solid-phase reagent, 2~8 DEG C of preservations.
(2) preparation of the squamous cell carcinoma-related antigen monoclonal antibody of acridinium ester label
500ug antibody is put into centrifuge tube, guarantees that antibody is located at centrifuge tube bottom position (centrifuge room temperature is centrifuged 20s)
After phosphate buffer solution is added, mix well, the DMF solution of 5 μ l 2mg/mL acridinium esters be added after mixing, with centrifuge room temperature
Under the conditions of be centrifuged 0.5 minute.It will be centrifuged to be put into after effective sealed membrane seals and be protected from light in magazine, magazine is put into gas bath constant temperature later
Oscillator (25 DEG C) mixes 4 hours.20% lysine confining liquid of 1mL is added, is put into gas bath constant temperature oscillator (25 DEG C), middling speed
It mixes, off-period 1h.By 250 column purification of sephadex G on the antibody closed, flow velocity 3ml/min is buffered with PB
Liquid elution, fraction collection.The antibody-solutions gathered are placed in 2~8 DEG C of preservations.It is when use that scaly epithelium after purification is thin
Born of the same parents' cancer antigen antibody concentrated solution 100mM Bis-tris propane, 0.05% tween, 0.05%Proclin300, pH6.0's is slow
Fliud flushing is diluted to final concentration of 0.2 μ g/ml, 2~8 DEG C of preservations.
(3) preparation of biotin coupling label squamous cell carcinoma-related antigen antibody
500ug antibody is put into centrifuge tube, guarantees that antibody is located at centrifuge tube bottom position (centrifuge room temperature is centrifuged 20s)
After phosphate buffer solution is added, mix well, after mixing be added 2ml 2mg/mL biotin DMF solution, with centrifuge room temperature
Under the conditions of be centrifuged 0.5 minute.2-8 DEG C mixes 4 hours.20% lysine confining liquid of 1mL is added, is put into gas bath constant temperature oscillator
(25 DEG C), middling speed mix, off-period 1h.It is pure that AKTA on the antibody closed is purified into instrument (250 column of sephadex G)
Change, flow velocity 3ml/min, is eluted with PB buffer, fraction collection.The antibody-solutions gathered are placed in 2~8 DEG C of preservations.
When use by the 100mM Bis-tris propane of squamous cell carcinoma-related antigen antibody concentrated solution after purification, 0.05% tween,
The buffer of 0.05%Proclin300, pH6.0 are diluted to final concentration of 0.7 μ g/ml, 2~8 DEG C of preservations.
The preparation of 2 squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit of embodiment
(1) preparation of Streptavidin magnetic particle suspension liquid
Taking concentration is 0.72 milliliter of the Streptavidin magnetic particle solution (72mg) of 100mg/ml, and the TBST that 10mL is added is molten
It after liquid mixes well 12 minutes, is placed on magnetic separator, until supernatant abandons supernatant, leave and take magnetic particle without muddiness.Repeated washing
Being made into magnetic bead concentration in 50mM MES, 0.05% tween, 0.05%Proclin300, the buffer of pH7.4 after 3 times is
0.072% solid-phase reagent, 2~8 DEG C of preservations.
(2) preparation of the squamous cell carcinoma-related antigen monoclonal antibody of acridinium ester label
500ug antibody is put into centrifuge tube, guarantees that antibody is located at centrifuge tube bottom position (centrifuge room temperature is centrifuged 30s)
After phosphate buffer solution is added, mix well, the DMF solution of 5 μ l 2mg/mL acridinium esters be added after mixing, with centrifuge room temperature
Under the conditions of be centrifuged 3 minutes.It will be centrifuged to be put into after effective sealed membrane seals and be protected from light in magazine, magazine is put into the vibration of gas bath constant temperature later
It swings device (25 DEG C), mixes 2 hours.20% lysine confining liquid of 1mL is added, is put into gas bath constant temperature oscillator (25 DEG C), middling speed is mixed
It is even, off-period 2h.By 250 column purification of sephadex G on the antibody closed, flow velocity 3ml/min, with PB buffer
Elution, fraction collection.The antibody-solutions gathered are placed in 2~8 DEG C of preservations.By squamous cell after purification when use
Cancer antigen antibody concentrated solution 100mM Bis-tris propane, 0.05% tween, 0.05%Proclin300, the buffering of pH6.0
Liquid is diluted to final concentration of 0.2 μ g/ml, 2~8 DEG C of preservations.
(3) preparation of biotin coupling label squamous cell carcinoma-related antigen antibody
500ug antibody is put into centrifuge tube, guarantees that antibody is located at centrifuge tube bottom position (centrifuge room temperature is centrifuged 30s)
After phosphate buffer solution is added, mix well, after mixing be added 2ml 2mg/mL biotin DMF solution, with centrifuge room temperature
Under the conditions of be centrifuged 3 minutes.2-8 DEG C mixes 2 hours.20% lysine confining liquid of 1mL is added, is put into gas bath constant temperature oscillator (25
DEG C), middling speed mixes, off-period 2h.AKTA on the antibody closed is purified into instrument (250 column of sephadex G) purifying, stream
Speed is 3ml/min, is eluted with PB buffer, fraction collection.The antibody-solutions gathered are placed in 2~8 DEG C of preservations.When use
By squamous cell carcinoma-related antigen antibody concentrated solution after purification 100mM Bis-tris propane, 0.05% tween, 0.05%
The buffer of Proclin300, pH6.0 are diluted to final concentration of 0.7 μ g/ml, 2~8 DEG C of preservations.
The preparation of 3 squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit of embodiment
(1) preparation of Streptavidin magnetic particle suspension liquid
Taking concentration is 0.72 milliliter of the Streptavidin magnetic particle solution (72mg) of 100mg/ml, and the TBST that 10mL is added is molten
It after liquid mixes well 12 minutes, is placed on magnetic separator, until supernatant abandons supernatant, leave and take magnetic particle without muddiness.Repeated washing
Being made into magnetic bead concentration in 50mM MES, 0.05% tween, 0.05%Proclin300, the buffer of pH7.4 after 3 times is
0.072% solid-phase reagent, 2~8 DEG C of preservations.
(2) preparation of the squamous cell carcinoma-related antigen monoclonal antibody of acridinium ester label
500ug antibody is put into centrifuge tube, guarantees that antibody is located at centrifuge tube bottom position (centrifuge room temperature is centrifuged 20s)
After phosphate buffer solution is added, mix well, the DMF solution of 5 μ l 2mg/mL acridinium esters be added after mixing, with centrifuge room temperature
Under the conditions of be centrifuged 2 minutes.It will be centrifuged to be put into after effective sealed membrane seals and be protected from light in magazine, magazine is put into the vibration of gas bath constant temperature later
It swings device (25 DEG C), mixes 3 hours.20% lysine confining liquid of 1mL is added, is put into gas bath constant temperature oscillator (25 DEG C), middling speed is mixed
It is even, off-period 1h.By 250 column purification of sephadex G on the antibody closed, flow velocity 3ml/min, with PB buffer
Elution, fraction collection.The antibody-solutions gathered are placed in 2~8 DEG C of preservations.By squamous cell after purification when use
Cancer antigen antibody concentrated solution 100mM Bis-tris propane, 0.05% tween, 0.05%Proclin300, the buffering of pH6.0
Liquid is diluted to final concentration of 0.2 μ g/ml, 2~8 DEG C of preservations.
(3) preparation of biotin coupling label squamous cell carcinoma-related antigen antibody
500ug antibody is put into centrifuge tube, guarantees that antibody is located at centrifuge tube bottom position (centrifuge room temperature is centrifuged 20s)
After phosphate buffer solution is added, mix well, after mixing be added 2ml 2mg/mL biotin DMF solution, with centrifuge room temperature
Under the conditions of be centrifuged 2 minutes.2-8 DEG C mixes 3 hours.20% lysine confining liquid of 1mL is added, is put into gas bath constant temperature oscillator (25
DEG C), middling speed mixes, off-period 1h.AKTA on the antibody closed is purified into instrument (250 column of sephadex G) purifying, stream
Speed is 3ml/min, is eluted with PB buffer, fraction collection.The antibody-solutions gathered are placed in 2~8 DEG C of preservations.When use
By squamous cell carcinoma-related antigen antibody concentrated solution after purification 100mM Bis-tris propane, 0.05% tween, 0.05%
The buffer of Proclin300, pH6.0 are diluted to final concentration of 0.7 μ g/ml, 2~8 DEG C of preservations.
4 squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit detection method of embodiment:
With Full-automatic chemiluminescence immunoassay analysis meter (CM180) for detection instrument, methodology mode is sandwich method, i.e. instrument
Sequentially add the sample of 25 μ L, the squamous cell carcinoma-related antigen monoclonal antibody of 50 μ L acridinium ester labels, 50 μ L biotin marks
The squamous cell carcinoma-related antigen analog of note and 40 μ L Streptavidin magnetic particles.Reaction after twenty minutes, carries out Magneto separate.Instrument
Reactant is sent into darkroom by device, primary that luminous substrate liquid A liquid (HNO is added3Solution) and B liquid (NaOH solution) reacted, most
Light quantum number is recorded afterwards.
5 squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit performance evaluation of embodiment
(1) linear detection: will be diluted to by a certain percentage 7 kinds of concentration close to the high level sample of the range of linearity upper limit, right
The sample standard deviation of each concentration repeats detection 2 times, calculates average value, result average value and dilution ratio are carried out with least square method
Straight line fitting calculates linearly dependent coefficient r, obtains linearly dependent coefficient r=0.9999, range of linearity 0.01-500.00ng/
mL.As a result as shown in Figure 2 and Table 1:
Table 1
(2) lowest detection line: the definition of lowest detection line is to carry out 20 measurement light quantity subnumbers to zero calibration object, takes it
Average value subtracts twice of standard deviation, and bringing into is sensitivity obtained by standard curve；Calculate squamous cell carcinoma-related antigen chemistry
The lowest detection line < 0.01ng/mL of electrochemiluminescent immunoassay detection kit, the results are shown in Table 2:
Table 2
(3) accuracy: the dermoid cancer for being about 50ng/mL (its concentration deviation is allowed to be ± 20%) for concentration is anti-
Raw sample (A) is added in the sample (B) of corresponding matrix, and the volume ratio that (A)/(B) is added is 1:9, is calculated the rate of recovery and is
97.80%, as shown in table 3:
Table 3
(4) repeated: respectively to repeat detection 10 times, two with the sample that concentration is 5 ± 2ng/mL and 50 ± 10ng/mL respectively
The sample repeatability coefficient of variation (CV) is respectively 1.78% and 1.33%, and the results are shown in Table 4:
Table 4
(5) anti-interference specificity experiments: for the mitomycin C of 1000ng/mL, adriamycin and fluorouracil test result
Less than 0.5ng/mL；20mg/dL bilirubin, 500mg/dL hemoglobin, 3000mg/dL triglycerides, 12g/dL total protein, RF
The sample of sample is not significantly affected by the measurement (± 10%) of squamous cell carcinoma-related antigen.
(6) stability: reagent is placed in 37 DEG C of baking ovens 7 days and 14 days, and calibration object and clinical sample is used to detect 7 days and 14 days
High temperature reagent and zero-time reagent light quantity subnumber attenuation rate, attenuation rate < 15%, the results are shown in Table 5:
Table 5
Claims (10)
1. a kind of squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit, which is characterized in that the kit includes:
Streptavidin magnetic particle suspension liquid, chemiluminescent labels label squamous cell carcinoma-related antigen monoclonal antibody and
It is coupled the squamous cell carcinoma-related antigen antibody of label.
2. a kind of squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit according to claim 1, feature
It is, in the kit, the mass fraction of Streptavidin magnetic particle is 0.01%~1%.
3. a kind of squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit according to claim 1, feature
It is, in the Streptavidin magnetic particle suspension liquid, the partial size of Streptavidin magnetic particle is 0.05~3 μm.
4. a kind of squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit according to claim 1, feature
It is, in the squamous cell carcinoma-related antigen monoclonal antibody of the chemiluminescent labels label, dermoid cancer is anti-
Former monoclonal antibody and the molar ratio of chemiluminescent labels are 1:3~20.
5. a kind of squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit according to claim 4, feature
It is, the chemiluminescent labels are acridinium ester, luminol, different luminol and tris (bipyridine) ruthenium.
6. a kind of squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit according to claim 1, feature
It is, in the squamous cell carcinoma-related antigen antibody of the coupling label, squamous cell carcinoma-related antigen antibody and coupling are marked
The molar ratio of object is 1:5~20.
7. a kind of squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit according to claim 6, feature
It is, the coupling marker is biotin or antigen.
8. a kind of squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit according to claim 1, feature
It is, the kit further includes Chemoluminescent substrate, and the Chemoluminescent substrate includes A liquid and B liquid, the A liquid
For nitric acid solution, B liquid is sodium hydroxide solution.
9. a kind of squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit according to claim 1, feature
It is, the kit further includes squamous cell carcinoma-related antigen calibration object, the squamous cell carcinoma-related antigen calibration object
Concentration be respectively 0.00ng/mL, 0.5ng/mL, 1.00ng/mL, 10ng/mL, 50ng/mL, 250ng/mL and 500ng/mL
Squamous cell carcinoma-related antigen solution.
10. a kind of preparation of squamous cell carcinoma-related antigen chemiluminescence immune detection reagent kit according to claim 1
Method, which comprises the steps of:
Step 1: the preparation of Streptavidin magnetic particle suspension liquid
After Streptavidin magnetic particle solution and TBST solution are mixed, it is placed on magnetic separator, until supernatant is without muddiness,
Supernatant is abandoned, magnetic particle is left and taken, is made into solid-phase reagent after cleaning in buffer, obtain Streptavidin magnetic particle suspension liquid；
Step 2: the preparation of the squamous cell carcinoma-related antigen monoclonal antibody of chemiluminescent labels label
SCC antibody is put into centrifuge tube and is centrifuged, phosphate buffer is then added, chemiluminescent labels solution is added after mixing
Centrifugation is protected from light being put into after the centrifugation seal of tube in magazine, then magazine is put into gas bath constant temperature oscillator and is mixed, closing is added
Liquid is put into gas bath constant temperature oscillator and mixes, and by the antibody closed by purifying, collection, is then placed in buffer and dilutes,
It saves；
Step 3: it is coupled the preparation of the squamous cell carcinoma-related antigen antibody of label
SCC antibody is put into centrifuge tube and is centrifuged, phosphate buffer is then added, after mixing be added coupling marker solution from
The heart is added confining liquid, is put into gas bath constant temperature oscillator and mixes, and by the antibody closed by purifying, collection, is then placed in slow
It dilutes, saves in fliud flushing.
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Cited By (3)
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| CN110780079A (en) * | 2019-11-28 | 2020-02-11 | 南京迪安医学检验所有限公司 | Squamous cell carcinoma antigen detection reagent |
| CN112129941A (en) * | 2020-09-22 | 2020-12-25 | 武汉生之源生物科技股份有限公司 | Chemiluminescence kit for detecting squamous cell carcinoma antigen |
| CN113109558A (en) * | 2021-04-21 | 2021-07-13 | 山东康华生物医疗科技股份有限公司 | Magnetic particle chemiluminescence kit for quantitatively detecting CYFRA21-1 and manufacturing method thereof |
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| CN102435740A (en) * | 2011-08-31 | 2012-05-02 | 内蒙古科慧生物科技有限责任公司 | Kit for quantitative measurement of squamous cell carcinoma antigen (SCC) and its detection method |
| CN102565405A (en) * | 2011-08-24 | 2012-07-11 | 苏州长光华医生物试剂有限公司 | Method for immunological detection by combining acridinium ester labeling technology with general magnetic particles |
| CN108169486A (en) * | 2017-12-07 | 2018-06-15 | 江苏泽成生物技术有限公司 | A kind of kit and its test method for measuring squamous cell carcinoma-related antigen content |
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| CN102565405A (en) * | 2011-08-24 | 2012-07-11 | 苏州长光华医生物试剂有限公司 | Method for immunological detection by combining acridinium ester labeling technology with general magnetic particles |
| CN102435740A (en) * | 2011-08-31 | 2012-05-02 | 内蒙古科慧生物科技有限责任公司 | Kit for quantitative measurement of squamous cell carcinoma antigen (SCC) and its detection method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110780079A (en) * | 2019-11-28 | 2020-02-11 | 南京迪安医学检验所有限公司 | Squamous cell carcinoma antigen detection reagent |
| CN112129941A (en) * | 2020-09-22 | 2020-12-25 | 武汉生之源生物科技股份有限公司 | Chemiluminescence kit for detecting squamous cell carcinoma antigen |
| CN113109558A (en) * | 2021-04-21 | 2021-07-13 | 山东康华生物医疗科技股份有限公司 | Magnetic particle chemiluminescence kit for quantitatively detecting CYFRA21-1 and manufacturing method thereof |
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