CN115541886A - Quantitative detection reagent and detection method for trace protein - Google Patents
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Abstract
The invention provides a quantitative detection reagent for trace protein, which comprises a DNA fragment marked by an affinity molecule A and an affinity molecule B; the affinity molecule B can mark a protein to be detected, and the affinity molecule A and the affinity molecule B can be specifically combined with each other; the DNA fragment sequence is shown in SEQ ID NO. 1. The invention also provides a method for quantitatively detecting the trace protein, which utilizes the affinity molecule A to mark the protein to be detected, utilizes the affinity molecule B to mark the artificially synthesized DNA segment, utilizes the characteristic that the affinity molecule A and the affinity molecule B can be specifically combined with each other, utilizes the quantitative PCR technology to detect the DNA segment, and reflects the amount of the protein to be detected through the relative amount of the DNA. The method is rapid and simple to operate, and can sensitively and accurately quantitatively detect the trace protein.
Description
Technical Field
The invention relates to a trace protein detection technology with simple operation and accurate quantification, belonging to the technical field of molecular biology detection.
Background
Among the techniques for quantitative detection of proteins, enzyme linked immunosorbent assay (ELISA) and Radioimmunoassay (RIA) are sensitive. The ELISA is characterized in that firstly, an antigen or an antibody is bonded to the surface of a certain solid phase carrier and keeps the immunocompetence, then, an enzyme-labeled antibody (formed by covalently connecting the antibody and a certain enzyme and keeping the immunocompetence and the activity of the enzyme) is specifically bonded with the antigen or the antibody adsorbed on the solid phase carrier, so that the enzyme-antibody-antigen is combined together to form a compound, after the unbound enzyme is cleaned, a substrate solution is dripped, a color reaction can occur to the substrate under the action of the enzyme, the shade of the color reaction is in positive correlation with the amount of the corresponding antibody or antigen in a specimen, and the shade of the color reaction is in direct proportion in a certain concentration range (linear range). Therefore, qualitative or quantitative analysis can be performed according to the shade of the color reaction. Because the catalytic efficiency of the enzyme is very high, the reaction effect can be greatly amplified, so that the determination method achieves higher sensitivity, and the ELISA method becomes a specific and sensitive detection method. However, it is difficult to accurately quantitatively detect an antigen at an extremely low concentration because the linear range of detection by the ELISA technique is narrow.
Radioimmunoassay RIA is an in vitro microanalysis method using the sensitivity of radioisotopes and specific binding of antigen-antibody reactions, and the principle is to competitively bind a radioisotope-labeled antigen and an unlabeled antigen (analyte) to a non-sufficient amount of specific antibody, separate an antigen-antibody complex after reaction, and determine the amount of unlabeled antigen by measuring radioactivity. RIA is also called competitive radiosaturation assay because RIA is the theoretical basis for binding of radiolabel to antibody in competition with unlabeled antigen. The RIA method has advantages of sensitivity, specificity, simplicity, convenience, and a small amount of sample, but the sensitivity is only picomolar. In addition, RIA also has the defects of cross reaction, false positive reaction, incapability of inactivating degrading enzymes and salts, influence on results in some cases due to pH and the like, and even has radioactive pollution.
The Western Blot method employs polyacrylamide gel electrophoresis, and the detected substance is a protein, "probe" is an antibody, "and" secondary antibody for color development "is labeled. A protein sample separated by PAGE (polyacrylamide gel electrophoresis) is transferred to a solid phase carrier (such as a nitrocellulose membrane), and the protein is adsorbed by the solid phase carrier in a non-covalent bond form, and the type of the polypeptide separated by the electrophoresis and the biological activity of the polypeptide can be kept unchanged. Taking protein or polypeptide on a solid phase carrier as an antigen, carrying out immunoreaction with a corresponding antibody, then carrying out reaction with a second antibody marked by enzyme or isotope, and carrying out substrate chromogenic or autoradiography to detect protein components expressed by specific target genes separated by electrophoresis. This technique is also widely used to detect protein level expression. However, the WB technology is complex in operation, complex in steps and more in error factors in the experimental process.
Polymerase Chain Reaction (PCR) is the most sensitive nucleic acid detection technique in modern molecular biology techniques, and can theoretically detect 1 copy of template DNA. The fluorescent quantitative PCR (FQ-PCR) technology is based on the PCR technology, and the fluorescence change of free fluorescein released by degradation of a TaqMan probe marked by fluorescein in the DNA synthesis process accompanying the PCR is detected in real time, so that the quantity of the initial DNA template can be accurately quantified. The technology is characterized in that heat-resistant DNA polymerase, specific primers, sequence specificity TaqMan probes, dNTPs substrates, template DNA, magnesium ions and the like are placed in the same buffer reaction system to carry out repeated high-temperature, low-temperature and medium-temperature thermal cycles, so that the template DNA is denatured, the primers and the templates are renatured, the primers extend along the template DNA and degrade the TaqMan probes, fluorescein is released, the fluorescence intensity detection and recording are repeatedly carried out, the cycle number (Ct value) when the fluorescence value reaches the fluorescence threshold value is obtained according to the fluorescence intensity change curve while the target DNA fragment is amplified by 2n times (wherein n is the number of thermal cycles), the Ct value is in a linear relation with the logarithm of the initial template number of FQ-PCR, and the linear range is extremely large, so that accurate quantitative analysis can be carried out on the initial template number of almost various concentration ranges according to the obtained Ct value. Although FQ-PCR is the highest sensitivity and accuracy technique among the current molecular detection techniques, it cannot be directly used for detecting proteins because it can only detect DNA molecules or RNA molecules (which need to be reverse-transcribed into cDNA and then detected), and thus cannot be effectively used in the field of quantitative detection of proteins.
The immuno-PCR technology is a new protein detection system formed by combining an immunoadsorption test (ELISA) with a PCR system. The existing immuno-PCR technology mainly comprises two parts, wherein the immunoreaction of the first part is similar to the ordinary enzyme-linked measuring process, firstly, an antigen to be measured (such as Bovine Serum Albumin (BSA)) is used for coating a microtiter plate hole, a corresponding specific antibody is added, then the antibody is combined with the antigen on a solid phase to form an antigen-antibody complex, a Protein A part in a Protein A-Streptavidin (Protein A-Streptavidin) chimera (recombinant fusion Protein) can be combined with an antibody IgG in the antigen-antibody complex on the solid phase, and a Streptavidin part can react with Biotin in biotinylated pUC19 (plasmid DNA) (Biotin-pUC 19), so that specific DNA is indirectly adsorbed to the solid phase; the second part, which is a conventional PCR assay, is that the pUC19 plasmid DNA adsorbed to the solid phase in the first step can be amplified by PCR for several millions of times in several hours in the presence of the corresponding primers, and the amount of PCR product is positively correlated with the amount of antigen on the solid phase, so that the condition of the antigen molecule can be examined based on the PCR result. However, since there is a plateau phase of PCR amplification, and the number of cycles required for PCR reactions with different concentrations of each template to reach the plateau phase is different, the amount of PCR product is not proportional to the number of starting templates, and thus the quantification is not accurate. In addition, the existing immuno-PCR needs to coat the antigen to be detected of each sample independently, and because the sample conditions are different, the coating efficiency and the immunoadsorption efficiency of the antigen are not uniform, and meanwhile, the uniformity of the adopted biotinylated plasmid is insufficient, the operation is more complicated, the quantification is not accurate enough, and the application is less.
Therefore, a detection technique for trace protein, which is simple to operate and can be accurately quantified, is still lacking at present.
Disclosure of Invention
The invention aims to provide a detection reagent and a method which are simple to operate and can accurately quantify trace protein.
The invention provides a quantitative detection reagent for trace protein, which comprises a DNA fragment marked by an affinity molecule A and an affinity molecule B; the affinity molecule B can mark a protein to be detected, and the affinity molecule A and the affinity molecule B can be specifically combined with each other; the DNA fragment sequence is shown in SEQ ID NO. 1.
Further, the affinity molecule a is avidin, and the affinity molecule B is biotin.
Further, the affinity molecule a is biotin, and the affinity molecule B is avidin.
Furthermore, the reagent also comprises a secondary antibody of the protein antibody to be detected connected with the magnetic beads.
The invention also provides a method for quantitatively detecting the trace protein, which comprises the following steps:
5. a method for quantitatively detecting a trace amount of protein, which comprises the steps of: the method comprises the following steps:
(1) Adding a secondary antibody of the protein antibody to be detected, which is connected with the magnetic bead, into the sample to be detected to obtain a magnetic bead-antibody-antigen compound;
(2) Separating the magnetic bead-antibody-antigen complexes using a magnetic separator;
(3) Adding excessive affinity molecule B for reaction to obtain a magnetic bead-antibody-antigen-affinity molecule B compound;
(4) Adding excessive DNA fragments marked by the affinity molecules A for reaction to obtain a magnetic bead-antibody-antigen-affinity molecule B-affinity molecule A-DNA compound;
(5) Separating the magnetic bead-antibody-antigen-affinity molecule B-affinity molecule A-DNA compound by using a magnetic separator;
(6) Detecting the DNA fragments by using a quantitative PCR technology, and calculating the amount of the protein to be detected according to the relative amount of the DNA fragments;
the affinity molecule A and the affinity molecule B are specifically combined with each other; the DNA fragment sequence is shown in SEQ ID NO. 1.
Further, the affinity molecule A is avidin, and the affinity molecule B is biotin; or the affinity molecule A is biotin and the affinity molecule B is avidin.
Further, the reactions in the above-mentioned step (1), step (3) and step (4) are carried out in a reaction solution which is PBS containing 1% BSA.
Further, the magnetic bead-antibody-antigen complex of the step (1) and the magnetic bead-antibody-antigen-affinity molecule B-affinity molecule a-DNA complex of the step (4) are immobilized by an immobilization reagent, and the amount of the immobilization reagent is 0.5 to 1.5 times, preferably 1 time, the volume of the reaction solution;
the immobilized reagent is 0.5-5 (v/v)% paraformaldehyde solution prepared by PBS; or 3-6 (v/v)% formaldehyde solution prepared by PBS; or 1-3 (v/v)% glutaraldehyde solution prepared by PBS; preferably, the concentration of the paraformaldehyde solution prepared by PBS is 1 (v/v)%; the concentration of the formaldehyde solution prepared by PBS is 4 (v/v)%; the concentration of the glutaraldehyde solution prepared by PBS is 1.2 (v/v)%.
Further, the step (1) further comprises a step of washing with a PBS solution to remove excess magnetic bead-antibody; the step (5) further comprises a step of washing the uncomplexed DNA fragments labeled with affinity molecule B and affinity molecule A with a washing solution containing 1mM EDTA in PBS.
Further, the above-mentioned quantitative PCR technique is a real-time fluorescent quantitative PCR reaction, and the real-time fluorescent quantitative PCR reaction system comprises 0.2-0.4umol/L upstream primer GAGCCCACTGGACTTCT, most preferably 0.25umol/L,0.2-0.4umol/L downstream primer GTGATGTTGCGGGTCTG, most preferably 0.25umol/L,0.2-0.4umol/L TaqMan probe GGAGGGCTGTGTCAAG, most preferably 0.25umol/L, 1-3U/50ul Taq DNA polymerase, most preferably 1.5U/50ul Taq DNA polymerase, most preferably 0.25mmol/L dNTPs 0.2-0.5mmol/L and PCR reaction buffer.
Furthermore, the PCR reaction buffer solution comprises 50 to 75mmol/L KCl, 10 to 15mmol/L Tris.HCl PH8.3 and 0.01 to 0.015 percent of gelatin; preferably, the PCR reaction buffer comprises 50mmol/L KCl, 10mmol/L Tris.HCl pH8.3, 0.01% gelatin
Furthermore, the real-time fluorescent quantitative PCR reaction is carried out under the reaction conditions of 94-96 ℃ 2min,94 ℃ 10s → 50 ℃ 10s → 60 ℃ 20s and 45 cycles, the fluorescence is detected and recorded at 60 ℃ for 20s, ct values of the PCR reactions are read, and the content of the protein to be detected is calculated according to the following formula:
Ct＝-1/lg(1+E x )*lgX 0 +lgN/lg(1+E x )
X 0 ＝Y 0
further, the above X 0 As initial template amount, E x For amplification efficiency, N is the amount of amplification product at which the fluorescence amplification signal reaches a threshold intensity, Y 0 The amount of the protein to be detected.
The invention has the following beneficial effects: the method is simple and quick to operate, and can accurately and quantitatively determine the content of the protein by detecting the fluorescence value of the DNA.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Detailed Description
The raw materials and equipment used in the invention are known products, and can be obtained by purchasing commercial products.
Example 1 quantitative detection of Trace proteins according to the invention
Step 1,
Preparation of a magnetic bead-secondary antibody Complex, 400. Mu.l of the magnetic bead was washed 3 times with 400. Mu.l of 30mM MES (pH 6.5), 50. Mu.l of 60mg/ml NHS solution and 50. Mu.l of solution containing 60mg/ml EDC were added, incubation was carried out at 36 ℃ for 1 hour, washing was carried out 2 times with 400. Mu.l of 30mM MES (pH 6.5), 80. Mu.l of the secondary antibody was added, 120. Mu.l of 30mM MES (pH 6.5) was added, and the mixture was left at 2 to 6 ℃ for 3 hours, and further washed 4 times with 400. Mu.l of PBS (containing 1 BSA), and stored at 2 to 6 ℃;
the resultant magnetic bead-secondary antibody complex was resuspended in a reaction solution containing 1% BSA in PBS, a small amount of the protein sample to be tested was mixed with the reaction solution and allowed to react to produce a magnetic bead-antibody-antigen complex, and the magnetic bead-antibody-antigen complex was immobilized with an immobilizing reagent so as to withstand washing repeated strictly. The immobilizing agent is 0.5-5 (v/v)% paraformaldehyde in PBS, and more preferably 1 (v/v)% paraformaldehyde. Other useful immobilizing agents are 3-6 (v/v)% formaldehyde in PBS, with a more preferred concentration of 4 (v/v)%; 1-3 (v/v)% glutaraldehyde solution in PBS, more preferably 1.2 (v/v)%. The dosage of the cell immobilization reagent is as follows: the immobilizing reagent is added to the reaction solution in an amount of 0.5 to 1.5 times by volume, more preferably 1 time by volume. And separating by a magnetic separator, washing redundant magnetic beads-antibodies which do not form magnetic bead-antibody-antigen complexes by washing liquid, and fully washing the magnetic beads-antibody-antigen complexes obtained by separation by adopting a large amount of PBS.
Adding excessive biotin, and reacting to generate a magnetic bead-antibody-antigen-biotin compound;
step 2,
And centrifuging the avidin at low temperature, adding the avidin into a solution containing specific primer DNA, and combining the 5 tail end of the primer with the avidin to obtain avidin-labeled DNA.
The DNA sequence of the avidin-labeled primer is GTGATGTTGCGGGTCTGGGAGGGGCTGTGTCAAGGGATGCTGAAGAAGAAGT CCAGTGGGCTC. The biotin-labeled DNA was dissolved in a buffer of 0.5mg/ml denatured salmon sperm DNA,20mM Tris,10mM EDTA (pH 7.5);
and (2) adding reaction liquid and excessive avidin-labeled DNA into the product obtained in the step (1), reacting to generate a magnetic bead-antibody-antigen-biotin-avidin-DNA compound, and fixing the magnetic bead-antibody-antigen-biotin-avidin-DNA compound by adopting an immobilization reagent. And separating by a magnetic separator, washing away excessive DNA which does not form a magnetic bead-antibody-antigen-biotin-avidin-DNA complex by using a washing solution, suspending in ultrapure water, and centrifugally collecting the DNA complex for later use. The wash was a PBS solution containing 1mM EDTA.
Step 3,
And detecting the content of the separated DNA by quantitative PCR so as to obtain the content of the protein to be detected.
A real-time fluorescent quantitative PCR reaction system comprises 0.2-0.4umol/L of an upstream primer GAGCCCACTGGACTTCT, most preferably 0.25umol/L,0.2-0.4umol/L of a downstream primer GTGATGTTGCGGGTCTG, most preferably 0.25umol/L,0.2-0.4umol/L of a TaqMan probe GGAGGGCTGTCAAG, most preferably 0.25umol/L, 1-3U/50ul of Taq DNA polymerase, most preferably 1.5U/50ul,0.2-0.5mmol/L of dNTPs, most preferably 0.25mmol/L, and PCR reaction buffer solution.
Real-time fluorescent quantitative PCR reaction, wherein the reaction conditions are 94-96 ℃ for 2min,94 ℃ for 10s → 50 ℃ for 10s → 60 ℃ for 20s, and 45 cycles, and the fluorescence is detected and recorded at 60 ℃ for 20 s. Ct values of each PCR reaction were read and used as the basis for quantification.
Ct＝-1/lg(1+E x )*lgX 0 +lgN/lg(1+E x )；
X 0 ＝Y 0 。
X is above 0 As initial template amount, E x For amplification efficiency, N is the amount of amplification product at which the fluorescence amplification signal reaches a threshold intensity, Y 0 Is the amount of protein to be measured.
By E x For amplification efficiency, N is the amount of amplified product and Ct value when the fluorescence amplification signal reaches the threshold intensity 0 And acquiring initial DNA content for initial template amount, and acquiring the amount of the protein to be detected according to the relative amount of the initial DNA.
Wherein the 1XPCR buffer comprises 50mmol/L KCl, 10mmol/L Tris.HCl pH8.3, 0.01% gelatin, and most preferably 1XPCR buffer.
In conclusion, the invention provides a reagent for quantitatively detecting trace protein, which can accurately and quantitatively determine the content of protein by detecting the fluorescence value of DNA through a simple and quick method, and has good application prospect in the field of trace protein detection.
SEQUENCE LISTING
<110> institute of medical science of Chengdu
<120> quantitative detection reagent and detection method for trace protein
<130> GY044-2021P0113330CCU
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 59
<212> DNA
<213> Artificial sequence
<400> 1
gtgatgttgc gggtctggga gggctgtgtc aagggatgct gaagaagtcc agtgggctc 59
Claims (12)
1. The trace protein quantitative detection reagent is characterized by comprising a DNA fragment marked by an affinity molecule A and an affinity molecule B; the affinity molecule B can mark a protein to be detected, and the affinity molecule A and the affinity molecule B can be specifically combined with each other; the DNA fragment sequence is shown in SEQ ID NO. 1.
2. The detection reagent of claim 1, wherein the affinity molecule a is avidin and the affinity molecule B is biotin.
3. The detection reagent of claim 1, wherein the affinity molecule a is biotin and the affinity molecule B is avidin.
4. The detection reagent according to any one of claims 1 to 3, further comprising a secondary antibody to the antibody against the test protein, which is linked to the magnetic beads.
5. A method for quantitatively detecting trace protein, which is characterized by comprising the following steps: the method comprises the following steps:
(1) And adding the secondary antibody of the protein antibody to be detected connected with the magnetic bead into the sample to be detected to obtain the magnetic bead-antibody-antigen compound.
(2) Separating the magnetic bead-antibody-antigen complexes using a magnetic separator
(3) Adding excessive affinity molecule B for reaction to obtain a magnetic bead-antibody-antigen-affinity molecule B compound;
(4) Adding excessive DNA fragments marked by the affinity molecule A for reaction to obtain a magnetic bead-antibody-antigen-affinity molecule B-affinity molecule A-DNA compound;
(5) Separating the magnetic bead-antibody-antigen-affinity molecule B-affinity molecule A-DNA compound by using a magnetic separator;
(6) Detecting the DNA fragments by using a quantitative PCR technology, and calculating the amount of the protein to be detected according to the relative amount of the DNA fragments;
the affinity molecule A and the affinity molecule B are specifically combined with each other; the sequence of the DNA fragment is shown as SEQ ID NO. 1.
6. The method of claim 5, wherein affinity molecule A is avidin and affinity molecule B is biotin; or the affinity molecule A is biotin and the affinity molecule B is avidin.
7. The method according to claim 5, wherein the reactions of step (1), step (3) and step (4) are carried out in a reaction solution comprising 1% BSA in PBS.
8. The method according to claim 5, wherein the magnetic bead-antibody-antigen complex of step (1) and the magnetic bead-antibody-antigen-affinity molecule B-affinity molecule A-DNA complex of step (4) are immobilized with an immobilization reagent in an amount of 0.5 to 1.5 times, preferably 1 time, the volume of the reaction solution;
the fixing agent is 0.5-5 (v/v)% paraformaldehyde solution prepared by PBS; or 3-6 (v/v)% formaldehyde solution prepared by PBS; or 1-3 (v/v)% glutaraldehyde solution prepared with PBS; preferably, the concentration of the paraformaldehyde solution prepared by PBS is 1 (v/v)%; the concentration of the formaldehyde solution prepared by PBS is 4 (v/v)%; the concentration of the glutaraldehyde solution prepared by PBS is 1.2 (v/v)%.
9. The method of claim 5, wherein step (1) further comprises the step of washing with a PBS solution to remove excess bead-antibody; the step (5) further comprises a step of washing the DNA fragments labeled with affinity molecule B and affinity molecule A, which have not formed a complex, with a washing solution which is a PBS solution containing 1mM EDTA.
10. The method of claim 5, wherein the quantitative PCR technique is a real-time fluorescent quantitative PCR reaction comprising 0.2-0.4umol/L upstream primer GAGCCCACTGGACTTCT, most preferably 0.25umol/L,0.2-0.4umol/L downstream primer GTGATGTTGCGGGTCTG, most preferably 0.25umol/L,0.2-0.4umol/L TaqMan probe GGGGGGGGCTGTCAAG, most preferably 0.25umol/L, taq DNA polymerase 1-3U/50ul, most preferably 1.5U/50ul,0.2-0.5mmol/L dNTPs, most preferably 0.25mmol/L, and PCR reaction buffer.
11. The method of claim 10, wherein the PCR reaction buffer comprises 50-75 mmol/L KCl, 10-15 mmol/L tris.hcl PH8.3, 0.01-0.015% gelatin; preferably, the PCR reaction buffer comprises 50mmol/L KCl, 10mmol/L Tris.HCl pH8.3, 0.01% gelatin.
12. The method of claim 10 or 11, wherein the real-time fluorescent quantitative PCR reaction is performed under the reaction conditions of 94-96 ℃ 2min,94 ℃ 10s → 50 ℃ 10s → 60 ℃ 20s, and 45 cycles, the fluorescence is detected and recorded at 60 ℃ 20s, the Ct value of each PCR reaction is read, and the content of the protein to be detected is calculated according to the following formula:
Ct＝-1/lg(1+E x )*lgX 0 +lgN/lg(1+E x )；
X 0 ＝Y 0 ；
said X 0 As an initial template amount, E x For amplification efficiency, N is the amount of amplification product at which the fluorescence amplification signal reaches a threshold intensity, Y 0 Is the amount of protein to be measured.
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