CN116355086A - Method for rapidly constructing pandwichELISA - Google Patents
Method for rapidly constructing pandwichELISA Download PDFInfo
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- CN116355086A CN116355086A CN202111632558.8A CN202111632558A CN116355086A CN 116355086 A CN116355086 A CN 116355086A CN 202111632558 A CN202111632558 A CN 202111632558A CN 116355086 A CN116355086 A CN 116355086A
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- C—CHEMISTRY; METALLURGY
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C12N15/09—Recombinant DNA-technology
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6872—Methods for sequencing involving mass spectrometry
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- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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Abstract
The invention discloses a method for quickly constructing a pandwich ELISA, which belongs to the technical field of biology, utilizes the principle of immune coprecipitation, utilizes the known monoclonal antibody to combine with a target antigen, directly screens in a polyclonal antibody and obtains a target antibody sequence, and simulates the combination of the pandwich ELISA antibody antigen in the whole process, thereby avoiding factors such as overlapping of two antibody recognition domains and interference of protein meta-resistance effect, and the like, and rapidly developing the pandwich ELISA and a corresponding kit in a time-saving and labor-saving manner.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for quickly constructing a pandwich ELISA.
Background
Enzyme-linked immunosorbent assay (ELISA) is a special type of Enzyme Immunoassay (EIA) that uses antibodies to quantify a target molecule. Antibodies are used to specifically detect analytes (e.g., peptides, proteins, antibodies, small molecules). An enzyme (e.g., horseradish peroxidase, HRP) is coupled directly or indirectly to the antibody, thereby providing a detection method and possibly amplifying the signal. The target molecule may be a toxin or other foreign substance that causes the animal's immune system to initiate a defensive response, such as influenza virus or environmental pollutants. The range of potential antigens is wide, and ELISA is used in many research and detection fields to detect and quantify antigens in various sample types. The ELISA is used for analyzing specific substances such as cell lysate, blood sample, food, etc., and is widely used in scientific research, medical diagnosis, and food sanitation and environmental safety monitoring.
At present, the enzyme-linked adsorption test is divided into: direct ELISA, indirect ELISA, competitive ELISA, and sandwich ELISA. Currently, the sensitivity and specificity of the pandwich ELISA are superior to other types of ELISA. The Sandwick ELISA is commonly called as a Sandwich ELISA, the target antigen is captured by using a capture antibody coated on a plate, and then the target molecule can be quantitatively detected in a sample by using a detection antibody to recognize the antigen. In the current pandwich ELISA, monoclonal antibodies and polyclonal antibodies can be used in combination with each other. Given the diversity of the components in polyclonal antibodies, which give higher non-specific signals, the pandwich ELISA is more prone to use of two monoclonal antibodies.
The core technology in the sandwick ELISA development process consists in developing two monoclonal antibodies that can be combined with each other. The conventional development scheme at present is to develop various monoclonal antibodies related to target molecules by utilizing a hybridoma technology, then combine the monoclonal antibodies one by one, and test the specificity and sensitivity of detection after combination. This process may be due to overlapping antibody recognition domains, the effect of steric effects between proteins, leading to failure in ELISA development. In addition, animal immunization, fusion and cell line selection, antibody combination testing, the time and economic costs for the whole procedure are enormous.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for quickly constructing a pandwich ELISA, which can effectively avoid the problems of time and labor waste in construction caused by factors such as overlapping of two antibody recognition domains and interference of protein meta-resistance effect.
In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:
the invention discloses a method for quickly constructing a pandwich ELISA, which comprises the following steps: through an immune coprecipitation method, an antibody sequence which can form immune enzyme-linked adsorption experiment double-antibody sandwich pairing with a monoclonal antibody of a known target antigen is screened out from the polyclonal antibodies of the known antigen, and then a prokaryotic expression or protein synthesis method is utilized to obtain the target antibody, namely the rapid construction of the sandwich ELISA is completed.
Preferably, the method for rapidly constructing the pandwich ELISA comprises the following steps:
1) Incubating the biotin-labeled monoclonal antibody with an antigen overnight to form a stable antibody-antigen complex;
2) Adding the polyclonal antibody to be screened into the antibody antigen complex obtained in the step 1), and continuing incubation to form a complex similar to a sandwich structure;
3) Adding a streptomycin-marked solid medium into the reaction system in the step 2), continuously incubating, separating the compound similar to a sandwich structure through centrifugation to obtain an antibody capable of combining an antigen with a monoclonal antibody, sequencing, expressing and purifying to obtain the target antibody.
Further preferably, in step 1), the molar ratio of monoclonal antibody to antigen is 1:1.
Further preferably, the molar concentration of the biotin-labeled monoclonal antibody used is greater than the molar concentration of the polyclonal antibody.
Further preferably, in step 3), the solid medium is a gel particle.
Still more preferably, the gel particles are subjected to a blocking treatment with 1% to 3% bovine serum albumin prior to use.
More preferably, 1% -3% bovine serum albumin is prepared by adding 0.1-0.3 mg BSA powder to 10ml PBS-T solution.
Still more preferably, in step 3), the specific sequencing procedure is as follows:
firstly, washing gel particles, eluting positive antibodies on the gel particles by using ammonia water, freeze-drying the eluted liquid, recovering the adhesive tape containing the target strip by gel electrophoresis, and carrying out mass spectrum sequencing to obtain the light chain variable region sequence of the target antibody.
Still more preferably, the gel particles are washed with 150mM ammonium hydrogen phosphate at pH 7.4 and the positive antibodies on the gel particles are eluted with 150mM ammonia.
Further preferably, in step 3), the specific expression and purification operations are as follows:
cloning the sequenced sequence into a plasmid vector by using a gene synthesis method, adding a His tag on the N section of the sequence, converting the synthesized plasmid into escherichia coli expression bacteria, inducing by using IPTG, crushing the induced bacterial liquid to obtain total protein, and purifying by using a His tag purification method to obtain the target protein.
Compared with the prior art, the invention has the following beneficial effects:
the method disclosed by the invention utilizes the principle of co-immunoprecipitation, utilizes the known monoclonal antibody to bind with a target antigen, directly screens and obtains a target antibody sequence in a polyclonal antibody, simulates the binding of the sandwich ELISA antibody antigen in the whole process, directly carries out mutual pairing of two antibodies in a multi-antibody stage, avoids factors such as overlapping of two antibody recognition domains and interference of a protein meta-resistance effect, and the like, thereby saving time and economic cost required for developing monoclonal antibodies, and being capable of rapidly developing the sandwich ELISA and a corresponding kit in a time-saving and labor-saving manner. The concrete advantages are as follows:
firstly, the invention can save the cost of developing the pandwich ELISA time, and under the condition that the target antigen has monoclonal antibodies and polyclonal antibodies, the antibody combination is directly carried out by using an immunoprecipitation method, and the proper antibody combination is obtained by screening, and the development is carried out before and after about 2-3 months;
secondly, the invention can save the antigen cost of developing the pandwich ELISA, and the greatest bottleneck is lack of target protein aiming at the pandwich ELISA development of rare proteins, which causes difficulty for antibody development, and the pandwich ELISA can be developed by only using about 100ug antigen;
thirdly, the invention can save the economic cost of developing the pandwich ELISA, and the development of the conventional method needs to consume a great deal of economic cost for antibody development, but the invention has low dosage of the antigen or the antibody used in the process; in addition, mass spectrum sequencing, gene synthesis and prokaryotic expression are all routine experimental operations, and the economic cost is low.
Further, the sandwich complex of diabody and antigen is separated from the solution using labeled gel particles or other solid medium.
Further, blocking with BSA solution and washing with PBST solution at varying concentrations reduced non-specific adsorption during co-immunoprecipitation.
Furthermore, the combined antibody-antigen complex is eluted by ammonia water, so that the later-stage direct freeze-drying sequencing is facilitated, and the combination of biotin and streptomycin is prevented from being damaged in a large amount.
Drawings
FIG. 1 is a flow chart of the technical scheme of the invention;
FIG. 2 is a first partial co-immunoprecipitation flow diagram;
FIG. 3 shows a plasmid vector with N-segment His-tag pET-30a (+) added; .
FIG. 4 is a graph showing the comparison result of western blot of the experimental group and the control group after co-immunoprecipitation;
FIG. 5 is a graph showing the comparison of coomassie brilliant blue staining of the experimental group and the control group after co-immunoprecipitation;
FIG. 6 is a sequencing result analysis;
FIG. 7 shows prokaryotic expression results;
FIG. 8 shows the results of an indirect ELISA.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention is described in further detail below with reference to the attached drawing figures:
as shown in the flow chart of fig. 1, the technical scheme of the invention is divided into four parts:
a first part: the steps of co-immunoprecipitation and screening for a suitable polyclonal antibody are shown in FIG. 2, and specifically include the steps of:
1) The stable antibody-antigen complex was formed by first incubating the biotin-labeled monoclonal antibody (1.33 mM) with the antigen Abeta 1-42 (1.35 mM) in 100ul of phosphate buffer overnight. To increase efficiency, the molar ratio of monoclonal antibody to antigen is close to 1:1;
2) Polyclonal antibodies to be screened (1.13 mM) were then added to the antibody antigen complex after the previous incubation and incubated in 100. Mu.L of phosphate buffer for two hours at room temperature to form a complex resembling a "sandwich" structure. To avoid competition of the polyclonal antibody for the labeled mab from the binding site of the antigen, it is ensured that the molar concentration of biotin-labeled mab is greater than that of polyclonal antibody.
3) Adding the gel particles marked by streptomycin into the solution in the previous step, incubating for 1.5h in 1ml of phosphate buffer solution at room temperature, screening the combined positive antibodies by using a centrifugal mode, and taking care to gently shake the sample in the incubation process so as to avoid precipitation of the gel particles. In order to avoid false positive caused by non-specific adsorption, gel particles are firstly blocked by 1% -3% of unequal bovine serum albumin, the precipitate after centrifugation is washed three times by PBS-T (0.05% tween), and meanwhile, negative control is required to be set (antigen is not added in the whole co-immunoprecipitation process), so that false positive sequences can be conveniently removed from the sequencing result in the later period.
A second part: antibody sequencing and data analysis
The method comprises the following steps:
1) The gel particles were first washed with 1ml 150mm ammonium bicarbonate (ph=7.4) to wash off residual PBST.
2) The positive antibodies on the gel particles are eluted by using 150mM ammonia water, and the connection between biotin and streptomycin is hardly broken by using 150mM ammonia water, so that most of the eluted antibodies are positive antibodies obtained by screening.
3) The eluted liquid was lyophilized and run on SDS-PAGE non-reducing gel.
4) And (3) recovering the adhesive tape containing the target band, and carrying out mass spectrum sequencing to obtain the light chain variable region sequence of the target antibody.
Third section: expression and purification of antibodies
The method comprises the following steps:
1) Cloning the sequenced sequence into a plasmid vector of pET-30a (+) by using a gene synthesis method, and adding a His tag to the N segment of the sequence, as shown in figure 3;
2) The synthesized plasmid is transformed into escherichia coli expression bacteria (BL 21 strain), and IPTG induction is utilized, wherein the induction conditions (time, temperature and IPTG concentration) depend on proteins;
3) The induced bacterial liquid is treated by ultrasonic or high-pressure crushing to obtain total protein, and the target protein is purified by using a His tag purification strategy.
Fourth part: and (3) verifying results: verification of purified antibodies by ELISA
The method comprises the following steps:
1) Verifying the affinity of the purified antibodies to the antigen by indirect ELISA;
2) Detection sensitivity after pairing of the two antibodies was verified using a pandwich ELISA.
Specifically, the invention is proved to be feasible through preliminary simulation experiments, and comprises the following steps:
1) Co-immunoprecipitation
After incubation of the complex with the biotin-labeled Abeta 1-42 monoclonal antibody 4G8 (1.33 mM) and Abeta 1-42 (1.35 mM) in 100. Mu.L of phosphate buffer, a "sandwich-like" complex was formed by incubation with Abeta 1-42 polyclonal antibody S98 (1.13 mM); the "sandwich" complex was separated from the solution using streptomycin-labeled gel particles, and to avoid the effects of non-specific adsorption, a control group (no aβ1-42 added) was set and the gel particles were blocked with BSA and washed with PBST, then eluted with ammonia, and verified using western blot and coomassie brilliant blue staining.
The results are shown in FIG. 4 and FIG. 5. In FIG. 4, the result of immunoprecipitation was verified by using a western blot, and the experimental group after co-immunoprecipitation was compared with the control group by using a western blot: the experimental group screens out the S98 antibody which can be successfully paired with 4G8, and the control group has no obvious antibody band. Therefore, the experimental group can be seen to be capable of specifically screening out the proper antibody, and the control group can be seen to have very low influence of nonspecific adsorption.
In fig. 5, coomassie blue staining was performed on the supernatant after elution of the separated gel particles with ammonia, in comparison with coomassie blue staining of the experimental group and the control group after co-immunoprecipitation. In fig. 5 (a), the experimental group screened S98 antibody successfully paired with 4G8, and the control group had no distinct antibody bands; in FIG. 5 (b) it is shown that the most biotin-labeled 4G8 (4G 8 b) was not eluted by comparing the eluted supernatant with the gel particles.
2) Sequencing and data analysis
The strips of the experimental and control groups were subjected to mass spectrometry, and later analysis was performed in combination with Uniprot, IMGT database and sequence alignment tools to find the Complementarity Determining Regions (CDRs) and Framework Regions (FR) of the light chain variable region, and the data results are shown in fig. 6.
3) Gene synthesis and prokaryotic expression
The target sequence obtained by sequencing is cloned into a plasmid vector of pET-30a (+) (as shown in figure 3), expressed in an expression bacterium of BL21, and purified by His-tag at the later stage, and the result is shown in figure 7, and the prokaryotic expression result shows that the sequence of one item is cloned into the vector, so that the target sequence can be expressed in a large amount in escherichia coli.
4) And (3) verifying results: verification of purified antibodies by ELISA
The results of the indirect ELISA are shown in FIG. 8, and it can be seen that the VL fragments obtained by prokaryotic expression can recognize the antigen, and have strong affinity with the antigen compared with the negative control.
In summary, the invention is to find the antibody sequence capable of forming the double-antibody sandwich pairing of the immune enzyme-linked adsorption experiment with the known monoclonal antibody in the polyclonal antibody of the known antigen by utilizing the principle of co-immunoprecipitation and obtain the target antibody by utilizing the prokaryotic expression or protein synthesis experimental method, thereby realizing the aim of developing the pandwich ELISA.
The invention can be used for developing monoclonal antibodies, and the monoclonal antibodies of known target antigens are utilized to find antibody sequences which can form immune enzyme-linked adsorption experiment double-antibody sandwich pairing with the known monoclonal antibodies in the polyclonal antibodies of the known antigens by utilizing the principle of co-immunoprecipitation, and conventional monoclonal antibodies or humanized antibodies and the like are obtained by utilizing antibody engineering.
The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (9)
1. A method for rapidly constructing a pandwich ELISA comprising: through an immune coprecipitation method, an antibody sequence which can form immune enzyme-linked adsorption experiment double-antibody sandwich pairing with a monoclonal antibody of a known target antigen is screened out from the polyclonal antibodies of the known antigen, and then a prokaryotic expression or protein synthesis method is utilized to obtain the target antibody, namely the rapid construction of the sandwich ELISA is completed.
2. The method for rapid construction of a pandwich ELISA according to claim 1 comprising the steps of:
1) Incubating the biotin-labeled monoclonal antibody with an antigen overnight to form a stable antibody-antigen complex;
2) Adding the polyclonal antibody to be screened into the antibody antigen complex obtained in the step 1), and continuing incubation to form a complex similar to a sandwich structure;
3) Adding a streptomycin-marked solid medium into the reaction system in the step 2), continuously incubating, separating the compound similar to a sandwich structure through centrifugation to obtain an antibody capable of combining an antigen with a monoclonal antibody, sequencing, and expressing and purifying to obtain the target antibody.
3. The method of rapid construction of a pandwich ELISA according to claim 2 wherein in step 1) the molar ratio of monoclonal antibody to antigen is 1:1.
4. The method of rapid construction of a pandwich ELISA according to claim 2 wherein the molar concentration of biotin-labeled monoclonal antibody used is greater than the molar concentration of polyclonal antibody.
5. The method of rapid construction of a pandwich ELISA according to claim 2 wherein in step 3) the solid medium is a gel particle.
6. The method of rapid construction of a pandwich ELISA according to claim 5 wherein the gel particles are blocked with 1% to 3% bovine serum albumin prior to use.
7. The method for rapid construction of a pandwich ELISA according to claim 5, characterized in that in step 3), the sequencing is performed as follows:
firstly, washing gel particles, eluting positive antibodies on the gel particles by using ammonia water, freeze-drying the eluted liquid, recovering the adhesive tape containing the target strip by gel electrophoresis, and carrying out mass spectrum sequencing to obtain the light chain variable region sequence of the target antibody.
8. The method of rapid construction of a pandwich ELISA according to claim 7 wherein the gel particles are washed with 150mM ammonium hydrogen phosphate at pH 7.4 and the positive antibodies on the gel particles are eluted with 150mM aqueous ammonia.
9. The method for rapid construction of a pandwich ELISA according to claim 2, characterized in that in step 3), the expression and purification are performed as follows:
cloning the sequenced sequence into a plasmid vector by using a gene synthesis method, adding a His tag on the N section of the sequence, converting the synthesized plasmid into escherichia coli expression bacteria, inducing by using IPTG, crushing the induced bacterial liquid to obtain total protein, and purifying by using a His tag purification method to obtain the target protein.
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| US20080254551A1 (en) * | 2004-12-30 | 2008-10-16 | Achsel Tilmann | Immunoprecipitaion-Based Method to Purify and Characterise Biological Macromolecular Complexes |
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| CN106918710A (en) * | 2015-12-28 | 2017-07-04 | 西藏自治区人民医院 | A kind of double-antibody sandwich elisa detection kit of human apolipoprotein C- III and application |
| CN109678962B (en) * | 2019-01-22 | 2020-05-08 | 山西农业大学 | Cdk5 nano antibody and screening method |
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