CN102023216A - Suspension chip detection method for simultaneously detecting hepatitis B virus, hepatitis C virus, treponema pallidum and human immunodeficiency virus pathogens - Google Patents
Suspension chip detection method for simultaneously detecting hepatitis B virus, hepatitis C virus, treponema pallidum and human immunodeficiency virus pathogens Download PDFInfo
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Abstract
The invention relates to a suspension chip detection method for simultaneously detecting hepatitis B virus, hepatitis C virus, treponema pallidum and human immunodeficiency virus pathogens. Plastic color coded microspheres with diameter of between 5.5 and 6.5 mu m are taken as vectors for a suspension chip; and the carriers are coated with monoclonal hepatitis B virus surface antibodies, HIV1, gP41 antigens, HCV NS3 antigens or treponema pallidum TP15 antigens. Through the suspension chip, the hepatitis B virus, hepatitis C virus, treponema pallidum and human immunodeficiency virus pathogens in a sample to be detected can be very conveniently and quickly detected at the same time; and the method has the advantages of accurate and reliable detection result, high specificity, small using amount of samples and high-throughput detection.
Description
Technical field
The invention belongs to the immunology detection technical field, promptly a kind of method for detecting suspension chip that hepatitis B, third liver, syphilis, acquired immune deficiency syndrome (AIDS) substance are carried out common inspection.
Background technology
Along with continuing to increase of China's open degree, the people-to-people contacts of entry and exit increase significantly, and the task of frontier port monitoring of infectious disease is more and more heavier; With SARS and bird flu is the public health security system formation stern challenge of typical new discovery infectious disease to China, and the territory health quarantine defence line bears immense pressure and risk especially.Reality is urgent call not only accurately and reliably but also sensitivity efficiently detection technique play a role, construct safe and reliable territory health quarantine defence line, the suspending chip technology satisfies the ideal of this actual demand just and selects.We aim at infectious disease hepatitis B, hepatitis C, syphilis, the AIDS of frontier port routine monitoring, developed the suspension protein chip system that many pathogen detect simultaneously, the inspection and quarantine personnel are freed from heavy experiment, promote the technical merit of China's health quarantine, hold the gateway of a country, ensure the people's life security, promote reform and opening-up, service foreign trade and economic development.TP (syphilis) confirms with TRUST primary dcreening operation TPPA at present; HIV (AIDS) uses the ELISA primary dcreening operation, confirms with WB; HBV (hepatitis B) and HCV (third liver) detect with ELISA.These methods once can only detect a kind of specific cause of disease, and the program complexity, workload is big, time-consuming, insufficient sensitivity is high.
Summary of the invention
The objective of the invention is to overcome the former body detecting method program of PI complexity, workload is big, time-consuming, insufficient sensitivity is high defective.
Technical solution of the present invention is: a kind of suspending chip that hepatitis B, third liver, syphilis, acquired immune deficiency syndrome (AIDS) substance are carried out common inspection, its special character is, this suspending chip is to be that the plastics colour code coding microball of 5.5～6.5 μ m is a carrier with diameter, Sheet clone hepatitis B surface antibody, HIV1 gP41 antigen, HCVNS3 antigen or syphilis TP15 antigen on this carrier.
A kind of method for preparing above-mentioned suspending chip may further comprise the steps:
1) activation coding microball:
1.1) at first get 3000-5000 coding microball in the 1.5mL centrifuge tube, the centrifugal 3-5min of 12000-14000g, careful sucking-off supernatant also discards;
1.2) adding 100 μ L pH 7.4 again, the PBS damping fluid of 0.05%TWEEN-20 suspends, shake after 30 seconds ultrasonic 30 seconds, the centrifugal 3-5min of 12000-14000g, carefully the sucking-off supernatant also discards;
1.3) then add 80 μ L 3g NaH
2PO
4, the microballoon of 5N NaOH 40drops/250mL H2O activation damping fluid shook after 30 seconds ultrasonic 30 seconds;
1.4) add the EDC of the 50mg/mL of the fresh configuration of 10 μ L again, and then add the Sulfo-NHS of the 50mg/mL of the fresh configuration of 10 μ L, shake at a high speed after 30 seconds with the aluminium foil parcel, jolt 700rpm in room temperature, 15-25min;
1.5) add the PBS of the pH7.4 of 150 μ L again, shake after 10 seconds, the centrifugal 3-5min of 12000-14000g, careful sucking-off supernatant also discards;
1.6) add the PBS of the pH7.4 of 150 μ L again, shake after 10 seconds, the centrifugal 3-5min of 12000-14000g, careful sucking-off supernatant also discards;
1.7) at last, adding the PBS suspended coding microballoon of the pH7.4 of 100 μ L, 300rpm is after 30 seconds in concussion, ultrasonic 15 seconds, finishes the activation of coding microball;
2) the bag quilt of activation coding microball:
2.1) get monoclonal hepatitis B surface antibody, HIV1 gP41 antigen, HCV NS3 antigen, syphilis TP15 antigen 5-50 μ g and join respectively in the microballoon of corresponding codes separately after the activation, PBS with pH7.4 is settled to 500 μ L from about 120 μ L, the aluminium foil parcel, after room temperature jolts 700rpm 2h, the centrifugal 3-5min of 12000-14000g, careful sucking-off supernatant also discards;
2.2) wash once with the PBS of the pH7.4 of 500 μ L, the centrifugal 3-5min of 12000-14000g, careful sucking-off supernatant also discards;
2.3) add the PBS of 500 μ L pH7.4 again, 1%BSA, the sealing damping fluid suspended coding microballoon of 0.05%Azide, 300rpm is after 15 seconds in concussion, with the aluminium foil parcel, jolts 30min in room temperature, the centrifugal 3-5min of 12000-14000g, careful sucking-off supernatant also discards;
2.4) then adding the PBS of 500 μ L pH7.4,1%BSA, 0.02%TWEEN, the microballoon of 0.05%Azide preserve liquid washing coding microball, the centrifugal 5-7min of 15000-17000g, careful sucking-off supernatant also discards;
2.5) preserve liquid suspended coding microballoon with the microballoon of 150 μ L at last, finish the bag quilt of activation coding microball.
Utilize above-mentioned suspending chip to detect the method for hepatitis B in the serum to be checked, third liver, syphilis, acquired immune deficiency syndrome (AIDS) substance, may further comprise the steps:
1) every hole adds the detection damping fluid of 150 μ L, and pre-wet hole is drained with vacuum pump;
2) every hole adds the working fluid that 50 μ L contain the corresponding encoded microballoon, adds 100 μ L washing lotions again, washes 2 times, drains with vacuum pump;
3) every hole adds 50 μ L serum to be checked, and blank well adds PBS, 700rpm 30 seconds, and the room temperature lucifuge jolts 300rpm 30min, drains with vacuum pump;
4) every hole adds 100 μ L washing lotions, washes 3 times, drains with vacuum pump;
5) it is anti-that every hole adds 1: 2500 biotinylation of 50 μ L two, added the antibody of biotinylated anti-surface antigen with monoclonal hepatitis B surface antibody bag by the hole of microballoon, 700rpm 30 seconds, and room temperature lucifuge 300rpm25-35min drains with vacuum pump;
6) every hole adds 100 μ L washing lotions, washes 3 times, drains with vacuum pump;
7) every hole adds the SA-PE of 50 μ L, 700rpm 30 seconds, and room temperature lucifuge 300rpm 8-12min drains with vacuum pump;
8) every hole adds 100 μ L washing lotions, washes 3 times, drains with vacuum pump
9) every hole adds the detection damping fluid of 125 μ L, 700rpm 30 seconds;
10) read FMI numerical value with the Bio-Plex suspension chip system;
11) with the FMI value and the contrast of cut-off value of every duplicate samples, it is positive to be higher than cut-off value person, and it is negative to be lower than cut-off value person; Wherein, described cut-off value is that average by the FMI of 33 parts of healthy human serums adds 2 times of standard deviations and gets.
Can detect hepatitis B, third liver, syphilis, acquired immune deficiency syndrome (AIDS) substance in the sample to be checked easily, efficiently simultaneously by method for detecting suspension chip of the present invention; Its testing result accurately and reliably, high specificity; But the little high throughput testing of amount of samples.
Description of drawings
Fig. 1 is the canonical plotting that single mass system detects HBsAg.
Embodiment
The present invention is a carrier with the plastics coding microball of purchasing in U.S. Luminex company (diameter is the plastics coding microball of 5.6 μ m), monoclonal hepatitis B surface antibody, HIV1 gP41 antigen, HCV NS3 antigen, syphilis TP15 antigen are wrapped respectively by on the microballoon of 4 kinds of different codings, and it is standby to make 4 kinds of suspending chips.
Method for detecting suspension chip of the present invention comprises following content:
At first prepare above-mentioned suspending chip, specifically may further comprise the steps:
1) activation coding microball:
1.1) at first getting 3000-5000 coding microball in the 1.5mL centrifuge tube, the centrifugal 3-5min of 12000-14000g is preferably the centrifugal 4min of 14000g, and careful sucking-off supernatant also discards;
1.2) adding 100 μ L pH 7.4 again, the PBS damping fluid of 0.05%TWEEN-20 suspends, shake after 30 seconds ultrasonic 30 seconds, the centrifugal 3-5min of 12000-14000g, the centrifugal 4min of preferable 14000g, carefully the sucking-off supernatant also discards;
1.3) then add 80 μ L 3g NaH
2PO
4, the microballoon of 5N NaOH 40drops/250mL H2O activation damping fluid shook after 30 seconds ultrasonic 30 seconds;
1.4) add the EDC of the 50mg/mL of the fresh configuration of 10 μ L again, and then add the Sulfo-NHS of the 50mg/mL of the fresh configuration of 10 μ L, shake at a high speed after 30 seconds with the aluminium foil parcel, jolt 700rpm in room temperature, 15-25min;
1.5) add the PBS of the pH7.4 of 150 μ L again, shake after 10 seconds, the centrifugal 3-5min of 12000-14000g, the centrifugal 4min of preferable 14000g, careful sucking-off supernatant also discards;
1.6) add the PBS of the pH7.4 of 150 μ L again, shake after 10 seconds, the centrifugal 3-5min of 12000-14000g, the centrifugal 4min of preferable 14000g, careful sucking-off supernatant also discards;
1.7) at last, adding the PBS suspended coding microballoon of the pH7.4 of 100 μ L, 300rpm is after 30 seconds in concussion, ultrasonic 15 seconds, finishes the activation of coding microball;
2) the bag quilt of activation coding microball:
2.1) get monoclonal hepatitis B surface antibody, HIV1 gP41 antigen, HCV NS3 antigen, syphilis TP15 antigen 5-50 μ g (the preferable 10 μ g that get) and join respectively in the microballoon of corresponding codes separately after the activation, PBS with pH7.4 is settled to 500 μ L from about 120 μ L, the aluminium foil parcel, after room temperature jolts 700rpm 2h, the centrifugal 3-5min of 12000-14000g, the centrifugal 4min of preferable 14000g, careful sucking-off supernatant also discards;
2.2) wash once with the PBS of the pH7.4 of 500 μ L, the centrifugal 3-5min of 12000-14000g, the centrifugal 4min of preferable 14000g, careful sucking-off supernatant also discards;
2.3) add the PBS of 500 μ L pH7.4,1%BSA, the sealing damping fluid suspended coding microballoon of 0.05%Azide again, 300rpm is after 15 seconds in concussion, with the aluminium foil parcel, jolts 30min in room temperature, the centrifugal 3-5min of 12000-14000g, the centrifugal 4min of preferable 14000g, careful sucking-off supernatant also discards;
2.4) then adding the PBS of 500 μ L pH7.4,1%BSA, 0.02%TWEEN, the microballoon of 0.05%Azide preserve liquid washing coding microball, the centrifugal 5-7min of 15000-17000g, the centrifugal 6min of preferable 16000g, careful sucking-off supernatant also discards;
2.5) preserve liquid suspended coding microballoon with the microballoon of 150 μ L at last, finish the bag quilt of activation coding microball.
Utilize above-mentioned suspending chip to detect the method for hepatitis B in the serum to be checked, third liver, syphilis, acquired immune deficiency syndrome (AIDS) substance, may further comprise the steps:
1) every hole adds the detection damping fluid of 150 μ L, and pre-wet hole is drained with vacuum pump;
2) every hole adds the working fluid that 50 μ L contain the corresponding encoded microballoon, adds 100 μ L washing lotions again, washes 2 times, drains with vacuum pump;
3) every hole adds 50 μ L serum to be checked, and blank well adds PBS, 700rpm 30 seconds, and the room temperature lucifuge jolts 300rpm 25-35min, drains with vacuum pump;
4) every hole adds 100 μ L washing lotions, washes 3 times, drains with vacuum pump;
5) it is anti-that every hole adds the biotinylation two of 50 μ L 1: 2500 (volume ratio), the antibody that is added biotinylated anti-surface antigen with monoclonal hepatitis B surface antibody bag by the hole of microballoon, 700rpm 30 seconds, room temperature lucifuge 300rpm 25-35min drains with vacuum pump;
6) every hole adds 100 μ L washing lotions, washes 3 times, drains with vacuum pump;
7) every hole adds the SA-PE of 50 μ L, 700rpm 30 seconds, and room temperature lucifuge 300rpm 8-12min drains with vacuum pump;
8) every hole adds 100 μ L washing lotions, washes 3 times, drains with vacuum pump
9) every hole adds the detection damping fluid of 125 μ L, 700rpm 30 seconds;
10) read FMI numerical value with the Bio-Plex suspension chip system;
11) with the FMI value and the contrast of cut-off value of every duplicate samples, it is positive to be higher than cut-off value person, and it is negative to be lower than cut-off value person; Wherein, described cut-off value is that average by the FMI of 33 parts of healthy human serums adds 2 times of standard deviations and gets.
Utilize this method to detect that antigen and detection of antibodies the results are shown in Table 1 in each sample:
Annotate :+positive ,-negative.
Now the sensitivity and the specificity of the inventive method are analyzed by following example or experiment:
One, sensitivity analysis: detecting HBsAg with single mass system is example, wrap by the 27# microballoon with monoclonal HBsAb, to detect with the HBsAg standard items and be diluted to 10 gradient concentrations by 1: 4 (volume ratio) equity, measure fluorescent value, its results are shown in Table 2 and accompanying drawing 1 (horizontal ordinate is represented concentration among the figure: μ g, ng, pg/ml; Ordinate is represented fluorescence intensity FI value):
Table 2 detects the FI value of HBsAg with No. 27 microballoons
According to following formula:
FI=-0.261878+ (6556.56+0.261878)/((1+ (Conc/1501.03) ^-1.33191)) ^0.803477 obtains sensitivity=2.9305ng/ml, can reach the nanogram level; The sensitivity of existing HBsAg ELISA detection kit can reach Gamma Magnitude, 1000 times of the highly sensitive detection sensitivities in ELISA that detection method of the present invention (suspension microballoon detection method) is described.
Two, specificity analyses:
Utilize the specificity analyses of 4 kinds of cause of diseases of detection method synchronous detection of the present invention (hepatitis B, third liver, syphilis, AIDS) as follows:
Specificity analyses (HCV) MFI of 4 kinds of cause of diseases of the multiple suspension microballoon of table 3 method synchronous detection
Through between variance analysis F group=17.088, F value table F looked into
0.01(n
13, n
221)=4.87 former P＜0.01, group difference has highly significant;
In the F group=0.94, F value table F looked into
0.01(n
17, n
221)=3.64 so P＞0.01, difference does not have conspicuousness or conspicuousness not obvious in the group; Difference to 4 groups compares in twos, tries to achieve the Q value, HCV group and other 3 groups Q value relatively, and Q1, Q2, Q3 are respectively 26.04,26.39,26.86; The Q value that other 3 groups compare in twos, Q4, Q5, Q6 are respectively 0.35,0.82,0.47; Look into Q value table, Q
0.01(20,4)=5.02, Q
0.05(20,4)=3.96, P＜0.01 that HCV group and other 3 groups compare in twos, and P＞0.01 that other 3 groups compare in twos, and＞0.05, prove that further the specificity of 4 kinds of microballoon synchronous detection HCV is more intense.
Specificity analyses (TP) MFI of 4 kinds of cause of diseases of the multiple suspension microballoon of table 4 method synchronous detection
Through between variance analysis F group=13.33, F value table (n looked into
13, n
221)=4.87 former P＜0.01, group difference has highly significant;
In the F group=1.57, F value table (n looked into
17, n
221)=3.64 so P＞0.01, difference does not have conspicuousness or conspicuousness not obvious in the group; Difference to 4 groups compares in twos, tries to achieve the Q value, the Q value that TP group and other 3 groups compare in twos, and Q1, Q2, Q3 are respectively 6.75,7.50,7.55; The Q value that other 3 groups compare in twos, Q4, Q5, Q6 are respectively 0.75,0.80,0.05; Look into Q value table, Q
0.01(20,4)=5.02, Q
0.05(20,4)=3.96, P＜0.01 that TP group and other 3 groups compare in twos, and P＞0.01 that other 3 groups compare in twos, and＞0.05, prove that more specifically the specificity of 4 kinds of microballoon synchronous detection TP is more intense.
Specificity analyses (HIV) MFI of 4 kinds of cause of diseases of the multiple suspension microballoon of table 5 method synchronous detection
Through between variance analysis F group=5.19, F value table (n looked into
13, n
221)=4.87 former P＜0.01, group difference has highly significant;
In the F group=2.56, F value table (n looked into
17, n
221)=3.64 so P＞0.01, difference does not have conspicuousness or conspicuousness not obvious in the group; Difference to 4 groups compares in twos, tries to achieve the Q value, the Q value that HIV group and other 3 groups compare in twos, and Q1, Q2, Q3 are respectively 4.43,4.44,4.76; The Q value that other 3 groups compare in twos, Q4, Q5, Q6 are respectively 0.008,0.33,0.33; Look into Q value table ,=Q
0.01(20,2)=4.02, Q
0.01(20,3)=4.64, Q
0.01(20,4)=5.02, Q
0.05(20,2)=2.95, Q
0.05(20,3)=3.58, Q
0.05P＜0.01 that compare in twos (20,4)=3.96, HIV group and other 3 groups or＜0.05, P＞0.01 that other 3 groups compare in twos, and＞0.05, prove that further the specificity of 4 kinds of microballoon synchronous detection HIV is more intense.
Specificity analyses (HBV) MFI of 4 kinds of cause of diseases of the multiple suspension microballoon of table 6 method synchronous detection
Through between variance analysis F group=13.88, F value table (n looked into
13, n
212)=5.95,, prove that the specificity of 4 kinds of microballoon synchronous detection HBV is more intense so P＜0.01 group difference has highly significant;
In the F group=9.02, F value table (n looked into
14, n
212)=5.41, so P＜0.01, difference has highly significant in the group, and the high low amplitude of data is bigger in the explanation group, and this experimental implementation precision has much room for improvement.Difference to 4 groups compares in twos, tries to achieve the Q value, the Q value that HBV group and other 3 groups compare in twos, and Q1, Q2, Q3 are respectively 6.86,7.12,8.13; The Q value that other 3 groups compare in twos, Q4, Q5, Q6 are respectively 0.26,1.26,1.01; Look into Q value table, Q
0.01(20,4)=5.02, Q
0.05(20,4)=3.96, P＜0.01 that HBV group and other 3 groups compare in twos, P＞0.01 that other 3 groups compare in twos, and＞0.05, prove that further the specificity of 4 kinds of microballoon synchronous detection HBV is more intense.
The comparison of 4 kinds of zymads of detection method of the present invention and traditional technique in measuring (HBsAg, HCV, HIV, TP), see the following form:
In summary, detection method of the present invention has high specificity, amount of samples is little, but high throughput testing, and it is quick to save time.
Claims (3)
1. suspending chip that hepatitis B, third liver, syphilis, acquired immune deficiency syndrome (AIDS) substance are carried out common inspection, it is characterized in that: this suspending chip is to be that the plastics colour code coding microball of 5.5～6.5 μ m is a carrier with diameter, Sheet clone hepatitis B surface antibody, HIV1 gP41 antigen, HCVNS3 antigen or syphilis TP15 antigen on this carrier.
2. method for preparing the described suspending chip of claim 1, it is characterized in that: this method may further comprise the steps:
1) activation coding microball:
1.1) at first get 3000-5000 coding microball in the 1.5mL centrifuge tube, the centrifugal 3-5min of 12000-14000g, careful sucking-off supernatant also discards;
1.2) adding 100 μ L pH 7.4 again, the PBS damping fluid of 0.05%TWEEN-20 suspends, shake after 30 seconds ultrasonic 30 seconds, the centrifugal 3-5min of 12000-14000g, carefully the sucking-off supernatant also discards;
1.3) then add 80 μ L 3g NaH
2PO
4, the microballoon of 5N NaOH 40drops/250mL H2O activation damping fluid shook after 30 seconds ultrasonic 30 seconds;
1.4) add the EDC of the 50mg/mL of the fresh configuration of 10 μ L again, and then add the Sulfo-NHS of the 50mg/mL of the fresh configuration of 10 μ L, shake at a high speed after 30 seconds with the aluminium foil parcel, jolt 700rpm in room temperature, 15-25min;
1.5) add the PBS of the pH7.4 of 150 μ L again, shake after 10 seconds, the centrifugal 3-5min of 12000-14000g, careful sucking-off supernatant also discards;
1.6) add the PBS of the pH7.4 of 150 μ L again, shake after 10 seconds, the centrifugal 3-5min of 12000-14000g, careful sucking-off supernatant also discards;
1.7) at last, adding the PBS suspended coding microballoon of the pH7.4 of 100 μ L, 300rpm is after 30 seconds in concussion, ultrasonic 15 seconds, finishes the activation of coding microball;
2) the bag quilt of activation coding microball:
2.1) get monoclonal hepatitis B surface antibody, HIV1gP41 antigen, HCV NS3 antigen, syphilis TP15 antigen 5-50 μ g and join respectively in the microballoon of corresponding codes separately after the activation, PBS with pH7.4 is settled to 500 μ L from about 120 μ L, the aluminium foil parcel, after room temperature jolts 700rpm 2h, the centrifugal 3-5min of 12000-14000g, careful sucking-off supernatant also discards;
2.2) wash once with the PBS of the pH7.4 of 500 μ L, the centrifugal 3-5min of 12000-14000g, careful sucking-off supernatant also discards;
2.3) add the PBS of 500 μ L pH7.4 again, 1%BSA, the sealing damping fluid suspended coding microballoon of 0.05%Azide, 300rpm is after 15 seconds in concussion, with the aluminium foil parcel, jolts 30min in room temperature, the centrifugal 3-5min of 12000-14000g, careful sucking-off supernatant also discards;
2.4) then adding the PBS of 500 μ L pH7.4,1%BSA, 0.02%TWEEN, the microballoon of 0.05%Azide preserve liquid washing coding microball, the centrifugal 5-7min of 15000-17000g, careful sucking-off supernatant also discards;
2.5) preserve liquid suspended coding microballoon with the microballoon of 150 μ L at last, finish the bag quilt of activation coding microball.
3. method of utilizing the described suspending chip of claim 1 to detect hepatitis B in the serum to be checked, third liver, syphilis, acquired immune deficiency syndrome (AIDS) substance is characterized in that this method may further comprise the steps:
1) every hole adds the detection damping fluid of 150 μ L, and pre-wet hole is drained with vacuum pump;
2) every hole adds the working fluid that 50 μ L contain the corresponding encoded microballoon, adds 100 μ L washing lotions again, washes 2 times, drains with vacuum pump;
3) every hole adds 50 μ L serum to be checked, and blank well adds PBS, 700rpm 30 seconds, and the room temperature lucifuge jolts 300rpm 30min, drains with vacuum pump;
4) every hole adds 100 μ L washing lotions, washes 3 times, drains with vacuum pump;
5) it is anti-that every hole adds 1: 2500 biotinylation of 50 μ L two, added the antibody of biotinylated anti-surface antigen with monoclonal hepatitis B surface antibody bag by the hole of microballoon, 700rpm 30 seconds, and room temperature lucifuge 300rpm25-35min drains with vacuum pump;
6) every hole adds 100 μ L washing lotions, washes 3 times, drains with vacuum pump;
7) every hole adds the SA-PE of 50 μ L, 700rpm 30 seconds, and room temperature lucifuge 300rpm 8-12min drains with vacuum pump;
8) every hole adds 100 μ L washing lotions, washes 3 times, drains with vacuum pump
9) every hole adds the detection damping fluid of 125 μ L, 700rpm 30 seconds;
10) read FMI numerical value with the Bio-Plex suspension chip system;
11) with the FMI value and the contrast of cut-off value of every duplicate samples, it is positive to be higher than cut-off value person, and it is negative to be lower than cut-off value person; Wherein, described cut-off value is that average by the FMI of 33 parts of healthy human serums adds 2 times of standard deviations and gets.
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