immuno-PCR

Summary

Immuno PCR (Im-PCR) is a technique for the detection of trace antigens that utilizes the specificity of the antigen-antibody reaction and the high sensitivity of the PCR amplification reaction. It uses a known DNA molecule labeled with an antibody as a probe, which reacts with the antigen to be tested, PCR amplifies the DNA molecule adhering to the antigen-antibody complex, and electrophoresis characterizes the DNA molecule, and determines whether the antigen to be tested is present or not based on the presence or absence of a specific PCR product.

Operation method

immuno-PCR

Principle

The first part of the immunoreaction is similar to the common ELISA procedure; the second part is the usual PCR assay, where the amount of antigen molecules is ultimately reflected by the amount of PCR product. In the first step, the wells of the microtitre plate are first coated with the antigen to be tested (e.g., bovine serum albumin (ESA)), and then the corresponding specific antibody is added, so the antibody binds to the antigen on the solid phase to form an antigen-antibody complex, and the protein A portion of the protein A-streptavidin chimera (recombinant fusion protein) binds to the IgG portion of the antibody in the solid phase antigen-antibody complex, and the antibody IgG portion of the solid-phase antibody binds to the IgG portion of the solid-phase antibody. The protein A portion of the Protein A-Streptavidin chimera (recombinant fusion protein) binds to the antibody IgG in the antigen-antibody complex on the solid phase, while the streptavidin portion reacts with the biotin in biotinylated pUC19 (plasmid DNA) (Biotin-pUC19) to indirectly adsorb a specific piece of DNA to the solid phase. The next step is the PCR process in the second step. pUC19 plasmid DNA adsorbed in the solid phase in the first step can be amplified millions of times in a few hours by PCR in the presence of the appropriate primers, and the amount of PCR product is directly proportional to the amount of antigen in the solid phase. PCR consists of the following five components: (1) the antigen to be tested; (2) a biotinylated antibody; (3) affinities (ligand molecules); (4) biotinylated DNA; and (5) a biotinylated antibody. ) biotinylated DNA; (5) PCR amplification. Main procedures: (1) Antigen + biotinylated antibody → antigen-biotinylated antibody complex; (2) Addition of affinity element → antigen-biotinylated antibody-affinity element complex; (3) Addition of biotinylated DNA → antigen-biotinylated antibody-affinity element-biotinylated DNA; (4) PCR amplification of the biotinylated DNA part.

Materials and Instruments

Antigen Samples
Coating buffer Wash solution Dilution solution Agarose gel
Microtitre plate Electrophoresis apparatus Electrophoresis tank

Move

I. Preparation of biotinylated DNA
A 280 bp DNa segment containing T3 and T7 primer sequences was prepared by PCR amplification of the phage BLuescript skt, with biotin-labeled M13 primers, as biotinylated DNA.
II. Immuno-PCR model
1. Reagents
Coating buffer: 20 mmol/L Tris-HCI (pH 9.5) with 150 mmol/L NaCl and 0.02% _NaN3;
Wash solution: 20 mmol/L Tris-HCI (pH 7.5) with 150 mmol/L NaCl 0.1 mmol/L EDTA and 0.1% Tween20;
Diluent: 20 mmol/L Tris-HCI (pH 7.5) with 150 mmol/L NaCl 0.45% skim milk and denatured salmon sperm DNA (0.1 mg/ml).
2. Operations
(1) Envelope
The antigen (BSA) was diluted with inclusion buffer, added to the microtitre plate (45 μl wells), overnight at 4°C (about 15 h), and the plate was washed 3 times × 5 min with washing solution.
(2) Closure
Each well was spiked with 200 μl of 20 mmol/L Tris-Hcl (pH 7.5) containing 4.5% skimmed milk and denatured salmon sperm DNA (1 mg/ml), 0.1 mmol/L EDTA (containing 150 mmol/L NaCl buffer, incubated at 37°C for 80 min, and the plates were washed several times.
(3) Antigen-antibody reaction
Dilute monoclonal anti-BSA antibody with release solution 1:8 000, add 50 μl to each well, and wash the plate wells 15 times × 10 min at room temperature (22°C) for 45 min to remove unbound antibody molecules.
(4) Chain-affinity protein A binding reaction
Add 50 μl of streptavidin-protein A chimera that has been bound to biotin-pUC19 diluted with diluent per well, room temperature (22°C) for 50 min, so that the chimera-pUC19 binds to the antigen-antibody complex in the solid phase, and then wash the plate 15 times × 10 min, and then wash it 3 times with NaN3-free TBS, then the microtitre plate can be used for the later PCR reaction.
(5) PCR
Experimental conditions 50 mmol/L KCI, 10 mmol/L Tris-HCI (pH 8.3 at 20°C), 1.5 mmol/L MgCl₂, gelatin (10 μg/ml), 0.8 mmol/L dNTPs (0.2 mM each), 2 μM primers (1 μM each), and TaqDNA polymerase (50 U/ml).
III. PCR cycle
Before PCR, the above reaction mixture was irradiated with ultraviolet (UV) light (254 nm), and then it was added to the microtitre plate wells, 40 μl per well, and covered with 20 μl of UV-irradiated paraffin wax, and the PCR cycle was performed on a PCR instrument according to the following temperatures: starting denaturation at 94°C for 5 min; 30 cycles: denaturation at 94°C for 5 min, annealing at 58°C for 1 min, extension at 72°C for 1 min, final extension 72°C for 5 min, and the PCR product obtained was a specific 261 bp fragment.
1. Dilute the bacterial solution to a certain concentration with 0.85% NaCl;
2. Add 50 μl to the microtiter plate and leave at 4°C overnight. Also set up a negative control (add 50 μl of 0.85% NaCl in another well);
3. With 400 μl of 0.05% warm 20pBS (hereafter referred to as TPBS), wash five times;
4. Add 2.25% of 100 μl of normal goat serum (NGS) PBS closed for 2 hours;

Carefully wash 3 times with TPBS containing 0.15% NGS;
6. Add 100 μl of monoclonal antibody (containing 100 μg of fresh fish sperm DNA) appropriately diluted with 0.75% NGS and incubate for 30 min at room temperature;
7. Wash five times with TPBS;
8. Add 50 μl of appropriately diluted biotinylated anti-mouse IgG antibody and incubate for 30 min at room temperature;
9. Wash five times with TPBS;
10. Add 60 μl of appropriately diluted biotinylated DNA and affinity protein and incubate for 30 min at room temperature;
11. Wash five times with TPBS and three times with HPLC grade water;
12. PCR amplification: add 50 μl of PCR reaction solution (containing T3 and T7 primers), 95 ℃ for 60 s, 50 ℃ for 110 s, 72 ℃ for 110 s, cycle 30 times. Take 10 μl of PCR product on 1.7% agarose gel for electrophoresis, and compare with the molecular weight standard of nucleic acid, the electrophoretic bands in the corresponding position are positive. The electrophoresis results were photographed when necessary for quantitative analysis.

Common Problems

I. Development

Immuno-PCR technology is still in the research stage, there is not a very mature and satisfactory method, supporting reagents are still lacking, so there are not many applications, in the several methods reported are tested with some known standards.Sano, Ruzicka and Hong zhou used bovine serum albumin, mouse IgG and recombinant human proto-oncogene product ETS; protein as the antigen to be tested for immuno-PCR, their results all showed that immuno-PCR is 105 times more sensitive than ELISA, and the background signal generated by PCR is very weak and can detect several hundred molecules. The antigen to be detected was subjected to immuno-PCR, and their results all showed that the sensitivity of immuno-PCR was 105 times higher than that of ELISA, and the background signal produced by PCR was so weak that several hundred molecules of antigen could be detected, and in theory immuno-PCR could detect one molecule of antigen, so immuno-PCR is particularly suitable for the detection of antigenic molecules of which the content is particularly small. There are some disadvantages of immuno-PCR, in the reported literature are used to detect the antigen directly adsorbed solid-phase, so that the homogeneity of the solid-phase will inevitably have a great impact on the results; at the same time, the detection of the sample solution of other components can also be adsorbed in the solid-phase, is very easy to produce a background of too high or decreased accuracy. In addition, some difficult to absorb the solid phase of the antigen can not be detected by immuno-PCR, the specificity and homogeneity of the connecting molecules have a great impact on the PCR, from the theoretical point of view of Hong Zhou's biotin-labeled antibody and DNA and free chain affinity to connect the antibody and DNA is a more ideal method, but if we can do the biotinylated antibody, affinity and biotinylated DNA 3 molecules The PCR amplification process is relatively simple, but if a microplate is used as the solid phase, a matching PCR instrument must be used, or it must be moved into the reaction tube, which inevitably leads to a large error, and the amplification can produce significant differences. The solid phase can also be used directly in some PCR reaction tubes, and can be amplified directly with a general PCR instrument, but the rinsing process is relatively complex. Immuno-PCR has a non-extensive application prospects, it is very necessary to further improve the experimental process of immuno-PCR and the development of supporting reagents.

Application

Fluorescence PCR is a hot technology for molecular diagnosis, which combines advanced quantitative PCR and real-time PCR technology. The domestic real-time fluorescence quantitative PCR instrument produced by Xi'an Tianlong provides advanced means for quantitative nucleic acid detection and molecular diagnosis of major diseases such as hepatitis and AIDS, as well as food safety detection of Salmonella sakazakii. Even in developed countries, fluorescence quantitative PCR and gene amplification instruments are widely used in clinical, biological and medical research. Due to their high sensitivity, wide detection range, accurate quantification, convenience, rapidity and no window period, the U.S. FDA and the Chinese national standard have specified quantitative PCR as an essential instrument for the diagnosis of infectious diseases such as avian influenza and food safety testing such as milk powder.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/