CN112415210A - Fish growth hormone double-antibody sandwich enzyme-linked immunosorbent assay reagent - Google Patents
Fish growth hormone double-antibody sandwich enzyme-linked immunosorbent assay reagent Download PDFInfo
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Abstract
The invention discloses a fish growth hormone double-antibody sandwich enzyme-linked immunosorbent assay reagent, which comprises the following components: the kit comprises an ELISA plate, a GH standard substance, a multi-hole plate coated with rabbit polyclonal antibodies, a detection antibody monoclonal antibody 1B5, a goat anti-mouse antibody marked by HRP and chromogenic substrates 3', 5' and 5' -tetramethyl benzidine (TMB), wherein the ELISA plate comprises a solid phase carrier and a coating layer, the solid phase carrier is provided with a plurality of micropores, and the coating layer is attached to the surface of the micropores of the solid phase carrier. The double-antibody sandwich enzyme-linked immunosorbent assay reagent for the fish growth hormone provided by the invention has the advantages of no radioactive pollution, simple and convenient operation, low cost and good stability, and can meet the requirements of common laboratories.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a double-antibody sandwich enzyme-linked immunosorbent assay reagent for fish growth hormone.
Background
The fish growth hormone is secreted by pituitary gland, and can activate receptor on target cell membrane to implement its biological effect, and the growth and development of fish whose pituitary gland is removed and synthesis of in vivo protein can be stopped, and after the purified growth hormone is injected, the fish whose pituitary gland is removed can recover growth, and the admission of exogenous growth hormone can show compensation effect for growth of fish body.
In terms of molecular weight and amino acid composition, the fish growth hormone has high homology with other growth hormones of higher vertebrates, the molecular weight is about 2.2ku, and the fish growth hormone consists of 173-190 amino acid residues. The content of leucine, glutamic acid and aspartic acid is higher, the content of tryptophan and methionine is lower, and the content of other amino acids is approximately equal. The primary structure of the fish growth hormone protein contains 4 or 5 cysteines, two disulfide bonds are formed in the molecule, and a characteristic large ring and a characteristic small ring are formed. In the spatial configuration, the alpha-helix constitutes 50% and is folded into 4 antiparallel helical segments, i.e., segments A, B, C, D, with at least one proline present between each segment. Sequence comparison shows that in the carboxyl terminal helix D region of the growth hormone protein, the amino acid conservation is stronger than that in other positions, and the hydrophobic amino acid in the region is buried in the protein to be used as a receptor binding site and plays an important role in maintaining the quaternary structure of the protein.
In teleost, growth hormone acts to promote growth primarily as a metabolic regulator, promoting more decomposition of fat as an energy source, and making more amino acids available for growth; the conversion efficiency of protein in the bait is improved, and the synthesis of the protein is promoted; the influence on the carbohydrate metabolism is shown in that the consumption of liver glycogen is promoted, and the utilization capacity of carbohydrate is enhanced. The growth hormone has the function of regulating the fish body in multiple layers, and effectively promotes the growth and development of the fish body. The content of the growth hormone in the fish blood is changed within 10-100 ng/mL. The exogenous growth hormone is injected to accelerate the growth of the receptor fish and improve the adaptability to the saline water environment, the mechanism is to improve the activity of Na + -K + ATP enzyme and increase the number of chloride cells, and the improvement of plasma permeability and ion concentration caused by the migration of the fish from fresh water to seawater is inhibited. The fish growth hormone is combined with liver membrane receptors, the expression of insulin-like growth factor-1 (IGF-1) mRNA in the liver is promoted, the content of IGF-1 in blood is increased, the IGF-1 directly promotes cell division, and the content of IGF-1 in the blood has a feedback regulation effect on pituitary GH secretion.
The principle of the fish GH radioimmunoassay method is the same as that of human, the GH pure product is marked by radioactive isotope 125I, the GH pure product and GH in a sample compete for a specific primary antibody together, a corresponding secondary antibody is added, a labeled antigen combined with an antibody is separated from an unlabeled antigen by methods such as chromatography, centrifugation and the like, the radioactivity specific activity of the labeled antigen combined with the antibody is measured, and the content of GH in the sample can be calculated according to a standard curve. This system has high sensitivity due to the use of radioiodine as the label, and good specificity due to the use of specific primary antibody in combination with antigen, but because of the use of radioactive labels, this system has significant disadvantages: high radiation harms human health and pollutes the environment, the high radiation also has damage to labeled protein and the half-life of radioactive iodine is short (60 days), so that the stability of the label is insufficient and the label needs to be labeled regularly; meanwhile, the gamma-ray energy emitted by 125I is very low and can be detected only by a sensitive gamma-ray detector, so that the release detection technology of GH is difficult to apply in a common laboratory, and the popularization of a release analysis method is limited.
Disclosure of Invention
The invention aims to: the fish growth hormone double-antibody sandwich enzyme-linked immunosorbent assay reagent is provided for solving the problems that the radioimmunoassay technology of GH is difficult to apply in common laboratories and the popularization of radioimmunoassay methods is limited.
In order to achieve the purpose, the invention adopts the following technical scheme:
a fish growth hormone diabody sandwich enzyme-linked immunosorbent assay reagent comprises:
an ELISA plate, a GH standard substance, a multi-hole plate coated with rabbit polyclonal antibody, a detection antibody monoclonal antibody 1B5, a goat anti-mouse antibody marked by HRP and chromogenic substrates 3', 5' and 5' -tetramethyl benzidine (TMB).
As a further description of the above technical solution:
the ELISA plate comprises a solid phase carrier and a coating layer, wherein a plurality of micropores are formed in the solid phase carrier, and the coating layer is attached to the surface of the micropores of the solid phase carrier.
As a further description of the above technical solution:
the GH standard substance is GH recombinant protein.
As a further description of the above technical solution:
the coating contains a polyclonal antibody against fish growth hormone.
As a further description of the above technical solution:
the solid phase carrier is a polystyrene reagent plate.
As a further description of the above technical solution:
the method comprises the following steps:
a. preparing a GH standard substance;
b. preparing a coating antibody;
c. preparing a detection antibody;
as a further description of the above technical solution:
the step b is divided into three steps of protein expression, animal immunity and antiserum purification.
As a further description of the above technical solution:
and c, six steps of protein expression, animal immunity, antiserum purification, cell fusion and subcloning, ascites preparation and antibody purification.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
the double-antibody sandwich enzyme-linked immunosorbent assay reagent for the fish growth hormone provided by the invention has the advantages of no radioactive pollution, simple and convenient operation, low cost and good stability, and can meet the requirements of common laboratories.
Drawings
FIG. 1 is a schematic diagram of the carrier information structure in step a of the fish growth hormone diabody sandwich enzyme-linked immunosorbent assay reagent provided by the invention;
FIG. 2 is a schematic diagram of the carrier information structure in step b of the fish growth hormone diabody sandwich enzyme-linked immunosorbent assay reagent provided by the invention;
FIG. 3 is a schematic diagram of the carrier information structure in step c of the fish growth hormone diabody sandwich enzyme-linked immunosorbent assay reagent provided by the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Referring to FIGS. 1-3, a fish growth hormone diabody sandwich enzyme-linked immunosorbent assay reagent comprising:
the kit comprises an ELISA plate, a GH standard product, a multi-hole plate coated with rabbit polyclonal antibodies, a detection antibody monoclonal antibody 1B5, goat anti-mouse antibodies marked by HRP and chromogenic substrates 3', 5' and 5 '-Tetramethylbenzidine (TMB), wherein the detection antibody monoclonal antibody 1B5 is the mouse monoclonal antibody 1B5, and the GH standard product, the multi-hole plate coated with rabbit polyclonal antibodies, the detection antibody monoclonal antibody 1B5, the goat anti-mouse antibodies marked by HRP and the chromogenic substrates 3', 5 'and 5' -Tetramethylbenzidine (TMB) are placed in the same kit and are respectively and independently stored.
Please refer to FIG. 1, example 2
The preparation of the GH standard substance comprises the following steps:
1. vector construction
According to the protein sequence provided by the client, the target gene insertion expression vector is artificially synthesized according to the codon preference of Escherichia coli (Escherichia coli), and the target protein sequence is as follows:
Masenqrlfnnavirvqhlhqlaakmindfednllpeerrqlskifplsfcnsdsieaptgldetqkssmlkllrisfrlieswefpsqtlsgqvsnsltvgnpnqitekladlkvgisvlikgcldgqpnmddndslplpfedfyltmgesslresfrllacfkkdmhkvetylrvancrrsldsnctl；
inserting into a specified vector Pet32
The pET-32 vector is ampicillin resistant and contains a Trx tag and a 6XHis tag. The Trx thioredoxin label is beneficial to enhancing the soluble expression of protein, has the molecular weight of about 20kDa and is positioned at the N-terminal of a target gene in a pET-32 vector.
Inserting gene sequence.
And (3) positive clone identification: the synthesized sequence is correct through sequencing verification.
2. Expression in small sample
Transforming BL21DE3 competent cells with the constructed plasmid, inoculating a resistant LB plate culture medium, and growing overnight;
6 monoclonals of the transformation plate are selected and inoculated with 3ml of resistant liquid culture medium respectively;
culturing at 37 deg.C and 220RPM until OD600nm0.5-0.6, adding 0.5mM IPTG20 deg.C, and inducing expression for 3.5 hr;
and (4) centrifuging to collect thalli, carrying out ultrasonic disruption, and detecting the expression condition by SDS-PAGE.
Analysis of the small sample expression results: the protein is expressed in both the supernatant and the inclusion body, and can be continuously subjected to soluble expression and purification.
3. Expression in the macrospecies
And selecting a strain with good small sample expression for large sample expression.
60ul of the strain was inoculated into 200ml of the resistant medium and cultured overnight at 37 ℃ and 220 RPM.
Adding fresh resistant culture medium to 800ml the next day, and culturing for 1-2h to OD600nm 0.5-0.6.
Expression was induced for 3.5h by addition of 200ul of 1M IPTG (28 ℃ or 37 ℃).
The cells were collected by centrifugation at 4 ℃ 66 rpm 15min, the supernatant was discarded, 30ml of PBST suspended cells were added, 1mM PMSF was added to the final concentration, and the cells were disrupted by ultrasonic waves at 200W for 6min under ice bath conditions.
Incubate shaker at 4 ℃ for 1 h.
High speed centrifugation at 4 ℃ for 133 r/s.times.15 min, supernatant was taken and added to 400ul nickel column for binding overnight at 4 ℃.
The nickel column was collected (33 rpm. times.5 min), and beads were washed with 20mM Mimidazole wash to remove the contaminating proteins (1 mL. times.3 times).
300ul of 300mM Mimidazole eluate was added, the eluate was allowed to bind to the beads well for 1h, and the supernatant was collected by centrifugation. Adding 300ul of eluent into the beads again, eluting for 1h, centrifuging and collecting supernatant, and combining the two eluents into one tube.
The solution was changed by dialysis against PBS buffer.
SDS-PAGE identifies protein molecular weight, purity and concentration.
Analysis of large sample expression results: the target protein is expressed as a soluble supernatant, the total amount is 3mg, the predicted molecular weight is 40kDa, and the target protein is not degraded in the expression process.
Please refer to fig. 2, example 3
The preparation of the coating antibody comprises the following steps:
1. protein expression
1.1 vector construction
Synthesizing a target gene insertion expression vector, wherein the target protein sequence is as follows:
SENQRLFNNAVIRVQHLHQLAAKMINDFEDNLLPEERRQLSKIFPLSFCNSDSIEAPTGKDETQKSSMLKLLRISFRLIESWEFPSQTLSGAVSNSLTVGNPNQITEKLADLKVGISVLIKGCLDGQPNMDDNDSLPLPFEDFYLTMGESSLRESFRLLACFKKDMHKVETYLRVANCRRSLDSNCTL
pet32a insertion designation vector
The pET-32 vector is ampicillin resistant and contains a Trx tag and a 6XHis tag. The Trx thioredoxin label is beneficial to enhancing the soluble expression of protein, has the molecular weight of about 20kDa and is positioned at the N-terminal of a target gene in a pET-32 vector.
Insertion of gene sequences
And (3) positive clone identification: the synthesized sequence is correct through sequencing verification.
1.2 thumbnail expression
Selecting 6 monoclonals of the transformation plate, and respectively inoculating 3ml of resistant culture medium;
culturing to OD600nm0.5-0.6, adding 0.5mM IPTG, and inducing expression at 28 deg.C for 3.5 hr;
and (4) centrifuging to collect thalli, carrying out ultrasonic disruption, and detecting the expression condition by SDS-PAGE.
Analysis of the small sample expression results: the protein is expressed in both the supernatant and the inclusion body, and can be continuously subjected to soluble expression and purification.
1.3 Large sample expression
Selecting a small sample of a strain with good expression and inoculating the small sample of the strain to a resistant culture medium;
culturing to OD600nm0.5-0.6, adding 0.5mM IPTG, and inducing expression at 28 deg.C for 3.5 hr;
centrifugally collecting thalli, carrying out ultrasonication, adding a nickel column for incubation, eluting by 0.5Mimidazole, and collecting by tubes;
the collected peaks were pooled and dialyzed against PBS overnight;
2. animal immunization
2.1 animals: rabbits 2 were used for the test.
2.2 immunization protocol:
3. antiserum purification
The second set of tagged expressed proteins was covalently attached to a Sepharose column in a conventional manner.
Incubating 10ml of serum with an affinity purification column, eluting with pH2.5 HCl,0.15M glycine, and neutralizing with pH7.5PBS buffer;
ELISA compares antisera and affinity purified antibody titers.
Please refer to fig. 3, example 4
The preparation of the detection antibody comprises the following steps:
1. protein expression
1.1 vector construction
The insertion of the desired gene into the expression vector was artificially synthesized according to the codon preference of Escherichia coli (Escherichia coli) based on the protein sequence provided by the customer. The sequence of the protein of interest is as follows:
Masenqrlfnnavirvqhlhqlaakmindfednllpeerrqlskifplsfcnsdsieaptgldetqkssmlkllrisfrlieswefpsqtlsgqvsnsltvgnpnqitekladlkvgisvlikgcldgqpnmddndslplpfedfyltmgesslresfrllacfkkdmhkvetylrvancrrsldsnctl
inserting into a specified vector Pet32
The pET-32 vector is ampicillin resistant and contains a Trx tag and a 6XHis tag. The Trx thioredoxin label is beneficial to enhancing the soluble expression of protein, has the molecular weight of about 20kDa and is positioned at the N-terminal of a target gene in a pET-32 vector.
Insertion of gene sequences
And (3) positive clone identification: the synthesized sequence is correct through sequencing verification.
1.2 thumbnail expression
Transforming BL21DE3 competent cells with the constructed plasmid, inoculating a resistant LB plate culture medium, and growing overnight;
6 monoclonals of the transformation plate are selected and inoculated with 3ml of resistant liquid culture medium respectively;
culturing at 37 deg.C and 220RPM until OD600nm0.5 is 0.5-0.6, adding 0.5mM IPTG, and inducing expression at 20 deg.C for 3.5 hr;
and (4) centrifuging to collect thalli, carrying out ultrasonic disruption, and detecting the expression condition by SDS-PAGE.
Analysis of the small sample expression results: the protein is expressed in both the supernatant and the inclusion body, and can be continuously subjected to soluble expression and purification.
1.3 Large sample expression
And selecting a strain with good small sample expression for large sample expression.
60ul of the strain was inoculated into 200ml of the resistant medium and cultured overnight at 37 ℃ and 220 RPM.
Adding fresh resistant culture medium to 800ml the next day, and culturing for 1-2h to OD600nm 0.5-0.6.
Expression was induced for 3.5h by addition of 200ul of 1M IPTG (28 ℃ or 37 ℃).
The cells were collected by centrifugation at 4 ℃ 66 rpm 15min, the supernatant was discarded, 30ml of PBST suspended cells were added, 1mM PMSF was added to the final concentration, and the cells were disrupted by ultrasonic waves at 200W for 6min under ice bath conditions.
Incubate shaker at 4 ℃ for 1 h.
High speed centrifugation at 4 ℃ for 133 r/s.times.15 min, supernatant was taken and added to 400ul nickel column for binding overnight at 4 ℃.
The nickel column was collected (33 rpm. times.5 min), and beads were washed with 20mM Mimidazole wash to remove the contaminating proteins (1 mL. times.3 times).
300ul of 300mM Mimidazole eluate was added, the eluate was allowed to bind to the beads well for 1h, and the supernatant was collected by centrifugation. Adding 300ul of eluent into the beads again, eluting for 1h, centrifuging and collecting supernatant, and combining the two eluents into one tube.
The solution was changed by dialysis against PBS buffer.
SDS-PAGE identifies protein molecular weight, purity and concentration.
2. Animal immunization
2.1 animals
5-8 weeks old Balb/C mice 2.
2.2 adjuvants
The Freund complete adjuvant is used for the first main injection, and the Freund incomplete adjuvant is used for the later reinforcing injection, and the Freund incomplete adjuvant and the equal volume of antigen are fully and uniformly mixed and then injected.
2.3 immunization methods
Back multiple injection.
2.4 immunization amount
The main injection of 100ug antigen/experimental rabbit, and the boost injection of 50ug antigen/experimental rabbit.
2.5 immune cycle
| Animal No：M0068-1,2,3 | Date | Immune dose |
| Primary Immunization: | 2017-8-22 | 0.10mg |
| Boost 1: | 2017-9-5 | 0.05mg |
| Boost 2: | 2017-9-19 | 0.05mg |
| Boost 3: | 2017-10-3 | 0.05mg |
| Test Bleed: | 2017-10-13 | |
| Boost 4: | 2017-12-11 | 0.05mg |
| Fusion: | 2017-12-15 |
3. Antiserum detection
3.1 antiserum was prepared by taking a small amount of blood from the vein of the mouse tail.
3.2 Indirect ELISA method for the detection of antiserum titer.
4. Cell fusion and subcloning
Myeloma cell preparation
One week prior to fusion, SP2/0 cells were revived and cultured normally to log phase.
Spleen cell preparation
Mice to be fused were selected, sacrificed on the day of fusion by cervical dislocation, spleens were removed, splenocytes collected and counted in a standard procedure.
Cell fusion
According to the proportion of 1: 3-1: 10, standard protocol for cell fusion followed by culture in HAT DMEM complete medium, hybridoma cells were visualized 3 days after fusion, 1/2HAT complete medium on day 7 and 1/2HT medium on day 8. Screening assays were started about 10 days after fusion.
Cell fusion results: after fusion, HAT selective medium is used for culture, and observation is carried out under a microscope, a plurality of growing hybridoma cells are observed, and the success of the fusion operation is proved.
Fusion screening
Cell supernatants were aspirated at 100 ul/well for indirect ELISA detection. According to the ELISA result, positive wells were judged. And (4) picking and checking the positive holes detected by the whole plate by using a single-channel pipettor, and performing secondary recheck to further confirm the positive holes.
Subcloning
Two rounds of subcloning were performed on the rescreened positive well cells. (since the positive well cell line obtained by the first subcloning is not stable yet and may contain multiple hybridoma cells, it is generally accepted that the hybridoma cells are a single cell line after the second subcloning and are determined to be positive).
Subcloning cells in the positive hole for the first time, adding an HT DMEM culture medium into the multiple holes for culture, observing under a microscope for about 7 days, detecting the hole with clone growth by indirect ELISA, and taking the hole with a high OD value as the positive hole; and (3) selecting the cells of the positive holes for secondary subcloning, detecting the stable and positive hybridoma cell strains as the cells for finally preparing the monoclonal antibody, and performing expanded culture.
Monoclonal antibody subtype identification
The subtype of each supernatant was determined separately using the monoclonal antibody subtype identification kit from Southern Biotech, USA. Strips coated with the immunogen protein were prepared, 50 ng/well, and 600ul of supernatant was collected from each clone and added dropwise to each of 6 enzyme-labeled wells for the corresponding protein, 100 ul/well. Incubation at 37 ℃ for 1h, PBST washing for three times, adding the diluted antibodies of the typing secondary antibodies, namely anti-IgM, IgA, IgG1, IgG2a, IgG2b and IgG3, into 6 holes, incubation at 37 ℃ for 1h, PBST washing for three times and TMB color development. The identified secondary antibody subtype corresponding to the signal reaction hole is the subtype of the antibody.
After two rounds of subcloning and retesting, positive cell strains and subtypes are determined.
5. Ascites production and antibody purification
5.1 preparation of ascites
The positive cells are subjected to amplification culture and injected into the abdominal cavity of a Balb/C mouse (sensitized by Freund's incomplete adjuvant), and ascites is generated when the abdominal bulge of the mouse is seen in 7-10 days generally. When the mouse has obvious ascites, the ascites is extracted in time.
5.2 purification of ascites
And purifying the ascites of the cells, wherein the purity of the purified antibody is more than 90%. The purification method comprises the following steps:
ammonium caprylate + DEAE ion column purification (IgG1, IgG2a, IgG2b, IgG3 subtype antibody):
the ascites was centrifuged, the volume of the pale yellow liquid was aspirated and calculated, diluted 1:3 with 4 volumes of 60mM acetic acid buffer (pH4.0), octanoic acid was added dropwise (final concentration of diluted ascites was 25. mu.l/ml), stirred at room temperature for 30min, and then allowed to stand at 4 ℃ for 2 hours or more to allow sufficient precipitation.
10000r/min, 4 ℃, 20min, the supernatant was collected and 1/10 volumes of 10 × PBS (0.1M pH7.4) were added. 0.277g of solid ammonium sulfate (0.291 g/ml of 45% saturated ammonium sulfate at 0 ℃) was added to each ml of the mixture, and the mixture was allowed to stand for at least 60 minutes.
10000r/min, 4 ℃, 20min, discarding the supernatant, and dissolving the precipitate in a small amount of PBS. Dialyzed against PBS and overnight at 4 ℃.
5.3 after detecting the purity of the concentration, adjusting the concentration to be beneficial to preservation.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (8)
1. The fish growth hormone double-antibody sandwich enzyme-linked immunosorbent assay reagent is characterized by comprising the following components in parts by weight:
an ELISA plate, a GH standard substance, a multi-hole plate coated with rabbit polyclonal antibody, a detection antibody monoclonal antibody 1B5, a goat anti-mouse antibody marked by HRP and chromogenic substrates 3', 5' and 5' -tetramethyl benzidine (TMB).
2. The fish growth hormone diabody sandwich enzyme-linked immunosorbent assay reagent as claimed in claim 1, wherein said microplate comprises a solid phase carrier and a coating layer, said solid phase carrier is provided with a plurality of micropores, and said coating layer is attached to the surface of the micropores of said solid phase carrier.
3. The fish growth hormone diabody sandwich enzyme-linked immunosorbent assay reagent of claim 1, wherein the GH standard is GH recombinant protein.
4. The fish growth hormone diabody sandwich enzyme-linked immunosorbent assay reagent according to claim 2, wherein said coating layer contains a polyclonal antibody against fish growth hormone.
5. The fish growth hormone diabody sandwich enzyme-linked immunosorbent assay reagent according to claim 2, wherein said solid support is a polystyrene reagent plate.
6. The production method of the fish growth hormone double-antibody sandwich enzyme-linked immunosorbent assay reagent comprises the following steps:
a. preparing a GH standard substance;
b. preparing a coating antibody;
c. and preparing a detection antibody.
7. The method for producing a reagent for measuring fish growth hormone diabody sandwich enzyme-linked immunosorbent assay according to claim 6, wherein the step b is divided into three steps of protein expression, animal immunization and antiserum purification.
8. The method for producing a reagent for measuring fish growth hormone diabody sandwich enzyme-linked immunosorbent assay according to claim 6, wherein the six steps of protein expression, animal immunization, antiserum purification, cell fusion and subcloning, ascites preparation and antibody purification are performed in step c.
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