AU2021103744A4 - Time-resolved fluorescence immunochromatographic method and its application for detecting Nicarbazine - Google Patents

Time-resolved fluorescence immunochromatographic method and its application for detecting Nicarbazine Download PDF

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AU2021103744A4
AU2021103744A4 AU2021103744A AU2021103744A AU2021103744A4 AU 2021103744 A4 AU2021103744 A4 AU 2021103744A4 AU 2021103744 A AU2021103744 A AU 2021103744A AU 2021103744 A AU2021103744 A AU 2021103744A AU 2021103744 A4 AU2021103744 A4 AU 2021103744A4
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nicarbazine
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Xiao Liang
Wanpeng Yu
Qidi Zhang
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Qingdao Agricultural University
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Abstract

The present invention provides a time-resolved fluorescence immunochromatographic method and its application for detecting Nicarbazine. The method of the present invention uses Nicarbazine-coated antigen, Nicarbazine monoclonal antibody, and Eu-fluorescent nanospheres as the detection system. Using Eu-fluorescent nanoparticles as the tracer to label the antibody, the TRFIA immunochromatographic method of Nicarbazine was established. After a series of optimization of conditions, a standard curve of Nicarbazine was established. The IC50 of the method was 0.4ng/mL, linear detection The range is 0.1 1.3ng/mL, and there is no obvious crossover with other anticoccidial drugs, and the specificity is good. It was applied to the detection of Nicarbazine in chicken meat. The LOD was 1.4ptg/kg, the recovery rate was 77.3%~92.7%, and the coefficient of variation was less than 7.0%. The accuracy and precision of this method were good, and it could be used for the determination of Nicarbazine. Residual detection. The method of the invention has simple operation, high efficiency, and can complete detection in a short time. 1/2 FIGURES sample pad NC film T line C line absorbent pad (A) negative Immunochromatographicdirection " positive OuNP NICprub N' mNI j Al u~tcick# IgG .014 Y E APchenkc IgY probe CoaR Antigen C (hickCn IY ErUE&inospheC Figure 1 NIC 0 0.02 0.06 0.19 0.57 1.67 5 15 (ng/mL) Figure 2

Description

1/2
FIGURES
sample pad NC film T line C line absorbent pad
(A) negative
Immunochromatographicdirection
" positive
OuNP NICprub N' mNI j Al u~tcick# IgG .014 Y E APchenkc IgY probe CoaR Antigen C (hickCn IY ErUE&inospheC
Figure 1
NIC 0 0.02 0.06 0.19 0.57 1.67 5 15 (ng/mL)
Figure 2
Time-resolved fluorescence immunochromatographic method and its application
for detecting Nicarbazine
TECHNICAL FIELD
The invention belongs to the technical field of veterinary drug residue detection,
and specifically relates to a time-resolved fluorescence immunochromatographic
method and its application for detecting Nicarbazine.
BACKGROUND
Chicken is the second largest meat product in my country except pork, and
coccidiosis is a common and serious parasitic disease in the poultry breeding industry.
Nicarbazin (NIC) is a good medicine for preventing coccidiosis in chickens and
turkeys. It is widely used in the prevention and treatment of chicken and turkey
coccidiosis. It is composed of 4,4-dinitrophenylurea (4,4-Dinitrocarbanilide, DNC)
and 2-hydroxyl4,6-dimethylpyrimidine (2-hydroxyl4,6-dimethylpyrimidine, HDP)
composed of an isomolecular complex, of which DNC is a residual marker. Although
its toxicity is low, it has also been found to be toxic. At present, studies on the
physiological toxicity of NIC have shown that after long-term intake of 80 times the
normal dose, the rate of sperm aberration in male mice increases significantly. NIC
also has a certain impact on the reproductive performance of laying hens, so it is
forbidden to use Nicarbazine in laying hens clinically. Long-term consumption of
chicken meat with excessive Nicarbazine residues may pose potential risks to human
health. In recent years, the veterinary drug residue monitoring and testing data of the
Ministry of Agriculture of my country show that NIC is the only anticoccidial drug
that exceeds the standard among all the required drugs, and the positive rate of its detection is increasing year by year. Therefore, it is very important for chicken and other poultry by-products. NIC residue detection is very necessary.
The current methods for NIC residue detection mainly include instrumental
analysis and immunoassay detection methods. The instrumental analysis methods are
accurate, highly selective, fast and efficient, etc. However, the instrumental methods
are also expensive, time-consuming, and not suitable for grassroots on-site monitoring
and large-scale rapid screening and other shortcomings. The immunoassay method is
the most common detection method besides the instrument method. It is a rapid
detection and analysis technology with the basic characteristics of the specific binding
of antigen and antibody. It is characterized by strong specificity, short time, high
sensitivity, low cost, and high throughput. It can be applied to feed production,
breeding, livestock and poultry product circulation and sales, etc. It is currently
widely used in rapid detection of drug residues in animal foods. For example, 3
stimulants, sulfonamides, chloramphenicol, aminoglycosides, quinolones and other
drugs that have received widespread international attention have all established rapid
immunoassay detection methods. However, so far, there have been few reports on the
analysis methods of NIC residues in animal foods using immunoassay methods at
home and abroad, and it is difficult to meet the detection requirements of actual
sample screening for NIC.
At present, the main immunoassay methods for detecting NIC residues mainly
include enzyme-linked immunoassay (ELISA), immunoassay chromatography
method, surface plasmon resonance (SPR) and so on. Among them, most literature
reports are based on ELISA detection methods. The ELISA method is a solid-phase
immunoassay method. The detection process requires multiple separation and washing steps, which takes a long time. The entire reaction step takes 1.5h or even up to 2h to complete.
Therefore, it is imperative to establish a fast, sensitive and simple method for
detecting NIC residues. It is of great significance for our country to realize the rapid
screening and detection of NIC residues in animal food and its standardized use in
livestock and poultry production.
Time-resolved fluorescence immunochromatography (TRFIA) is developed on
the traditional colloidal gold immunochromatography technology. Compared with
traditional fluorescein labeling, it uses lanthanides and their chelates with unique
fluorescence characteristics. It has the advantages of high sensitivity, good specificity,
and no pollution of radioactive elements. Therefore, it is of great significance to
establish a TRFIA method for detecting NIC residues.
SUMMARY
Aiming at the disadvantages of the above-mentioned existing detection of NIC
immune technology such as complicated operation and time-consuming, the present
invention establishes a time-resolved fluorescence immunochromatographic method
for detecting Nicarbazine. The method is simple in operation, high in efficiency, and
can be detected in a short time. With good accuracy and precision, it can be used for
the residual detection of Nicarbazine.
Based on the above objective, the present invention provides a time-resolved
fluorescence immunochromatographic method for detecting Nicarbazine, the steps are
as follows:
(1) Preparation of time-resolved fluorescent microsphere-conjugated antibody
probes: Europium-fluorescent nanospheres with carboxyl groups on the surface are
used to activate the carboxyl groups on the surface of the microspheres by the active
ester method, and react with the amino groups on the surface of the nicarbazine
monoclonal antibody. Valence-bound Eu-nanosphere probe;
(2) Preparation of time-resolved immunochromatographic test strips: spray
Nicarbazine antigen DNC-4-BSA and goat anti-chicken IgG on the NC membrane in
sequence with the detection line T line and the quality control line C line position.
The spacing is 3mm in turn. Dry the drawn NC film, and stick the sample pad and
absorbent pad to the PVC bottom in turn; the sample pad and absorbent pad press the
NC film 2mm at both ends; cut into test papers with a width of 4mm Strip, installed in
the plastic card shell and assembled into a test card;
(3) Detection: add the sample to the microwell, then add the Eu-nanosphere
probe prepared in step 1, pipetting and mixing, and incubate at room temperature;
aspirate the mixed solution in the microwell, add the sample hole of the test card, and
chromatograph After that, the result is judged.
The result judgment includes:
Qualitative determination method: observe under the ultraviolet instrument, if the
T and C lines have colors, it is a negative sample that does not contain NIC; if the C
line has no color and the T line is colored, it is a positive sample that contains NIC;
Quantitative determination method: use a time-resolved immunoquantitative
analyzer to measure the fluorescence intensity under the T and C lines. T/C is the
ordinate, and the concentration of the NIC standard substance is added as the abscissa
to establish a standard curve for quantitative detection.
The specific method of step(1) for preparing time-resolved fluorescent
microsphere-coupled antibody probes is: adopt a one-step coupling method, that is,
after washing europium-fluorescent nanospheres with activation buffer, add EDC and
NHS for activation, and then activate again. Nicarbazine monoclonal antibody is
added for coupling, and the Eu-nanosphere probe is obtained by re-dissolving after
sealing and centrifugation. The dosage ratio of the europium-fluorescent nanospheres
and the Nicarbazine monoclonal antibody is 5:2.
The establishment of the standard curve: according to the standard gradients of 0,
0.07, 0.19, 0.56, 1.67, 1.37, 5, 15 ng/mL, establish the standard curve, repeat each
gradient 3 times, take the average value, and add the concentration as the abscissa,
take the T/C fluorescence ratio as the ordinate, fit the standard curve, calculate the IC
value and determine the linear range, the specific steps:
(1) Take 0.4uL Eu-nanosphere probe and dissolve it in 120L Nicarbazine
standard solution of different concentrations, and repeatedly pipette until all the white
solids in the micropores are dissolved;
(2) Incubate for 1 min at room temperature;
(3) Take 100tL of liquid in the microwell, add it to the sample pad at the bottom
of the test strip, and react accurately for 8 minutes;
(4) Insert the test strip into the reading of the time-resolved immunofluorescence
analyzer, calculate the NIC drug content in the sample, and establish a standard curve.
The IC 50 of the standard curve NIC established by the present invention is 0.43
ng/mL, the LOD is 0.07 ng/mL, and the detection range is 0.13-1.37 ng/mL.
The sample is the solution after the chicken is pre-treated; the method of the
chicken pre-treatment: weigh 2.00±0.05g homogenized chicken sample into a 10mL
centrifuge tube, add 2mL acetonitrile, vortex for 10min, centrifuge at 8000rpm for
min, take it Dilute 5 times with borate buffer solution.
In the time-resolved fluorescence immunochromatography method for detecting
Nicarbazine, the Nicarbazine monoclonal antibody used is diluted 10 times as the
working concentration, and the Nicarbazine antigen DNC-4-BSA used is diluted 2
times as the working concentration.
The present invention also provides the application of the time-resolved
fluorescence immunochromatographic method for detecting Nicarbazine in the
detection of the content of chicken meat or the preparation of the time-resolved
fluorescence immune reagent card for detecting Nicarbazine.
The present invention also provides a time-resolved fluorescence immunoassay
reagent card for Nicarbazine, which includes a bottom plate and a sample pad placed
thereon, a nitrocellulose membrane and a water-absorbing pad. The nitrocellulose
membrane uses Nicarbazine antigen DNC- 4-BSA and goat anti-chicken IgG are
assigned. The scribing method is: spraying the detection line T line and the quality
control line C line on the NC film with a scribing machine at a spraying speed of 0.7
[tL/cm. Position and spacing are 3mm in turn. Place the marked NC film in an oven at
37°C overnight.
At present, there are few reports on Nicarbazine Residue
Immunochromatography Method. In 2004, Hargen adopted a new one-step dry
chemistry method to establish a TRFIA method for Nicarbazine Residue in Liver and
Eggs. However, this method has a larger matrix effect and egg samples. Dilute 100 times and liver samples 200 times. The detection time takes 18 minutes. In 2018, Wu et al. established Nicarbazine Residual Colloidal Gold Immunochromatography. The test sample is milk. The matrix effect can be eliminated by diluting 10 times. The detection limit in the sample is 8.6ng/mL, and the cutoff value is 100ng/mL. In contrast, the TRFIA method established by the present invention is simpler and more sensitive.
In summary, the time-resolved fluorescence immunochromatography method for
detecting Nicarbazine established by the present invention has the following
beneficial effects compared with the above-mentioned existing methods:
(1) The present invention uses Eu-fluorescent nanoparticles as a tracer to label
antibodies, and establishes a TRFIA method for Nicarbazine detection. After a series
of optimization of conditions, a standard curve of Nicarbazine was established. The
IC50 of the method is 0.4ng/mL, the linear detection range is 0.13-1.37ng/mL, and
there is no obvious crossover with other anticoccidial drugs, and the specificity is
good.
(2) The method of the present invention is applied to the detection of
Nicarbazine in chicken, the LOD is 1.4 [g/kg, the recovery rate is 77.3%-92.7%, and
the coefficient of variation is less than 7.0%, indicating that the method of the present
invention has good accuracy and precision. , Can be used for the residual detection of
Nicarbazine.
(3) The TRFIA method established by the present invention is simpler and more
sensitive. The detection time is 9 minutes, the sensitivity and precision meet the
detection requirements, and it can be used for the residual detection of Nicarbazine.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a schematic diagram of the structure of a time-resolved fluorescent
immunoassay reagent card for Nicarbazine of the present invention and the TRFIA
chromatography process of Nicarbazine of the present invention; (A) negative result;
(B) positive result.
Figure 2 is a graph showing the chromatographic results of the NIC standard
curve in the TRFIA assay buffer in Example 2 of the present invention.
Figure 3 is the standard curve of NIC in the TRFIA assay buffer in Example 2 of
the present invention.
Figure 4 shows the matrix curve used in Example 2 for chicken meat detection.
DESCRIPTION OF THE INVENTION
The present invention will be described below through specific embodiments,
but the present invention is not limited thereto. The experimental methods used in the
following examples are conventional methods unless otherwise specified; the reagents,
biological materials, etc. used, unless otherwise specified, can be obtained from
commercial sources.
Example 1. Establishment of a time-resolved immunoassay method for the
detection of Nicarbazine in the present invention
1. Material
Nicarbazine, goat anti-chicken IgG polyclonal antibody, chicken IgY, Proclin
300, europium-fluorescent nanospheres (particle size 210nm), bovine serum albumin
(BSA, 99.9%), nitrocellulose membrane, PVC bottom plate, water absorption Pads,
etc. are all purchased commercially.
The DNC hapten, DNC antigen and Nicarbazine monoclonal antibody were gifts
from China Agricultural University; the DNC hapten (in the present invention is
represented by DNC-4), DNC antigen (in the present invention is represented by
DNC-4-BSA) and Nicarbazine Carbachol monoclonal antibody (represented by NIC
specific antibody 3B4 in the present invention) is described in patent number
ZL201910536490.X, named 4,4'-dinitrophenylurea hapten and artificial antigen, and
the invention of its preparation method and application Patent pending.
The DNC hapten DNC-4 is 4,4'-dinitrophenylurea hapten, and its structural
formula is shown in the following formula (I):
COOH
N02 0'
H H formula (1)
The DNC antigen (DNC-4-BSA) is obtained by coupling 4,4'-dinitrophenylurea
hapten and bovine serum albumin. Nicarbazine monoclonal antibody is prepared by
immunizing experimental animals (mice) with DNC-BSA artificial antigen using
hybridoma technology. The method used is a common method in the field.
Solution system:
(1) Coating buffer
0.0lmol/L , PH7.4 phosphate buffer
0.05mol/L,
0.01moLL Naj HPOj •2H 0.290g , PH7.4 NaH2POJ 2HD 0.024 g phosphate NaCI 0.800g buffer KCI 0.020g deIonized water dilute to 100mL .05rnomi, NaHCO, 0.293 9Na6CO 0.159 carboate coating deionized water dilute toIOOmL buffer
(2) Activation buffer: 0.05mol/L, pH 6.0 MES buffer
Morpholine ethanesulfonic acid 0.975g
deionized water dilute to 100mL
(3) Coupling buffer: 0.04mol/L, pH 8.0 Boric acid borax buffer (BB)
Na2B407 0.457g
H3B03 0.692g
deionized water dilute to 400mL
(4) Blocking buffer: 20% BSA
BSA 1og
deionized water dilute to 50mL
(5) Reconstituted solution:
I#: BSA I g deioized water 3g Prolin 100 2 pL
0.02M borate buffer dilute to 100mIL 2#: BSA Ig PEG 2000 0.05 g Prcn 300 21pL 0M M PBS (pH 74) dilute to 1OOmL 3# Tris *605g BSA S sucrose l0g trehalos;elg Prolin 300 10 pL deionized water dilute to 500mL
(6) Secondary antibody diluent:
BSA Ig Pr11in 300 20p.I 0.01 IPBS(pi17.4) dilute to 100mL
2 Test method
2.1 Preparation of time-resolved fluorescent microsphere conjugated antibody
probe
The Eu-time-resolved nanospheres (particle size 210nm) modified with carboxyl
groups on the surface are used to activate the carboxyl groups on the surface of the
microspheres by the active ester method, and react with the amino groups on the
antibody surface to form covalently bound Eu-nanosphere probes. Specific steps are
as follows:
(1) Wash the microspheres: take 20L of microspheres into a 1.5mL EP tube,
add 180pL of activation buffer, vortex for 5s, centrifuge 20000g, centrifuge for 15min,
and discard the supernatant.
(2) Activation: add 160 L activation buffer, sonicate for 2 min using an
ultrasonic instrument, add 20 L each of EDC and NHS, activate on a horizontal
shaker at room temperature for 15 min; then centrifuge at 20,000 g for 15 min, and
discard the supernatant.
(3) Wash the microspheres again: add 200L of coupling buffer (iX) to
reconstitute the microspheres, sonicate for 2min with an sonicator, divide into 4 EP
tubes, each 50[L, centrifuge 20,000g, centrifuge for 15min, discard the supernatant.
(4) Conjugation: add 200uL of coupling buffer to reconstitute the microspheres,
sonicate for 2min using an sonicator, divide into 4 EP tubes, each 50tL, centrifuge
,000g, centrifuge for 15min, discard the supernatant. Add 25uL of coupling buffer
to reconstitute the microspheres, sonicate for 2min, centrifuge for 5s, add the
reconstituted microspheres to 25L of diluted antibody (2 g) and mix well by
pipetting and mixing, at room temperature, on a horizontal shaker for 2h. Centrifuge
at 15000g for 15min, and discard the supernatant.
(5) Blocking: use 100pL of blocking buffer to reconstitute the microspheres,
sonicate for 2min in a sonicator, centrifuge for 2s in a handheld centrifuge, at room
temperature, on a horizontal shaker for 2h (or 4°C, overnight blocking); centrifuge at
15000g for 15min, discard the supernatant.
(6) Reconstitution: Add 50tL of reconstitution buffer to reconstitute the
microspheres, ultrasonic for 2min, centrifuge at 15000g, centrifuge for 15min, discard
the supernatant; again use 50tL of reconstitution buffer to reconstitute the
microspheres, ultrasonic for 2min, Centrifuge in a palm centrifuge for 2 seconds and
store at 4°C.
2.2 Establishment of NIC time-resolved immunoassay method
2.2.1 Preparation of time-resolved immunochromatographic test strips
DNC-4-BSA (1.5mg/mL) and goat anti-chicken IgG (0.2mg/mL) were sprayed
on the detection line (T line) and quality control on the NC film with a scribing
machine at a spray rate of 0.7 [L/cm. Line (C) line position, the spacing is 3mm in
turn, place the drawn NC film in an oven at 37°C overnight. Stick the dried NC film,
sample pad and absorbent pad on the PVC bottom plate in turn. The sample pad and
the absorbent pad are pressed against the NC film by 2 mm at both ends. The structure
of the chromatographic test strip is shown in Figure 1. Cut the processed slabs into
test strips with a width of 4.0mm with a strip cutter, install them in a plastic card case
and assemble them into a test card, and then place them in a dry and closed aluminum
foil bag for use.
2.2.2 Detection steps of time-resolved immunochromatographic test strips
Add the sample to be tested into the microwell, add a certain amount of Eu
nanosphere probe to the sample with a pipette, pipette to mix, and after incubating at
room temperature, take the mixed solution, add it to the spot hole of the test card, and
chromatograph. Make the result judgment.
Qualitative determination method: observe under the ultraviolet instrument. If
the T and C lines have colors, it is a negative sample that does not contain NIC; if the
C line has no color and the T line is colored, it is a positive sample that contains NIC.
The specific process is shown in Figure 1.
Quantitative determination method: use a time-resolved immunoquantitative
analyzer to measure the fluorescence intensity under the T and C lines. T/C is the ordinate, and the concentration of the NIC standard substance is added as the abscissa to establish a standard curve to achieve the purpose of quantitative detection.
2.3 Optimization of NIC time-resolved immunoassay method
Use the fluorescence intensity and inhibition rate of the control and spike buffer
to select the best parameters. Inhibition rate is calculated by the following formula:
Inhibition rate=(1-T/T 0 )x100% (T 0 and T are the fluorescence intensity of the
control and spiked buffer respectively), the concentration of NIC in the spiked buffer
is 5ng/ mL, the higher the inhibition rate, the more sensitive.
2.3.1 Selection of the amount of time-resolved fluorescent microspheres
Take 2.5, 5, 10, and 20 [L of time-resolved fluorescent microspheres,
respectively, and the coupling method is the same as above. Choose the best time
resolved amount of fluorescent microspheres according to the color development and
inhibition of the test strip under the UV instrument.
When the amount of time-resolved fluorescent microspheres gradually increased
from 2.5tL to 20jL, the fluorescence intensity of the T line also gradually increased.
When the amount of microspheres increased to 20L, the fluorescence intensity
became lower. When using 20 L microspheres to couple antibodies, the microspheres
are in an excessive state, and the fluorescence intensity is not only lower than the
coupling effect of 10 L microspheres, but also the chromatography is incomplete, the
background value increases, and the T-line boundary is not obvious. When 10 L of
microspheres are used to couple antibodies, the fluorescence intensity is significantly
higher, and the inhibitory effect is also significantly reduced. When 5 L of
microspheres are selected for antibody coupling, the fluorescence intensity and inhibition rate are both higher than the coupling effect of 2.5 L of microspheres, so 5 pL of microspheres is chosen for subsequent coupling.
2.3.2 Selection of EDC/NHS Dosage
Mix EDC and NHS into a 1.0 mg/mL mixed solution, add 2.5, 5, 10, and 20 L
of activated Eu-nanospheres respectively. The rest of the labeling steps are the same
as above. After coupling with the antibody, use the concentration of 0 and 5ng/mL of
the NIC standard product respectively and test it, the fluorescence intensity of the T
line and C-line of the test strip and the release of the antibody probe are observed
under the ultraviolet lamp, the fluorescence value is measured and the inhibition rate
is calculated.
EDC and NHS mainly activate the carboxyl group on the microsphere and
couple it with the amino group of the antibody, which can directly affect the coupling
efficiency of the antibody and the time-resolved fluorescent microsphere. The results
showed that when 20 L of activator was added, the chromatography was incomplete.
When the amount of EDC is too much, the antibodies of the system will be self
coupling, causing aggregation. Adding 10 L of activator, although the T-ray
fluorescence intensity increased and the coupling efficiency increased, the inhibition
rate decreased. The inhibition rate is obviously better when 2.5tL of activator is
added, so the best dosage of EDC/NHS is chosen to be 2.5[tL.
2.3.3 Orthogonal test of antibody and antigen
The antibodies were diluted 5, 10, 20, and 40 times respectively, and probes
were prepared by coupling with the microspheres according to the aforementioned
method. Screening is performed according to the color development intensity and
inhibition rate of the test strips. Then DNC-4-BSA was diluted 2, 4, 8 and 16 times, respectively, and sprayed on the NC membrane with a scribing machine to prepare test strips, and then the best antigen coating concentration was selected according to the color development intensity and inhibition rate.
The results are shown in the following Tables 1 and 2. As the dilution factor of
the antibody and the coating agent gradually increased, the fluorescence intensity of
the T line gradually decreased, and the inhibition rate gradually increased. However,
when the antibody dilution factor is less than 10 times, and the antigen dilution factor
is less than 2 times, the fluorescence intensity drops significantly. In terms of color
intensity and inhibition rate, the best conditions are when the antibody is diluted by 10
times and the antigen is diluted by 2 times.
Table 1 The effect of the amount of antibody on TRFIA
antibodydilutionfactor fhwrescereintesityoftheTline 5rg/gstandardinlibitionrate(%)
5X 52342 93.12
loX 24037 98.74
20X 7787 100.00
40X 435 100.00
Table 2 The effect of the amount of antigen on TRFIA
artibo dy dilution fador flurescerre intesityofteTline 5ng/g standard inhibition rate(%)
2X 25858 98.4
4X 6050 1000 X 2967 75.85
16X 298 1JAX)
2.3.4 Selection of probe dosage
The amount of fluorescent probe was 0.8[L, 0.6tL, 0.4tL, and 0.24L, and
screened according to the color intensity and inhibition rate of the test strip.
The results showed that with the decrease in the amount of Eu-nanosphere probe,
the fluorescence intensity of T-line gradually decreased, and the inhibition rate also
gradually increased. When the amount of Eu-nanosphere probe was less than 0.6[tL,
the line disappeared. The fluorescence intensity is controlled at around 12,000.
Therefore, we finally chose to add 0.4 LEu-nanomicrosphere probe to each sample
for subsequent experiments.
2.3.5 Choice of coating fluid
Dilute the coated antigen DNC-4-BSA with 0.02MPB (pH7.4), CB (pH9.6) and
0.02MPBS (pH7.4) according to the determined optimal antigen coating
concentration, spray the membrane, and detect the fluorescence intensity.And
calculate the inhibition rate to determine the best coating solution.
The results show that the fluorescence intensity of coating with PBS is the worst,
and the fluorescence intensity of the coating liquid PBS and CB is not much different,
but when PB is used as the coating liquid, the inhibition rate is the best, and PB is
selected as the best coating liquid.
2.3.6 Selection of NC film
Choose 4 commonly used NC membranes (Sartorius 95, Millipore 135, MDI 90
and Vivid 170), respectively scribing the membranes to assemble test strips, observe
the fluorescence intensity and the release of microspheres, and calculate the inhibition
rate to determine the best NC membrane model. NC membrane mainly affects the sensitivity of the test through membrane pores and protein binding force. The results showed that except for Millipore 135 membrane, the inhibition rate of the other three membranes was the same. But the fluorescence intensity of MDI 90 film is the lowest,
V film is second, and Sartorius 95 film has the best fluorescence intensity. So choose
Sartorius 95 membrane.
2.3.7 Selection of sample pad material
Three materials including SB08, blood filter and RB65 were selected,
respectively treat with the best sample pad treatment solution determined above, dry
and assemble test strips, observe the fluorescence intensity and the release of
microspheres, and calculate the inhibition rate to determine the best sample pad
material. The sample pad mainly affects the immunochromatographic results through
the chromatographic speed and release effect. The results showed that when the blood
filter membrane was used as the sample pad, the whole sample pad was reddish, the
release was incomplete and the chromatography speed was very slow, resulting in
very low fluorescence intensity. The release effect of RB65 is the second, the sample
pad is still red, and the release is not complete; SB08 is the best, the release effect is
the best, and the fluorescence intensity and inhibition are the best in comparison. So
choose SB08 as the sample pad.
2.3.8 Selection of probe compound solution
The common components of the complex solution, such as 0.02 MPB(pH7.4),
0.05 MBB(pH8.0), 0.05M Tris-HCl(pH9.0), surfactants (Tween-20, TritonX-100,
PVP), sucrose, trehalose and BSA, were made into three groups according to different
components, which were re-dissolved in the microsphere probe and tested The results
show that the fluorescence intensity of 1# complex solution is the strongest, while that of 2# and 3# complex solution is equivalent, but the inhibition rate of 2# is high, so 2# complex solution is chosen as the best complex solution.
2.3.9 Selection of sample diluent
Dilute the NIC standard respectively with 0.01M PBS, sample 1 and sample 2,
to detect the effects of different dilutions on the color and sensitivity of the test strips,
and determine the best diluent composition. The results show that PBS will affect the
release of Eu-nanosphere probes, and the background value is high.
2.3.10 Incubation time
After adding the sample solution and fluorescent probe to the microwells and
mixing them, incubate for 0, 1, 2, 3, 4, and 5 minutes, respectively, and then perform
immunochromatographic detection. Determine the best incubation time according to
the inhibition. Before loading the sample in immunochromatography, the standard and
Eu-nanosphere probe are fully reacted, which can make the inhibition more sufficient
and improve the sensitivity of the method. The research results show that the
incubation time has no major influence on the fluorescence intensity and inhibition,
and the best choice is to incubate for 1 min.
2.3.11 Color development time
After adding the sample, start from the 2nd minute, measure the fluorescence
ratio (T/C) of the test strip every 1 minute, and determine the time required for the test
strip reaction to balance as the best reaction time for the test strip.
The immunochromatographic time has a great influence on the results of the
method. When the immunochromatographic time is short, the probe in the solution is
not enough to fully react with the T line and C line, resulting in low color intensity; when the reaction time is too long, The probe in the solution is in full contact with the
T and C lines, resulting in too deep color development, which will affect the
inhibition and reduce the sensitivity of the method. In this experiment, the results
showed that the fluorescence intensity gradually deepened with the increase of time,
the inhibition situation was relatively stable in the first 8 minutes, and the inhibition
situation began to decrease gradually at 9 minutes. Choose 8min as the
chromatography time, at this time the fluorescence intensity is relatively high, and the
sensitivity is good.
Example 2: Evaluation of the performance of the time-resolved immunoassay
method for detecting Nicarbazine in the present invention
1 Establishment of standard curve in buffer
Using the above optimized conditions, establish a standard curve with standard
gradients of 0, 0.07, 0.19, 0.56, 1.67, 1.37, 5, 15 ng/mL, repeat each gradient 3 times,
take the average value, and add the concentration as On the abscissa, take the T/C
fluorescence ratio as the ordinate, fit the standard curve, calculate the IC 50 value and
determine the linear range.
The final determination of TRFIA's testing steps are as follows:
(1) Take 0.4tL of Eu-nanosphere-labeled antibody and dissolve it in 120tL of
standard solution of different concentrations, and repeatedly pipette until all the white
solids in the microwells are dissolved;
(2) Incubate for 1 min at room temperature;
(3) Take 100 L of liquid in the microwell, add it to the sample pad at the bottom
of the test strip, and react accurately for 8 minutes;
(4) Insert the test strip into the reading of the time-resolved immunofluorescence
analyzer, calculate the NIC drug content in the sample, and establish a standard curve.
After chromatography, the cut-off value is 5ng/mL by visual observation as
shown in Figure 2. As shown in Figure 3, the IC 50 of the method for the NIC is
0.43ng/mL, the LOD is 0.07ng/mL, and the detection range is 0.13-1.37ng/mL,
respectively.
2 specificity
Gradient addition of NIC and its structural analogue standards to the final
optimized buffer system, use test strips to detect, draw a standard curve, determine the
IC 50 value and calculate the cross-reaction rate. The results are shown in Table 3.
The cross-reaction rate of the method with other drugs is less than 0.1%, and there is
no cross-reaction with other structural analogs.
Table 3 Test results of TRFIA cross-reaction rate of the present invention
diugs structure C rnglmt c~a&-embon rite. (%)
O-Nitroamline io1
4-nitropropionic acid V <oa
H-iArohe-PNA l J1
4-ifitroplienethylanaine I ~irI ~ u
N-methyl-4-nitrophenethylamine no rMi
L-alanime-4-nitroanilme um140) Ai
k
N.N-dimethvl-4nitroaniline Do eI
L-glutmic acid p-nixoamlide --. i «
NKal ie It,
toltrazuril inq .1'
diphenylamie .. I
ronidazole som II~x I
3,5-dinaitrosaminae -uj I
3 Detection in chicken samples
3.1 Sample pretreatment
Method 1: Weigh 2.00±0.05g homogenized chicken sample into a 50mL
centrifuge tube, add 8mL acetonitrile, quickly vortex for 5min for extraction, sonicate
for 10min, and centrifuge at 5000rpm for 10min. Take 4 mL of the supernatant and
blow dry with nitrogen at 40-50°C. Add 1 mL of n-hexane to the residue, add 1 mL of
% methanol aqueous solution, vortex to mix, then centrifuge at 2000 rpm for 10
minutes, remove the lower layer of matrix solution and dilute it with buffer by 5 times, and use the established TRFIA method for detection and determination Then compare with the standard curve established in the buffer solution.
Method 2: Weigh 2.00±0.05g homogenized chicken sample into a lOmL
centrifuge tube, add 2mL acetonitrile, vortex for 10min, centrifuge at 8000rpm for
min, take the supernatant and dilute 5 times with BB buffer for TRFIA detection.
The concentrations of NIC added to the blank chicken samples were 1.5, 4, 10
pg/kg and 6, 16, 30 [g/kg, and each sample was repeated 5 times to calculate the
recovery rate, intra-assay variation and inter-assay variation of the method.
First, several organic reagents commonly used in the processing of chicken
samples were selected, and the sample processing was initially explored. After the
sample is processed, the matrix is uniformly diluted by 5 times, and the test reading is
performed. The results in Table 4 show that in addition to acetonitrile, the
fluorescence intensity drops sharply after processing the samples with other organic
solvents. Acetonitrile was preliminarily selected for subsequent treatment, and the
matrix after treatment with acetonitrile diluted 5 times still had a certain matrix effect,
so two pre-treatment methods were carried out with acetonitrile. Method 1, extract
with acetonitrile, degrease with n-hexane after nitrogen blowing, and dilute the matrix
solution 5 times; Method 2 extract directly with acetonitrile, and dilute the matrix 20
times. The two matrix curves obtained are shown in Figure 4, which are basically
consistent with the standard curve in the buffer, which can effectively reduce the
matrix effect and can be applied to actual detection. The IC 50 in the matrix curve is
0.714[tg/kg and 0.523tg/ respectively. kg. The LODs of the two methods for NIC in
chicken were 0.4jag/kg and 1.4jag/kg, and the detection ranges were 0.9-13.9ag/kg and
2.96-37.06jag/kg, respectively.
Table 4 Different methods of processing chicken samples
sample processing method dilution times fluorescence intensity of T line Bukr - iK23
acetonitrile 5 7707
methanol 5 3774
acetonitrile trichloroaeticacid (1: 3) 5 40 acetonifile gchloroacetacid (1: 1) 5 179
acetonitileAMichloroaccaid (3: 1) 5 25H4 ethyl acetate 5 0 dichloromethane 0
3.2 Adding recovery determination
Add targets of different concentrations to the chicken to determine the accuracy
and precision. The results are shown in Table 5 below. The recovery rate of method
one NIC is between 77.7%-93.9%, and the coefficient of variation is less than 14.9%;
the recovery rate of method two NIC is between 77.3%-92.7%, and the coefficient of
variation is less than 7.0%. It shows that the accuracy and precision of this method are
good. Taking into account the importance of time in rapid detection, the final choice
is method two, direct extraction with acetonitrile.
Table 5 Recovery rate of NIC addition in chicken sample extraction add amount intra-assav (n=33 mira-assay (n=3) method Fwp measurearecoveryrate V measured recoveryrate v chicken Method 1 '-5 .ils* A A. ir -* 4+34. 5.6% 4 O4.910in C73& 4 2A 2.N% .7ninu n.A%L5.9 A7%~ in K l lt 14.!AYkr4£A12. h.7tnta9 h3.5% I33 & IdII M1.
Method 2 6 a: ±s "1. 77. Y, I2.P 0-24 O±W m3.2 4.0% r6 aisa.403 I Al e tA% 4l+01 MM.%3 3.
% 3 7 1.751004 97.0%±6.1 f1% i35±0.(13 92.7%i.0 S.4%

Claims (10)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:
1. A time-resolved fluorescence immunochromatographic method for detecting
Nicarbazine, which is characterized in that the steps are as follows:
(1) Preparation of time-resolved fluorescent microsphere coupled antibody probe:
Europium-fluorescent nanospheres with carboxyl modification on the surface were
used to activate carboxyl on the surface of microspheres by active ester method, and
reacted with amino on the surface of nicarbazine monoclonal antibody to form
covalently bonded Eu- nanospheres probe.
(2) Preparation of time-resolved immunochromatographic test strips: spray
Nicarbazine antigen DNC-4-BSA and goat anti-chicken IgG on the NC membrane in
sequence with the detection line T line and the quality control line C line position.
Dry the marked NC film, and stick the sample pad and absorbent pad to the bottom
plate in turn; the sample pad and absorbent pad press the NC film 2mm at both ends;
cut into test strips with a width of 4mm and install them on the plastic card shell
Assemble it into a test card;
(3) Detection: add the sample to the microwell, then add the Eu-nanosphere
probe prepared in step 1, pipetting and mixing, and incubate at room temperature;
aspirate the mixed solution in the microwell, add the sample hole of the test card, and
chromatograph After that, the result is judged.
2. The time-resolved fluorescence immunochromatography method for detecting
Nicarbazine according to claim 1, the result determination comprises:
Qualitative determination method: observe under the ultraviolet instrument, if the
T and C lines have colors, it is a negative sample that does not contain NIC; if the C line has no color and the T line is colored, it is a positive sample that contains NIC;
Quantitative determination method: use a time-resolved immunoquantitative
analyzer to measure the fluorescence intensity under the T and C lines. T/C is the
ordinate, and the concentration of the NIC standard substance is added as the abscissa
to establish a standard curve for quantitative detection.
3. The time-resolved fluorescence immunochromatography method for detecting
Nicarbazine according to claim 1, the specific method for preparing the time-resolved
fluorescent microsphere-conjugated antibody probe in the step (1) is: adopting one
step coupling Combined method, that is, after washing the europium-fluorescent
nanospheres with activation buffer, adding EDC and NHS for activation, after
activation, adding Nicarbazine monoclonal antibody for coupling, sealing and
centrifugation, and re-dissolving to obtain Eu-nanomicrospheres needle.
4. The time-resolved fluorescence immunochromatography method for detecting
Nicarbazine according to claim 1 or 3, wherein the dosage ratio of the europium
fluorescent nanospheres and the Nicarbazine monoclonal antibody is 5 :2.
5. The time-resolved fluorescence immunochromatographic method for detecting
Nicarbazine according to claim 2, characterized in that the establishment of the
standard curve: press 0, 0.07, 0.19, 0.56, 1.67, 1.37, 5. l5ng/mL standard product
gradient, establish a standard curve, repeat each gradient 3 times, take the average
value, take the added concentration as the abscissa and the T/C fluorescence ratio as
the ordinate, fit the standard curve, and calculate the IC 50 value and determine the
linear range, the specific steps are as follows:
(1) Take 0.4pL of Eu-nanomicrosphere probe and dissolve it in 120IL of
Nicarbazine standard solution of different concentrations, and repeatedly pipette until all the white solids in the microwells are dissolved;
(2) Incubate for 1 min at room temperature;
(3) Take 100pL of liquid in the microwell, add it to the sample pad at the bottom
of the test strip, and react accurately for 8 minutes;
(4) Insert the test strip into the reading of the time-resolved immunofluorescence
analyzer, calculate the NIC drug content in the sample, and establish a standard curve.
6. The time-resolved fluorescence immunochromatography method for detecting
Nicarbazine according to claim 5, the standard curve: IC 50 of NIC is 0.43ng/mL,
LOD is 0.07ng/mL, The detection range is 0.13-1.37ng/mL, respectively.
7. The time-resolved fluorescence immunochromatography method for detecting
Nicarbazine according to any one of claims 1-6, the sample is a solution of chicken
meat after pretreatment; the chicken pretreatment method : Weigh 2.00±0.05g
homogenized chicken sample into a 10mL centrifuge tube, add 2mL acetonitrile,
vortex for 10min, centrifuge at 8000rpm for 10min, take the supernatant and dilute it
with borate buffer 5 times.
8. The application of the time-resolved fluorescence immunochromatographic
method for detecting Nicarbazine of any one of claims 1-7 in the detection of the
content of chicken meat or in the preparation of a time-resolved fluorescence
immunoassay reagent card for detecting Nicarbazine application.
9. A time-resolved fluorescence immunoassay reagent card for Nicarbazine,
comprising a bottom plate, a sample pad, a nitrocellulose membrane and a water
absorbing pad placed on it, characterized in that the nitrocellulose membrane is made
of DNC-4- BSA and goat anti-chicken IgG are delineated.
10. The time-resolved fluorescent immunoassay reagent card for Nicarbazine
according to claim 9, the method of scribing film is: using DNC-4-BSA and goat anti
chicken IgG with a scribing instrument at 0.7 pL/ The spraying speed of cm is
sprayed on the detection line T line and the quality control line C line position on the
NC film in sequence, and the spacing is 3mm in turn. Place the drawn NC film in a
37 °C oven overnight.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114264810A (en) * 2021-12-08 2022-04-01 南京诺唯赞医疗科技有限公司 Monitoring method of antibody coupled latex microspheres and application thereof
CN114608942A (en) * 2022-03-17 2022-06-10 浙江华南仪器设备有限公司 Improve universal tester that detects precision

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114264810A (en) * 2021-12-08 2022-04-01 南京诺唯赞医疗科技有限公司 Monitoring method of antibody coupled latex microspheres and application thereof
CN114264810B (en) * 2021-12-08 2024-03-15 南京诺唯赞医疗科技有限公司 Monitoring method of antibody-coupled latex microsphere and application thereof
CN114608942A (en) * 2022-03-17 2022-06-10 浙江华南仪器设备有限公司 Improve universal tester that detects precision
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