CN113912732B

CN113912732B - Method for detecting maduramycin or maduramycin content and single-chain antibody thereof

Info

Publication number: CN113912732B
Application number: CN202111251280.XA
Authority: CN
Inventors: 李建成; 黄婧洁; 李苗; 陈莹娴; 梁雪燕
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2021-10-26
Filing date: 2021-10-26
Publication date: 2023-06-20
Anticipated expiration: 2041-10-26
Also published as: CN113912732A

Abstract

The invention discloses a method for detecting the content of maduramycin or maduramycin and a single-chain antibody thereof. The single chain antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are composed of a determinant complementary region and a framework region; the determinant complementary regions consist of CDR1, CDR2 and CDR 3. The single-chain antibody has high sensitivity and strong specificity, can be applied to residue detection of maduramycin, and meets the requirements of practical application.

Description

Method for detecting maduramycin or maduramycin content and single-chain antibody thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for detecting the content of maduramycin or maduramycin and a single-chain antibody thereof.

Background

The Maduramicin (MAD) belongs to polyether ionophore antibiotics (polyether ionophore antibiotics, PIAs), has a wide anticoccidial spectrum, has anticoccidial activity on sporozoites and first generation merozoites, can effectively control 6 common chicken coccidiosis, has a good prevention effect on duck coccidiosis, is not easy to generate drug resistance, has antibacterial and growth promoting effects, and is widely applied to production as a feed additive. However, eating animal food containing the medicine residues can cause vasodilation and induce coronary artery diseases, and has potential harm to human and livestock. Therefore, the detection of the residual MAD is more and more important. The highest residual limit of maduramycin in animal foods approved in China is as follows: chicken muscle 0.24 μg/mL, chicken skin fat 0.48 μg/mL, chicken liver 0.72 μg/mL.

Currently, the common methods for detecting maduramycin residues mainly include microbiological detection methods, chromatographic methods, immunoassay methods and the like. The microbiological method is economical and simple, but has limited sensitivity. Because maduramycin lacks ultraviolet, fluorescent and electrochemical characteristics, it is derivatized to introduce chromophores based on HPLC, and although highly sensitive, the pretreatment process is cumbersome. The chromatography requires higher equipment requirements, is not suitable for high-throughput sample screening, has high instrument price, has higher requirements on operators, and is difficult to popularize. The immunoassay detection technology is a rapid, high-throughput and low-cost detection technology which is widely applied in the field of environmental and food safety monitoring in recent years, and has become one of the main methods for rapid screening and monitoring of toxic and harmful residues in various countries of the world, so that a new approach is provided for detection of chromatography.

The immune magnetic beads (immuno magnctic beads, IMB) are magnetic particles with specific antibodies fixed on the surfaces, so that target objects can be captured specifically, immune complexes can be separated under certain magnetic field intensity, the IMB is mainly covalent bond connection between primary amino groups on the antibodies and activated carboxyl groups on the magnetic beads, and is stable, but due to the fact that part of antibodies are combined with the magnetic beads in a physical adsorption (electrostatic attraction and the like) manner, the immune magnetic beads are infirm, and the immune magnetic beads are easy to separate from the magnetic beads in the subsequent antigen capturing and antigen eluting processes.

Disclosure of Invention

One of the objects to be solved by the present invention is: aiming at the current situation that a maduramycin antibody has not been developed in China, the invention provides a maduramycin single-chain antibody, a preparation method thereof and a method for detecting maduramycin by an immunomagnetic bead purification-enzyme-linked immunoassay. The maduramycin single-chain antibody provided by the invention has high sensitivity and strong specificity, can be applied to residue detection of maduramycin, and meets the requirements of practical application.

The present invention provides single chain antibodies or antigen binding portions thereof to maduramicin comprising a heavy chain variable region and a light chain variable region, both consisting of a determinant complementary region and a framework region; the determinant complementary regions are each comprised of CDR1, CDR2 and CDR 3;

the amino acid sequence of CDR1 of the heavy chain variable region is shown in 153-163 of SEQ ID No. 2;

the amino acid sequence of CDR2 of the heavy chain variable region is shown in 179-185 of SEQ ID No. 2;

the amino acid sequence of CDR3 of the heavy chain variable region is shown in positions 218-226 of SEQ ID No. 2;

the amino acid sequence of CDR1 of the light chain variable region is shown in 27-33 positions of SEQ ID No. 2;

The amino acid sequence of CDR2 of the light chain variable region is shown in positions 53-57 of SEQ ID No. 2;

the amino acid sequence of CDR3 of the light chain variable region is shown in positions 96-103 of SEQ ID No. 2.

Alternatively, according to the single chain antibody or antigen-binding portion thereof described above, the amino acid sequence of the heavy chain variable region is shown at positions 131-251 of SEQ ID No.2, and the amino acid sequence of the light chain variable region is shown at positions 2-115 of SEQ ID No. 2.

Alternatively, according to the single-chain antibody or the antigen-binding portion thereof described above, the amino acid sequence of the single-chain antibody or the antigen-binding portion thereof is shown in SEQ ID No. 2.

The single chain antibody or antigen binding portion of maduramicin may further comprise a protein tag. The protein tag (protein-tag) refers to a polypeptide or protein which is fused and expressed together with a target protein by using a DNA in-vitro recombination technology so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc. For example, the amino acid sequence of a single-chain antibody or antigen-binding portion thereof of maduramicin is a sequence obtained by ligating a protein tag encoding gene at the end of the sequence shown in SEQ ID No. 2.

The present invention also provides a biological material related to the above single chain antibody or antigen binding portion thereof, the biological material being any one of the following:

b1 A nucleic acid molecule encoding the single chain antibody or antigen binding portion thereof described above;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1);

b4 A recombinant vector comprising the expression cassette of B2);

b5 A recombinant microorganism comprising the nucleic acid molecule of B1);

b6 A recombinant microorganism comprising the expression cassette of B2);

b7 A recombinant microorganism containing the recombinant vector of B3);

b8 A recombinant microorganism containing the recombinant vector of B4).

Alternatively, according to the above biological material, B1) the nucleic acid molecule is a gene encoding the above single-chain antibody or antigen-binding portion thereof, and the gene is a DNA molecule as described in a) or B) below:

a) The coding gene sequence of CDR1 of the heavy chain variable region is shown in 457-489 positions in SEQ ID No. 1; the coding gene sequence of CDR2 of the heavy chain variable region is shown in 535-555 bits in SEQ ID No. 1; the coding gene sequence of CDR3 of the heavy chain variable region is shown in 652 th-678 th positions in SEQ ID No. 1; the coding gene sequence of CDR1 of the light chain variable region is shown in 79 th-99 th positions in SEQ ID No. 1; the coding gene sequence of CDR2 of the light chain variable region is shown in 157 th-171 th positions in SEQ ID No. 1; the coding gene sequence of CDR3 of the light chain variable region is shown in 286-309 th position in SEQ ID No. 1;

B) DNA which has more than 90% identity with the DNA molecule defined in A) and which encodes said single chain antibody or antigen binding portion thereof.

The coding gene sequence of the heavy chain variable region can be shown in the 388-753 th positions of SEQ ID No.1, and the coding gene sequence of the light chain variable region can be shown in the 4-342 th positions of SEQ ID No. 1. For example, the sequence of the nucleic acid molecule B1) is shown in SEQ ID No. 1.

The invention also provides an immune magnetic bead, and the surface of the magnetic bead of the immune magnetic bead is coupled with the single-chain antibody or the antigen binding part thereof. The magnetic beads may be manufactured by the company beaver, su, under the product of MB 004.

The preparation method of the immunomagnetic beads can comprise the steps of uniformly mixing the NHS activated magnetic beads and the single-chain antibody or the antigen binding part thereof by vortex for 30s, vertically mixing for 1.5h at room temperature, carrying out coupling reaction for 30min, and then, carrying out vortex for 15s every 5min, and finally, carrying out vortex for 15s every 15 min. The concentration of the single chain antibody or antigen binding portion thereof may be 0.4mg/mL. The mass ratio of the single-chain antibody or antigen-binding portion thereof to the magnetic beads may be 1:25. And (3) blocking the activated residues of the unconjugated single-chain antibody or the antigen binding portion of the magnetic bead surface by using a blocking solution. And obtaining the immune magnetic beads by adopting magnetic separation, enrichment and sealing of the magnetic beads.

The invention also provides a product for detecting maduramicin or the content of maduramicin, comprising the above single chain antibody or antigen-binding portion thereof. The product may also include immunomagnetic beads as described above.

The invention also provides a product for enriching maduramicin, comprising the immunomagnetic beads.

The invention also provides the use of the single chain antibody or antigen binding portion thereof, the biological material, the immunomagnetic beads, the product or the product described above in any of the following:

(1) Preparing a product for detecting the maduramycin or detecting the maduramycin;

(2) Preparing a product for detecting the content of the maduramycin or detecting the content of the maduramycin;

(3) Preparing a product enriched in maduramycin or enriching maduramycin.

The invention also provides a method for detecting the content of the maduramycin or the maduramycin, which comprises

(1) The immune magnetic beads are adopted to treat the sample to obtain an ICELISA sample to be detected;

(2) And (2) detecting the ICELISA to-be-detected sample obtained in the step (1) by adopting the maduramycin as a coating antigen and adopting the single-chain antibody or the antigen binding part thereof as a primary antibody, and determining whether the sample contains maduramycin or the maduramycin content in the sample.

Step (1) may comprise the steps of:

a. weighing 2.0g (+ -0.02 g) of a sample, adding 5mL of methanol, mixing for 10s at 24000rpm in a homogenizer, oscillating for 5min at 5000rpm in a vortex oscillator, centrifuging for 10min at 4 ℃, taking a supernatant, blowing and drying by nitrogen at 37 ℃, and re-dissolving with 2mL of 5% methanol-PBS solution (0.01M (pH 7.4) PBS as a solvent and 5% (volume percent) methanol as a solute in the solution) to obtain a sample to be detected;

b. adding 500 mu L of the prepared sample to be detected into 1mg of the immunomagnetic beads, uniformly mixing by vortex, reacting for 30min on a shaking table at 37 ℃, and washing the immunomagnetic beads for 3 times by using a PBST solution after magnetic separation;

c. adding 500 mu L of 50% methanol water (water is used as a solvent, and the solute and the content of the solute in the solution are 50% (volume percent) methanol) into the immunomagnetic beads, suspending the immunomagnetic beads, carrying out vortex mixing uniformly, carrying out reaction on a shaking table at 37 ℃ for 30min, carrying out magnetic separation, and preserving supernatant, wherein the supernatant is the sample to be detected by the ICELISA.

Step (2) may comprise sequentially coating, blocking, binding of the single chain antibody or antigen binding portion thereof to an iced ELISA sample, binding to horseradish peroxidase-labeled goat anti-mouse IgG antibody, developing, terminating and reading the OD of the reaction system with MAD-OVA as the coating antigen (e.g., coating antigen concentration of 1. Mu.g/mL), 5% skim milk (solvent of 0.01M (pH 7.4) PBS), solute and 5% of its content in the blocking solution of 5% skim milk.) as the blocking solution, diluting the single chain antibody or antigen binding portion thereof with 5% methanol-PBS solution ₄₅₀ Values.

The coating conditions may be incubation at 4℃for 12h. The blocking conditions may be incubation for 1h at 37 ℃. The dilution factor of the single-chain antibody or antigen-binding portion thereof (concentration of 1.5. Mu.g/ml) may be 1000-fold by volume. The binding condition of the single-chain antibody or antigen-binding portion thereof to the sample to be tested by the iceelisa can be incubation at 37 ℃ for 1h. The binding conditions for horseradish peroxidase-labeled goat anti-mouse IgG antibodies may be incubation at 37 ℃ for 1h.

The single-chain antibody has small molecular mass, strong tissue penetrating ability and low immunogenicity, and has strong advantages compared with mAb in tumor treatment; the single-chain antibody has no Fc segment, can reduce nonspecific adsorption, simultaneously maintains affinity and specificity of parent antibody, combines with hapten unit price, has higher sensitivity, and is also suitable for the field of immunodetection.

The icELISA method has the advantages of high detection speed, low cost, low instrument and equipment requirements, high sensitivity, strong selectivity and the like, and can be used for field inspection.

The immunomagnetic beads can enrich pure antigen target substances in a short time, so that the sample detection time is obviously shortened, the defect that immune complexes are difficult to separate from background solution in the immunoassay process is overcome, and the detection benefit and the sensitivity of the method are improved. The stability of the immunomagnetic beads prepared in the examples of the present invention was examined by 1M NaCl,

The value is 85.43%, the stability of the prepared immunomagnetic beads is good, and most single-chain antibodies (scFv-chain variable fragment) are combined with the magnetic beads through covalent bonds.

According to the invention, the prepared single-chain antibody is coupled with magnetic beads to prepare immunomagnetic beads, the immunomagnetic beads have the functions of enriching and purifying MAD molecules in a sample, further separating the MAD molecules through a magnetic separation technology, eluting MAD from the immunomagnetic beads through methanol, and establishing an immunomagnetic bead purification-enzyme-linked immunosorbent assay method to detect the residual quantity of the MAD.

The detection limit of chicken is 6.31 mug/kg, the addition recovery rate is between 72.93% and 89.51%, the daily and intra-day variation coefficients are not more than 15%, and the sensitivity and stability can meet the limit standard and detection requirement. The detection method has high separation efficiency and good stability, can improve the working efficiency, and can be used as an effective tool for screening residues of MAD in chicken.

Drawings

FIG. 1 is a structural formula of maducin.

FIG. 2 is a schematic diagram of a single chain antibody gene construction route.

FIG. 3 is a diagram of the abYsis search for heavy and light chain variable regions of example 1.

FIG. 4 shows the determination of optimal coating antigen and 3B4-scFv concentration by checkerboard titration.

FIG. 5 shows an indirect competition ELISA for single chain antibody IC ₅₀ Is a schematic diagram of the curve of (a).

FIG. 6 is a schematic diagram of the preparation route of immunomagnetic beads.

FIG. 7 shows the results of optimizing the conditions of immunomagnetic beads.

FIG. 8 is a comparison of MAD standard curves in chicken matrix and MAD standard curves in solvent.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Coli RV308 and pJB plasmid vectors were both stored in the laboratory (described in Wen K, nolke G, schillbergS, et al improved fluoroquinolone detection in ELISA through engineering of a broad-specific single-chain variable fragment binding simultaneously to 20fluoroquinolones [ J ]. Analytical and bioanalytical chemistry,2012, 403 (9): 2771-2783.)

The quantitative tests in the following examples were all set up in triplicate and the results averaged.

Maduramycin (MAD): ehrenstor, germany, CAS number 84878-61-5, has the structure shown in FIG. 1. Magnetic beads: the product number MB004 is available from the company beaver, suzhou. Bovine Serum Albumin (BSA): the company OXOID, uk, cat No. 37525. Chicken Ovalbumin (OVA): the company OXOID, uk. Horseradish peroxidase-labeled goat anti-mouse IgG antibody: beijing Soy reagent Co., ltd., product No. SA131. Mouse anti-His-tag monoclonal antibody, HRP-labeled: shanghai assist, saint Biotech Co., ltd., product number 30404ES60.TMB substrate color development liquid: sigma, U.S. Co., ltd., product number T0565.DMEM: gibco, inc., U.S. under the product number 8121475. Fetal bovine serum: gibco, inc., U.S. under the designation 10099-141. Green streptomycin mixed solution (100X): beijing Soy Bao reagent company, cat# P1400.EDC: thermo company, usa, cat: 22980.NHS: thermo company, usa, cat: 24500.2% (mass percent) of skimmed milk and 5% (mass percent) of skimmed milk were prepared from skimmed milk powder (Bomad Biotechnology Co., ltd., product number: PA 201-01) and 0.01M (pH 7.4) PBS.

H ₂ SO ₄ Stop solution: concentrated sulfuric acid 22.2mL, ultrapure water 177.8mL. Binding Buffer:1.0MTris-HCl,300mM sodium chloride, 10mM imidazole, 0.05% Tritonx-100, pH8.0.Washing Buffer:50mM Tris-HCl,300mM sodium chloride, 50mM imidazole, 0.05% Tritonx-100, pH8.0.Elution Buffer:50mM Tris-HCl,300mM sodium chloride, 300mM imidazole, pH8.0.MEST:100mM MES, pH 5.0, 20% Tween 20.Storage Buffer: PBST, PH 7.2,0.1%BSA,0.02%Procline 300.

EXAMPLE one preparation of Single chain antibody to maduramicin

1. Preparation of maduramycin single-chain antibody gene fragment

1. Resuscitation of hybridoma cells: and taking out the required hybridoma cell strain capable of stably secreting the MAD-3B4 antibody from the liquid nitrogen tank, immediately placing the frozen tube into a water bath kettle at 37 ℃ after taking out, and heating while shaking, so that only small icicles are left in the melted tube almost completely within one minute. Transfer to a 15mL centrifuge tube after autoclaving, add 10mL of incomplete medium (DMEM), and centrifuge at 1000rpm for 5min. The supernatant was discarded, and 2mL of complete culture solution (20% fetal bovine serum, 80% DMEM,1% penicillin mixed solution) was added to resuspend the cells, transferred to a 24-well cell culture plate, cultured in a 5% CO2 cell incubator at 37℃for a period of time, and when the cells were grown to 24 wells, the growth state was stable, the size was uniform, and the whole cell RNA extraction was performed.

2. Serotype identification: the single chain antibody immunoglobulin subclass was determined using a murine IgG subclass detection kit.

3. Extraction of hybridoma cell RNA: total RNA of 3B4 hybridoma cells was extracted according to the RNAminikit protocol, and the total RNA of the extracted cells was stored at-20 ℃. The whole operation process needs to avoid RNase pollution as much as possible.

4、cDNA-As0 ₄ Synthesis of linker: and (3) using the extracted total RNA of the cells as a template, and synthesizing cDNA by reverse transcription. Ligating a linker with known sequence to the cDNA As04 linker at T ₄ Under the action of RNALigase, as0 ₄ Linker and cDNA were ligated into cDNA-As0 ₄ linker. And purifying and recycling the reaction product by using a Promega gel recycling kit.

5. And (3) designing primers according to the single-chain antibody typing result in the step (2). With cDNA-As0 ₄ linker isTemplate, the heavy chain and light chain genes of the antibody are respectively amplified by adopting corresponding primers, and the primers used by the heavy chain of the antibody are As0 ₄ -F (sequence: TAGGATCCACCAAGCTTTCTGCAG) and PrimerIgG ₁ (SEQ ID NO: GTCGACTCATTTACCAGGAGAGTGGGAGAGG) the primer used for the light chain of the antibody was As0 ₄ F (SEQ ID NO: TAGGATCCACCAAGCTTTCTGCAG) and Primerkappa (SEQ ID NO: CTCGAGTCAACACTCATTCCTGTTGAAGCTCTTGAC).

6. And (3) determining the heavy chain and the light chain genes of the antibody amplified in the step (5) by using 1% agarose gel, and cutting the gel to recover the target fragment.

7. Determination of heavy and light chain variable regions: and (3) respectively connecting the heavy chain gene and the light chain gene of the antibody amplified in the step (6) with pGM-TFast vectors. The mixed reaction solution is placed at 22 ℃ for reaction for 30min, and then the reaction solution is transferred into competent cells of escherichia coli DH5 alpha by a heat shock method, and white and opaque colonies are cultured. The PCR was sequenced to identify the correct bacterial fluids and software was used to determine the antibody heavy and light chain gene variable region sequences based on the sequencing results.

2. Splicing of maduramicin single chain antibody (3B 4-scFv) gene fragments

The scFv gene construction route of 3B4 is shown in FIG. 2 by overlapping extension PCR technology, and the variable region (VH) of the heavy chain gene and the variable region (VL) of the light chain gene are connected by Linker by SOE PCR to obtain the ScFv gene.

The primers used for SOE PCR are shown in tables 1 and 2. First round SOE-PCR: extracting plasmids after amplifying and culturing the bacterial liquid with correct sequencing, and carrying out PCR amplification by taking the plasmid extracting liquid as a template. The PCR reaction system and the PCR reaction conditions of the second round and the third round of SOE-PCR are the same as those of the first round of SOE-PCR. The difference is that the reaction template of each round is the recovery product of the previous round of PCR amplification reaction, VH is subjected to three times of PCR amplification, and VL is subjected to two times of PCR amplification.

TABLE 1 primers required for SOE-PCR reactions

TABLE 2 primers required for amplification of scFv

The PCR reaction system is as follows:

plasmid/PCR recovery product (100 ng) 0.5. Mu.L

primer-F0.3. Mu.L

primer-R0.3. Mu.L

2×Fast Pfu mix 12.5μL

dd water 11.4μL

The total volume was 25. Mu.L.

PCR reaction procedure: 5min at 95℃for 30 cycles (1 min at 95℃for 50s at 55℃for 1min at 72 ℃) for 10min at 72 ℃. And (3) identifying the PCR product by 1% agarose gel electrophoresis, and cutting and recovering the gel to obtain the full-length scFv gene fragment. The dialyzed 3B4-scFv was mixed with a proper amount of Coupling buffer A and stored at 4℃for further use.

And purifying and recovering PCR products of the VH and the VL of the second round or the third round, and connecting the VH and the VL by taking the VH-R3 and the VL-F2 as primers to obtain the complete 3B4-ScFv gene fragment. The reaction system is as follows:

the PCR reaction conditions were identical to the first round SOE-PCR reaction conditions. The reaction products are identified by 1% agarose gel electrophoresis, and the complete 3B4-scFv gene fragment is recovered by cutting gel.

Sequencing results show that the 3B4-scFv gene fragment is 753bp (excluding a stop codon, a protective base, a SfiI enzyme cutting site and the like), the nucleotide sequence of the 3B4-scFv gene fragment is shown as SEQ ID No.1, and abYsis is used for searching a heavy chain variable region and a light chain variable region, and as shown in FIG. 3, VL and VH regions are respectively composed of 4 FR regions and 3 CDR regions embedded in the VL and VH regions, so that expected guess is met. Wherein, the 1 st to 3 rd bit is methionine sequence, the 4 th to 342 th bit is VL sequence, the 113 th amino acid is coded, the 343 th to 387 th bit is Linker sequence coding, the 15 th amino acid is coded, the 388 th to 753 th bit is VH sequence, the 122 th amino acid is coded, the 79 th to 99 th bit is CDR1 sequence of VL, the 157 th to 171 th bit is CDR2 sequence of VL, the 286 th to 309 th bit is CDR3 sequence of VL, the 457 th to 489 th bit is CDR1 sequence of VH, the 535 th to 555 th bit is CDR2 sequence of VH, and the 652 th to 678 th bit is CDR3 sequence of VH.

The amino acid sequence of the maduramicin single-chain antibody coded by the 3B4-scFv gene fragment is shown as SEQ ID No.2, wherein the 2 nd to 115 th positions are VL sequences, the 116 th to 130 th positions are Linker sequences, the 131 th to 251 th positions are VH sequences, the 27 th to 33 th positions are CDR1 sequences of VL, the 53 th to 57 th positions are CDR2 sequences of VL, the 96 th to 103 th positions are CDR3 sequences of VL, the 153 th to 163 th positions are CDR1 sequences of VH, the 179 th to 185 th positions are CDR2 sequences of VH, and the 218 th to 226 th positions are CDR3 sequences of VH.

3. Construction of RV308 expression System of 3B4-scFv

Inoculating frozen E.coli RV308 cells on an LB plate for streaking, culturing for 12-18h at 37 ℃ in an inverted mode, picking single colonies, inoculating the single colonies into 2mL of LB broth culture medium, and culturing for 12-18h at 37 ℃ at 250 rpm. The above 2mL of culture was added to 200mL of LB broth medium, and when the culture was carried out at 37℃and 250rpm until the OD600 was about 0.5, the flask was taken out, and immediately ice-cooled for 30min. Centrifuge at 3000rpm for 15min at 4 ℃.200mL ice-bath sterile water resuspended the cells, and the supernatant was blotted by centrifugation at 3000rpm for 20min at 4 ℃.150mL of 10% ice-bath glycerol-resuspended cells, centrifugation was repeated once after centrifugation at 4000rpm for 20min at 4℃and the supernatant was blotted off, 6mL of 10% ice-bath glycerol-resuspended cells were sub-packed and stored at-70 ℃.

4. Transformation, expression and purification of single chain antibodies

1. shock transformation of scFv-PJB33 expression vectors

The scFv gene and PJB33 plasmid were digested with SfiI, and the digested products were ligated with T4 DNA library to construct scFv-PJB33 expression vector. The scFv-PJB33 expression vector is a vector in which a fragment (small fragment) inserted between pelB and (His) 6tag recognition sites of the pJB33 plasmid vector is replaced with the 3B4-ScFv gene fragment described above, and the other nucleotide sequences of the pJB33 plasmid vector are kept unchanged.

The scFv-PJB33 expression vector is transferred into the escherichia coli RV308 by using an electric shock method to construct a 3B4 scFv escherichia coli expression system, and the steps are as follows:

the dialysis membrane was placed on top of deionized water and 3 μl of scFv-PJB33 expression vector was dialyzed onto the dialysis membrane for 30min, and the whole dialysis process was performed under ice bath conditions. Competent cells RV308 (100. Mu.L/tube) were removed from-70℃and placed on ice, and the dialyzed plasmid was added immediately after thawing, flicked and mixed. The above product was transferred to a pre-chilled, sterile, dry electric beaker. The electroporation cuvette was placed in an electroporation apparatus under conditions of 2.5kv,25uF,200Ω. Immediately after electrotransformation, the electrotransformation was washed out with 900. Mu.L of 2X YT (Yeast Tryptone) broth and shaken at 37℃for 1h with 200rpm to recover the bacteria. 100. Mu.L of the bacterial liquid was spread on a 2 XYT agar plate containing 34. Mu.g/mL chloramphenicol, and the plate was inverted to a 37℃incubator for overnight culture. 15 single colonies were picked from the plates to 2mL of 2 XYT broth (containing chloramphenicol 34. Mu.g/mL) for expansion culture at 37℃for overnight at 250rpm, and the bacterial solutions were subjected to PCR identification, with the following PCR reaction system:

PJB 33F sequence (5 '. Fwdarw.3'): GGCTTTACACTTTATGCTTCCG;

PJB 33R sequence (5 '. Fwdarw.3'): CGAGAAAGGAAGGGAAGAAAGC.

The PCR reaction conditions were the same as in example one, "two", and the products were identified by agarose gel electrophoresis. The correct bacteria were identified for sequencing to determine if the 3B4-scFv gene fragment was transferred correctly. The strain with correct sequencing is preserved at-80 ℃. Recombinant bacteria with correct sequencing express maduramicin single-chain antibody (3B 4-scFv).

2. Prokaryotic expression and purification of 3B4-scFv

Recombinant bacteria with correct sequencing are selected for expansion culture, and 2 mu L to 5mL of 2 XYT liquid culture medium (containing 34 mu g/mL chloramphenicol) with correct sequencing is sucked for culture at a constant temperature of 37 ℃ and 250rpm overnight. The overnight cultured broth was inoculated into 2 XYT broth (containing chloramphenicol 34. Mu.g/mL) at an inoculum size of 1%, and shaken at 250rpm and 37℃until OD600 was 0.6-0.8. Then, IPTG was added to the mixture to induce culture at 0.5mM, and the culture was induced at 20℃and 180rpm overnight. The flask was placed on ice for 5min pre-chilling, and the collected bacterial liquid was transferred to a 50mL centrifuge tube. The bacterial liquid is centrifuged at 8000rpm for 10min at 4 ℃ to collect bacterial cells. The cells were washed with pre-chilled PBS and centrifuged repeatedly. The cells were resuspended with pre-chilled binding buffer, and 5mL of binding buffer was added per gram of cells. 200W power ice bath ultrasonic, breaking thallus until suspension becomes clear, centrifuging at 10000rpm for 20min at 4deg.C, collecting supernatant to obtain 3B4-scFv extractive solution, and storing at 4deg.C for purification.

The 3B4-scFv after prokaryotic expression is provided with a 6xHis tag, and the 3B4-scFv containing the His tag is purified by a magnetic separation method, so that the purification purpose is achieved by gradient concentration elution of imidazole. The method mainly comprises the following steps:

(1) The reagent bottle with the magnetic beads is fully and evenly mixed, 200uL of the magnetic beads are sucked and added into a centrifuge tube containing 800uL of Binding Buffer (10 mM imidazole), the mixture is evenly mixed and placed on a magnetic separation frame for 10s, and the supernatant is discarded.

(2) Mixing 1mL of 3B4-scFv extract with 1mL Binding Buffer, adding into the centrifuge tube, mixing, shaking for 30min, magnetically separating on a magnetic separation frame, and discarding supernatant.

(3) 1mL Wash Buffer (50 mM imidazole) was added and washing was repeated to remove the heteroproteins on the beads, while the washing was performed with Nanodrop to determine the protein concentration until no protein was eluted.

(4) Finally, the target protein bound on the magnetic beads is washed by an enzyme Buffer (300 mM imidazole), and the eluent is collected, namely the purified 3B4-scFv. The elution was repeated while Nanodrop measures the protein concentration until no protein was eluted.

(5) The beads were washed thoroughly with Elutation Buffer and ultra pure water and stored at 4℃in 20% (v/v) ethanol for the next purification.

(6) And (3) dialyzing the eluent obtained in the step (4) for 48 hours by using PBS, and collecting target proteins to obtain purified 3B4-scFv, namely the maduramicin single-chain antibody solution.

(7) Finally, the protein concentration in the maduramicin single-chain antibody solution was 1.45mg/ml as measured by BCA kit.

Example two preparation of coating Material

(1) 20mg of maduramicin was taken and dissolved in 1.5mL of DMF, followed by the sequential addition of 25mg of EDC and 20mg of NHS, and shaking at room temperature until the solution was completely clear (4-5 h) to give reaction solution I.

(2) 35mg of OVA was dissolved in 4.5mL of 0.05M carbonate buffer (pH 9.6) to obtain a reaction solution II.

(3) The reaction solution I was added dropwise to the reaction solution II, followed by shaking at room temperature for 24 hours.

(4) And (3) putting the solution obtained in the step (3) into a dialysis bag, dialyzing for 3d in 0.01M PBS buffer solution (pH 7.4) (changing the solution twice a day), centrifuging at 10000rpm for 10min, collecting supernatant, namely coating raw solution (MAD-OVA), subpackaging, and storing at-20 ℃.

Example three, preparation of an iceelisa standard curve

1. MAD-OVA and 3B4-scFv antibody solution concentration optimization

The MAD-OVA synthesized in example II was diluted to an actual concentration of about 4. Mu.g/mL, 2. Mu.g/mL, 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL with PBS solution at pH 7.4.10 mM, and the 3B4-scFv solution prepared in example 1 was diluted 500, 1000, 2000, 4000, 8000 times, respectively.

The optimal concentration of MAD-OVA and 3B4-scFv solutions was determined by checkerboard titration. The specific method (basic procedure for iceelisa) is as follows.

1. MAD-OVA with different concentrations was added to 96-well plates at 100. Mu.L/well, incubated at 4℃for 12h, washed 3 times with 1 XPBST, and dried by pipetting;

2. adding blocking solution (5% skimmed milk), 280 μl/well, incubating at 37deg.C for 2h, washing with 1 XPBST for 3 times, and drying;

3. adding a maduramicin solution with the concentration of 10ng/mL, 50 mu L/hole, respectively adding 3B4-scFv solutions with different dilution factors, 50 mu L/hole, incubating for 1h at 37 ℃, washing for 3 times by 1 XPBST, and beating to dry;

4. HRP-anti-His antibody (1:5000 dilution) was added, 100. Mu.L/well incubated for 1h at 37℃and 1 XPBST washed 3 times, and the wells were patted dry;

5. adding TMB substrate color development solution, 100 mu L/hole, and reacting at 37 ℃ in a dark place for 10min and 10min;

6. 2M H2SO4 stop solution, 50 mu L/well, and an enzyme-labeled instrument are added to detect OD450 value.

As a result, as shown in FIG. 4, when the concentration of MAD-OVA was 2. Mu.g/mL, OD was measured ₄₅₀ No longer increases with increasing concentration, when OD ₄₅₀ The ELISA test sensitivity was high at about 1.5, so that the optimal dilution factor for the antibody was 1000, the concentration was 1.5. Mu.g/mL, and the concentration of the coating antigen was 1. Mu.g/mL.

2. Optimization of the icELISA method

The above has been initially established with an iceelisa method and the concentrations of optimal MAD-OVA, 3B4-scFv were established using a checkerboard titration method, and further optimized for iceelisa to establish a 3B4-scFv standard curve.

1. Coating conditions: optimal antigen-antibody concentrations were selected, and the coating conditions were set to 37 ℃ for 2h incubation or 4 ℃ overnight incubation, respectively, and the optimal coating conditions were selected in combination with OD450 values and IC50 of the zero standard wells.

2. Sealing liquid: the optimal antigen-antibody concentration and the optimal coating conditions are selected, BSA, OVA, 2% skim milk and 5% skim milk are used as blocking solutions, the rest steps are carried out according to the basic steps of the ICELISA, and the optimal blocking solution is selected by combining the OD450 value and the IC50 of the zero standard well.

3. Closing time: the optimal conditions of the steps are adopted, the rest steps are carried out according to the basic steps of the ICELISA, the closing time is respectively set to be 30min, 1h, 2h and 4h, and the optimal closing time is selected by combining the OD450 value and the IC50 of the zero standard well.

4. Buffer methanol content: with the optimal conditions for the above steps, the remaining steps were performed according to the basic step of the iceelisa, with 5%, 10%, 20% methanol-PBS dilution of MAD standard and 3B4-scFv, and with the combination of OD450 value and IC50 for zero standard well, the optimal methanol content was selected.

5. Buffer ionic strength: with the optimal conditions for each of the above steps, the remaining steps were performed according to the basic step of the iceelisa, with 10 mmbs, 20 mmbs, 30 mmbs, 50 mmbs diluted antibodies and standards, and the optimal ionic strength was determined in combination with the OD450 value and IC50 of the zero standard well.

6. Buffer pH: with the optimal conditions of the above steps, the rest steps are carried out according to the basic steps of the ICELISA, the pH values of the buffer solutions are 6.2, 6.8, 7.4 and 8.0 respectively, and the optimal reaction buffer solution pH value is selected by combining the OD450 value of the zero standard and the IC 50.

7. Competing reaction time: the optimal conditions for each of the above steps were used, with the remaining steps being performed according to the iceelisa basic procedure. The competing reaction time is set to be 30min, 45min and 60min, and the optimal reaction time of the competing reaction is selected by combining the OD450 value of the zero standard and the IC 50.

8. Secondary antibody reaction time: the optimal conditions of the steps are adopted, the rest steps are carried out according to the basic steps of the ICELISA, the secondary antibody time is respectively set to be 15min, 30min, 45min and 60min, the OD450 value of each hole is measured, and the zero standard OD is combined ₄₅₀ The values and IC50 determine the optimal reaction time for the secondary antibody.

According to IC ₅₀ And zero standard hole OD ₄₅₀ (B ₀ ) The condition of high sensitivity is selected as the optimum condition. The results of the steps are shown in Table 3, wherein the bold is the preferred condition. Overnight at 4℃was chosen as coating conditions, 5% skim milk was blocked for 1h, 3B4-scFv and MAD standard were diluted with pH 7.4 10mM PBS containing 5% methanol, competing for 1h, and secondary for 1h.

TABLE 3 optimization of condition detection results

3. Establishment of an iceELISA Standard Curve

An icELISA assay for MAD based on 3B4-scFv was established under the optimal conditions obtained in the above steps.

Optimal dilutions of the coating antigen and scFv were selected and maduramycin solutions of different concentrations were prepared using maduramycin and PBS buffer (hereinafter also abbreviated as 5% methanol-PBS solution) with pH7.4 and 0.01M containing 5% methanol. The concentration of maduramicin was 4. Mu.g/mL, 2. Mu.g/mL, 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL, respectively. A PBS buffer, pH7.4, 0.01M containing 5% methanol was used as a blank.

1. Diluting the coating raw material to 1 mug/mL by using coating liquid, adding 100 mug/hole into a 96-well plate, incubating for 12h at 4 ℃, washing the plate for 3 times by using 1 XPBST, and drying by beating;

2. adding 5% skimmed milk, 280 μl/well, incubating at 37deg.C for 1h, washing with 1 XPBST for 3 times, and drying;

3. sequentially adding the maduramicin solution with different concentrations after gradient dilution, 50 mu L/hole, adding 3B4-scFv solution with optimal working concentration of 1.5 mu g/mL into all holes, 50 mu L/hole, incubating for 1h at 37 ℃, washing for 3 times by 1 XPBST, and drying by beating;

5. adding TMB substrate color development solution, 100 mu L/hole, and reacting at 37 ℃ in a dark place for 10min;

6. Add 2M H ₂ SO ₄ Stop solution, 50 mu L/hole, and OD detection by enzyme-labeled instrument ₄₅₀ Values.

Mapping with origin8.5 to OD ₄₅₀ Values are plotted on the ordinate and the logarithm of the concentration of the maduramicin solution is plotted on the abscissa, and the inhibition standard curve is plotted by Origin software, see FIG. 5, for calculation of IC ₅₀ 。

Inhibition (%) = [ (B) ₀ -B)/B ₀ ]X 100%. B represents the OD value of the test well, B ₀ OD values representing blank wells. The concentration of the maduramicin solution at the inhibition rate of 50% is the IC of the monoclonal antibody ₅₀ Values. IC (integrated circuit) ₅₀ =15.43 ng/mL, linear range 5.41-43.99ng/mL.

Example IV preparation of MAD immunomagnetic beads

Immunomagnetic beads preparation principle As shown in FIG. 6, 3B4-scFv was coupled with carboxylated magnetic beads (2 μm) activated with EDC and NHS (N-hydroxysuccinimide) to prepare MAD immunomagnetic beads. The method comprises the following specific steps:

1. activation of magnetic beads

Mix the magnetic bead samples and draw 100 μl (i.e. 1 mg) into 3 1.5mL centrifuge tubes, vortex mix well, place on a magnetic separation rack for magnetic separation for 3min, and remove the supernatant. The magnetic bead sample was washed by adding 200. Mu.L of pre-chilled Washing Buffer at 4℃and vortexing for 15s, immediately magnetically separating and removing the supernatant. NHS (10 mg/ml) was added to 100. Mu.L of activated beads and the mixture was placed on a vertical mixer and activated at 25℃for 30min. Washing with MEST 200 μl, vortex mixing for 10s, magnetically separating on a magnetic separation rack for 3min, removing supernatant, washing repeatedly for 2 times, and removing supernatant for use.

2. Coupling of antibodies with magnetic beads (preparation of immunomagnetic beads)

1) The single-chain antibody solution of maduramicin obtained in example one was diluted with PBST solution (pH 7.4, 0.01M phosphate buffer containing 0.05% Tween-20) to obtain a single-chain antibody dilution with a protein concentration of 1.0 mg/mL.

2) To a centrifuge tube containing activated magnetic beads, 15. Mu.L of the single-chain antibody dilution of step 1) was added and vortexed for 30s. And (3) vertically and uniformly mixing for 1.5 hours at room temperature, and performing a coupling reaction of the magnetic beads and the 3B 4-scFv. The reaction was continued for 30min, vortexing for 15s every 5min, followed by vortexing for 15s every 15 min. The magnetic beads were enriched by a magnetic separation rack and the supernatant was saved for detection of the coupling effect.

3) Adding 200 mu L of sealing solution (3M ethanolamine) into the magnetic beads obtained in the step 2), swirling for 30s, enriching the magnetic beads through a magnetic separation frame, and discarding supernatant. Repeated 4 times.

4) Adding 200 mu L of sealing liquid obtained in the step 3) into a centrifuge tube, swirling for 30s, then placing into a vertical mixer for reaction at room temperature for 2h to seal activated residues of unconjugated 3B4-scFv, enriching the magnetic beads through a magnetic separation frame, and discarding supernatant.

5) Adding 200 mu L of ultrapure water into the centrifuge tube, fully mixing, and enriching the magnetic beads through a magnetic separation frame.

6) Adding 200 mu L of Storage Buffer into a centrifuge tube, fully mixing, enriching the magnetic beads through a magnetic separation frame, and discarding the supernatant. This operation was repeated 2 times.

7) And (3) adding the magnetic beads obtained in the step (6) into a centrifuge tube, fully mixing, and preserving at 4 ℃ for later use.

The prepared 3B4-scFv solution was mixed with PBST to a concentration of 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 100. Mu.L of each 3B4-scFv solution was coupled with 1mg of magnetic beads by the above method, the supernatant of step 2) was magnetically separated and retained, the concentration of 3B4-scFv in the supernatant was measured using the BCA kit, 3 parts of each supernatant was made in parallel, the average of the concentrations was taken, and the remaining amount of antibody in the supernatant was calculated from the volume of 100. Mu.L.

The coupling ratio of the 3B4-scFv and the magnetic beads is as follows: coupling ratio (Coupling ratio) = (antibody addition amount-supernatant antibody residual amount)/antibody addition amount, and the Coupling ratio of the 3B4-scFv solutions with different concentrations is calculated to be more than 80%, so that the effect is better. The results of the coupling ratios are shown in FIG. 7 (A), and when the amounts of the added antibody were 40. Mu.g and 50. Mu.g, the coupling amounts of the antibody were not large, and were 33.5. Mu.g and 34.3. Mu.g, respectively, but the coupling ratios were large, 83.8% and 68.6%, respectively. When the magnetic bead coupling antibody is not saturated, the addition amount of the antibody is positively correlated with the coupling rate, and when the concentration of the antibody is too high, the coupling site is saturated, and the rest of the antibody cannot be coupled, thereby affecting the coupling effect of the magnetic bead. Therefore, the optimal amount of antibody to be added was 40. Mu.g, and the amount of antibody coupling to the immunomagnetic beads prepared at this time was 33.1mg/g.

The optimized preparation method of the immunomagnetic beads is specifically as follows.

1) The single-chain antibody solution of maduramicin obtained in example one was diluted with PBST solution (pH 7.4, 0.01M phosphate buffer containing 0.05% Tween-20) to obtain a single-chain antibody dilution with a protein concentration of 0.4 mg/mL.

2) To a centrifuge tube containing activated magnetic beads, 15. Mu.L of the single-chain antibody dilution of step 1) was added and vortexed for 30s. And (3) vertically and uniformly mixing for 1.5 hours at room temperature, and performing a coupling reaction of the magnetic beads and the 3B 4-scFv. The reaction was continued for 30min, vortexing for 15s every 5min, followed by vortexing for 15s every 15 min. The magnetic beads are enriched by a magnetic separation rack.

7) And (3) adding 100 mu L of Storage Buffer into the centrifuge tube, fully mixing, and preserving at 4 ℃ for later use.

3. MAD and optimization in enrichment and separation samples of simulated immunomagnetic beads

The purification method of the immunomagnetic beads is specifically as follows.

1. Adding 500 mu L of maduramycin solution prepared from adsorption liquid and maduramycin standard into an immune magnetic bead centrifuge tube synthesized by the optimized preparation method of the immune magnetic beads, mixing uniformly by vortex, capturing antigen on a shaking table at 37 ℃ for 30min, enabling the antigen (namely maduramycin) to fully react with the immune magnetic beads, and reserving supernatant for measuring the antigen capturing rate after magnetic separation. The immunomagnetic beads were washed 3 times with PBST solution.

2. Adding 500 mu L of eluent into the magnetic beads, suspending the magnetic beads again, mixing uniformly by vortex, reacting for 30min on a shaking table at 37 ℃ to elute antigens, then carrying out magnetic separation by adopting a magnetic separation frame, and preserving supernatant, wherein the supernatant is the sample to be detected of the iceLSA.

3. To the centrifuge tube, 500. Mu.L of PBS pH7.4, 0.01M was added, washed, vortexed, and the supernatant removed by magnetic separation and repeated 3 times. Finally, the immunomagnetic beads were resuspended in centrifuge tubes using 100. Mu.L Storage Buffer and stored at 4℃for further use.

4. The 1:10 fold diluted supernatant was assayed using icELSA, which was identical to the icELSA method of example 2, except that the maduramicin solution at different concentrations after gradient dilution was replaced with the 1:10 fold diluted supernatant. The concentration of the supernatant after 1:10 dilution was obtained according to the standard curve prepared in example three. The remaining amount of antigen in the supernatant was calculated from the supernatant concentration after 1:10-fold dilution in step (1), and the adsorption rate= (antigen addition amount-remaining amount of antigen in the supernatant)/antigen addition amount. And (3) calculating the amount of antigen in the eluent according to the concentration of the supernatant fluid diluted 1:10 in the step (1), and calculating the amount of magnetic bead capture antigen according to the concentration of the supernatant fluid diluted 1:10 in the step (2), wherein the elution rate=the amount of antigen in the eluent/the amount of magnetic bead capture antigen.

The purification method of the immunomagnetic beads is optimized as follows.

1. Optimization of antigen capture time

The antigen is captured by adopting the method, the capturing time is 15min, 30min and 60min respectively, the adsorption solution is 5% methanol-PBS solution, the concentration of the maduramycin solution is 500ng/mL, and the eluent is 50% methanol water.

The supernatant obtained in step (1) was 1:10-fold diluted and then detected by ICELISA, and the relation between the Capture time (Capture time) and the antigen Capture efficiency was shown in FIG. 7 (B), and the OD was measured at a Capture time of 15min and 30min ₄₅₀ A significant increase, indicating that antigen was still not captured for a period of 15-30min, and an OD at 60min capture time ₄₅₀ Has no great difference with 30min. The antigen capture time was selected to be 30min.

2. Optimization of adsorption liquid

MAD is not readily soluble in water and is readily soluble in organic solvents. Therefore, the adsorption liquid is properly added with a solvent, which is favorable for dissolving MAD and improving the efficiency of capturing antigen by the immunomagnetic beads, while the biological activity of scFv is influenced by the excessive organic solvent, which is unfavorable for the specific combination of antigen and antibody. Methanol was used as the organic solvent, and thus the physical and chemical properties and the bioactivity of the antibody were less affected, and thus 0, 5%, 10%, and 20% methanol-PBS solutions were respectively selected as the adsorption solution, and the blocked blank magnetic beads were used as the blank control. The method is used for capturing the antigen, the capturing time is 30min, the adsorption liquid is respectively 0, 5%, 10% and 20% methanol-PBS solution, the concentration of the maduramicin solution is 500ng/mL, and the eluent is 50% methanol water.

The supernatant obtained in the step (2) was 1:10-fold diluted and then subjected to an iceELISA test, and as shown in FIG. 7 (C), a 5% methanol-PBS solution was used as the adsorption solution.

3. Optimization of antigen addition

The antigen is captured by adopting the method, the capture time is 30min, the adsorption solution is 5% methanol-PBS solution, the concentration of the maduramicin solution is 100ng/mL, 250ng/mL, 500ng/mL and 750ng/mL, and the closed blank magnetic beads are used as blank control, and the eluent is 50% methanol water.

The supernatant obtained in step (1) was 1:10-fold diluted and then subjected to ICELISA, and the results were shown in FIG. 7 (D), in which OD was increased with the increase of the antigen addition amount at 100ng/mL to 500ng/mL ₄₅₀ Without great change, OD when the added concentration of antigen is 750ng/mL ₄₅₀ The antigen concentration in the supernatant was significantly reduced, i.e., increased, so that the optimal antigen addition concentration was selected to be 500ng/mL, i.e., the antigen addition amount was 250ng. The immunomagnetic beads prepared by the invention are suitable for enriching samples to be detected, wherein the content of the maduramycin is less than or equal to 500 ng/mL.

4. Optimization of eluent methanol content

The antigen is captured by adopting the method, the capturing time is 30min, the adsorption solution is 5% methanol-PBS solution, the concentration of the maduramycin solution is 500ng/mL, and the eluents are water, 25% methanol water, 50% methanol water, 75% methanol water and pure methanol respectively.

The effect of the 1:10 dilution of the supernatant from step (2) on the elution was measured by ICELISA as shown in FIG. 7 (E), and the OD of the eluate was measured from 0 to 50% methanol ₄₅₀ At progressively lower OD of 50% to 100% methanol content of the confining liquid ₄₅₀ Without major changes. Based on the effect of high concentration organic solvents on antibody activity, 50% methanol water was chosen as the optimal eluent.

5. Reproducibility of immunomagnetic beads

With the repeated use of the immunomagnetic beads, part of the magnetic beads are lost in the magnetic separation process, so that the antigen capturing effect of the immunomagnetic beads is affected. The immunomagnetic beads continuously capture the antigen for 5 times by adopting the method, the capture time is 30min, the adsorption liquid is 5% methanol-PBS solution respectively, the concentration of the maduramycin solution is 500ng/mL, and the eluent is 50% methanol water.

The supernatant obtained in the step (1) is diluted 1:10 times and then detected by an icELISA method, the result is shown in fig. 7 (F), the antigen capturing rate is reduced along with the increase of the using times of the immunomagnetic beads, and the repeated using times of the immunomagnetic beads are selected to be 3 times in order to ensure that the immunomagnetic beads completely capture the antigen.

4. Isolation of MAD in sample by immunomagnetic bead enrichment

1. 2.0g (plus or minus 0.02 g) of the sample is weighed, 5mL of methanol is added, 24000rpm is mixed for 10s in a homogenizer, 5min is oscillated on a vortex oscillator, the mixture is centrifuged for 10min at 5000rpm at 4 ℃, the supernatant is taken, the mixture is blown to dryness by nitrogen at 37 ℃, and then 2mL of 5% methanol-PBS solution is used for redissolving, so that the sample to be detected is obtained.

2. Adding 500 mu L of the prepared sample to be detected into an immune magnetic bead centrifuge tube synthesized by the optimized preparation method of the immune magnetic beads, mixing uniformly by vortex, reacting for 30min on a shaking table at 37 ℃ to enable the sample to fully react with the immune magnetic beads, and washing the immune magnetic beads for 3 times by using a PBST solution after magnetic separation.

3. Adding 500 mu L of 50% methanol water into the magnetic beads, re-suspending the magnetic beads, uniformly mixing by vortex, reacting for 30min on a shaking table at 37 ℃ to elute antigens, then performing magnetic separation by using a magnetic separation frame, and preserving supernatant, wherein the supernatant is the sample to be detected by the ICELISA.

4. To the centrifuge tube, 500. Mu.L of PBS pH7.4, 0.01M was added, washed, vortexed, and the supernatant removed by magnetic separation and repeated 3 times. Finally, the immunomagnetic beads were resuspended in centrifuge tubes using 100. Mu.L Storage Buffer and stored at 4℃for further use.

Example five detection of maduramycin content in samples

1. Matrix effect

1. Sample pretreatment: weighing 2.0g (+ -0.02 g) of a blank chicken sample (i.e. a chicken sample without additional maduramycin), adding 5mL of methanol, mixing for 10s at 24000rpm in a homogenizer, vibrating for 5min on a vortex oscillator, centrifuging for 10min at 5000rpm at 4 ℃, taking the supernatant, blowing and drying at 37 ℃ with nitrogen, and re-dissolving with 2mL of 5% methanol-PBS solution.

2. And adding a proper amount of MAD standard substance into the pretreated sample to obtain a sample to be detected, wherein the concentration of the MAD in the sample to be detected is respectively 0ng/mL, 0.9ng/mL, 2.7ng/mL, 8.1ng/mL, 24.3ng/mL, 72.9ng/mL, 218.7ng/mL and 656.1ng/mL.

3. Adding 500 mu L of the prepared sample to be detected into an immune magnetic bead centrifuge tube synthesized by the optimized preparation method of the immune magnetic beads, mixing uniformly by vortex, reacting for 30min on a shaking table at 37 ℃ to enable the sample to fully react with the immune magnetic beads, and washing the immune magnetic beads for 3 times by using a PBST solution after magnetic separation.

4. Adding 500 mu L of 50% methanol water into the magnetic beads, re-suspending the magnetic beads, uniformly mixing by vortex, reacting for 30min on a shaking table at 37 ℃ to elute antigens, then performing magnetic separation by using a magnetic separation frame, and preserving supernatant, wherein the supernatant is the sample to be detected by the ICELISA.

5. The matrix addition curve was established by the "establishment of an iceELISA standard curve" method of example III, and was compared with the standard curve of 5% methanol-PBS in FIG. 5 to analyze the magnitude of matrix effect.

As a result, as shown in FIG. 8, the matrix was the standard curve of the matrix prepared as described above, and the solvent was the standard curve of 5% methanol-PBS in FIG. 5. The matrix labeling IC50 is 15.72ng/mL, the linear range is 5.06-48.78ng/mL, the matrix labeling IC50 and the linear range are basically consistent, the purification effect of the immunomagnetic beads is good, and the influence of the matrix on the ICELISA is negligible.

2. Accuracy and precision determination

1. Sample pretreatment: weighing 2.0g (+ -0.02 g) of a blank chicken sample (i.e. a chicken sample without the maduramycin), adding 5mL of methanol, mixing for 10s at 24000rpm in a homogenizer, adding an MAD standard diluted by 5% methanol-PBS solution, wherein the concentration (addition value) of the MAD standard in the mixture is 0ng/mL, 0.9ng/mL, 2.7ng/mL, 8.1ng/mL, 24.3ng/mL, 72.9ng/mL, 218.7ng/mL and 656.1ng/mL, vibrating the mixture on a vortex shaker for 5min at 4 ℃ for 10min, taking a supernatant, blowing and drying by nitrogen at 37 ℃, re-dissolving by 2mL of 5% methanol-PBS solution, and diluting 10 times by 5% methanol-PBS solution as a sample to be detected.

4. And (3) detecting the MAD concentration of the sample to be detected in the ICELISA by adopting the method of detecting MAD in the sample enriched in the magnetic beads by adopting the following (II) indirect competition ELISA, and further calculating the actual measurement concentration (actual measurement value) of the MAD standard added in the blank chicken sample.

5. The recovery rate and the coefficient of variation were calculated from the recovery rate (%) =actual measurement value/added value×100%, coefficient of variation=standard deviation/average value of recovery rate.

The detection result shows that the addition recovery rate is between 72.93 and 89.51 percent, the daily variation coefficient is not more than 15 percent, and the requirements of the veterinary drug residue test technical specification accuracy and precision are met, so that the method can be used for residue detection of actual samples.

3. Method for detecting maduramycin content in sample

MAD in magnetic bead enriched samples

1. 2.0g (plus or minus 0.02 g) of the sample is weighed, 5mL of methanol is added, 24000rpm is mixed for 10s in a homogenizer, 5min is oscillated on a vortex oscillator, the mixture is centrifuged for 10min at 5000rpm at 4 ℃, the supernatant is taken, the mixture is blown to dryness by nitrogen at 37 ℃, and then 2mL of 5% methanol-PBS solution is used for redissolving to obtain the sample to be detected.

(II) detection of MAD in magnetic bead enriched samples by indirect competitive ELISA

1. The MAD-OVA coating stock solution was diluted with PBS (phosphate buffer solution) having a pH of 7.4 and containing 5% methanol to give a coating solution having a coating stock concentration of 1. Mu.g/mL (based on the amount of protein), and the coating solution was added to the ELISA plate, incubated at 100. Mu.L/well at 4℃for 12 hours, and then the supernatant was discarded, washed once with 1 XPBST solution, and then the plate was dried.

2. Blocking solution (5% skim milk) was added, 150. Mu.L/well incubated at 37℃for 1h, the supernatant was discarded, washed 3 times with 1 XPBST, and patted dry.

3. 50. Mu.L of the sample to be tested in step (I) and 50. Mu.L of 1.5. Mu.g/mL single-chain antibody dilution (the single-chain antibody solution of maduramycin obtained in example I was diluted to 1000 volumes with PBS solution of pH 7.410 containing 5% methanol, at a concentration of 1.5. Mu.g/mL) were added to each well, incubated at 37℃for 30min, plate washed 3 times with 1 XPBST solution, and blotted with absorbent paper.

4. 100. Mu.L of horseradish peroxidase-labeled goat anti-mouse IgG antibody (5000-fold dilution) was added to each well, incubated at 37℃for 1h, plates were washed 3 times with 1 XPBST solution, and blotted dry with absorbent paper.

5. 100 mu L of TMB color developing solution is added into each hole, and the reaction is carried out for 10min at 37 ℃ in a dark place.

6. 50 mu L of 2M H are added to each well ₂ SO ₄ Termination liquid and detection OD of enzyme label instrument ₄₅₀ Values.

7. And (5) obtaining the maduramicin content in the sample according to the prepared matrix standard curve and the reading of the enzyme-labeled instrument obtained in the step 5.

4. Detection limit determination

And detecting chicken samples by using the established method, taking 20 blank chicken samples, performing ICELISA detection after pretreatment, and taking the average value of OD450 minus 3 times of standard deviation, wherein the concentration on the corresponding matrix standard curve is the detection Limit (LOD).

The detection result shows that the detection limit of chicken is 6.31 mug/kg, and MAD samples exceeding the detection limit (1 ng/mL) of the conventional immunodetection method are detected.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

<110> Chinese university of agriculture

<120> method for detecting maduramycin or maduramycin content and single-chain antibody thereof

<130> 212199

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 753

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

atggacatta ttctcaccca atctccacct tctttggctg tgtctctagg gcagagagcc 60

accgtctcct gcagagccag tgaaagtgtt gaatattttg gcacaagttt aatgcagtgg 120

taccaacaga aacccggaca gccacccaag ctcctcatct ttgctgcatc caacgtaaat 180

tctggggtcc ctgccagatt tagtggcagt gggtctggga cagacttcag cctcaacatc 240

catcctgtgg aggaggatga tattacaatg tatttctgtc accaaagtag gaaatttccg 300

ttcacgttcg gaggggggac caaactggaa ataaaacggg ctggtggtgg tggttctggc 360

ggcggcggct ccggtggtgg tggatccgat gtgcagcttc aggaatcagg acctgacctg 420

gtgaaacctt ctcagtcact ttcactcacc tgcactgtca ctggctattc catctccagt 480

ggttatacct ggcactggat ccggcagttt cctagaaaca cactggaatg tatgggttat 540

atacattaca gtggtaccac taattacagc ccatctctca aaagtcgaat ctctatcact 600

cgagacacat ccaaaaacca gttcttcctg cagttgaatt ctgtgactac tgaggacaca 660

gccacatatt tctgtgcaag tttctactat ggtgactcct cttactatgg tttggactac 720

tggggtcagg gaacctcagt caccgtctcc tcc 753

<210> 2

<211> 251

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Met Asp Ile Ile Leu Thr Gln Ser Pro Pro Ser Leu Ala Val Ser Leu

1 5 10 15

Gly Gln Arg Ala Thr Val Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr

20 25 30

Phe Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro

35 40 45

Pro Lys Leu Leu Ile Phe Ala Ala Ser Asn Val Asn Ser Gly Val Pro

50 55 60

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile

65 70 75 80

His Pro Val Glu Glu Asp Asp Ile Thr Met Tyr Phe Cys His Gln Ser

85 90 95

Arg Lys Phe Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser

130 135 140

Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Ser Ser

145 150 155 160

Gly Tyr Thr Trp His Trp Ile Arg Gln Phe Pro Arg Asn Thr Leu Glu

165 170 175

Cys Met Gly Tyr Ile His Tyr Ser Gly Thr Thr Asn Tyr Ser Pro Ser

180 185 190

Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe

195 200 205

Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe

210 215 220

Cys Ala Ser Phe Tyr Tyr Gly Asp Ser Ser Tyr Tyr Gly Leu Asp Tyr

225 230 235 240

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

245 250

Claims

1. A single chain antibody or antigen binding portion thereof to maduramicin, characterized in that: the single chain antibody or antigen binding portion thereof comprises a heavy chain variable region and a light chain variable region, both of which consist of a determinant complementary region and a framework region; the determinant complementary regions are each comprised of CDR1, CDR2 and CDR 3;

2. The single chain antibody or antigen-binding portion thereof of claim 1, wherein: the amino acid sequence of the single-chain antibody or the antigen binding portion thereof is shown as SEQ ID No. 2.

3. A biological material associated with the single chain antibody or antigen binding portion thereof of claim 1 or 2, said biological material being any one of the following:

b1 A nucleic acid molecule encoding the single chain antibody or antigen binding portion thereof of claim 1 or 2;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1);

b4 A recombinant vector comprising the expression cassette of B2);

b5 A recombinant microorganism comprising the nucleic acid molecule of B1);

b6 A recombinant microorganism comprising the expression cassette of B2);

b7 A recombinant microorganism containing the recombinant vector of B3);

b8 A recombinant microorganism containing the recombinant vector of B4).

4. A biomaterial according to claim 3, wherein: b1 The nucleic acid molecule is a gene encoding the single-chain antibody or antigen-binding portion thereof according to claim 1 or 2, which is a DNA molecule according to a) or B) as follows:

5. Immune magnetic bead, its characterized in that: the single-chain antibody or the antigen binding portion thereof according to claim 1 or 2 is coupled to the magnetic bead surface of the immunomagnetic bead.

6. A product for detecting maduramycin or maduramycin content, characterized in that: comprising the single chain antibody or antigen binding portion thereof of claim 1 or 2.

7. A product for enriching maduramycin, characterized in that: comprising the immunomagnetic bead of claim 5.

8. Use of the single chain antibody or antigen binding portion thereof of claim 1 or 2, the biomaterial of claim 3 or 4, the immunomagnetic bead of claim 5, the product of claim 6 or the product of claim 7 in any of the following:

(3) Preparing a product enriched in maduramycin or enriched in maduramycin;

the application is for non-disease diagnosis and therapeutic purposes.

9. The method for detecting the content of the maduramycin is characterized by comprising the following steps of: comprising

(1) Treating a sample with the immunomagnetic beads according to claim 5 to obtain an iceelisa sample;

(2) Using maduramicin as a coating antigen, using the single-chain antibody or antigen binding portion thereof as primary antibody according to claim 1 or 2, detecting the sample to be detected of the ICELISA obtained in the step (1) through indirect enzyme-linked immunosorbent assay, and determining whether the sample contains maduramicin or the maduramicin content in the sample;

the methods are for non-disease diagnosis and treatment purposes.