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Home > Technical services > Immunoassay

It is an analytical method wherein a protein sample is electrophoresed on an SDS-PAGE and electrotransferred onto nitrocellulose membrane. The transferred protein is detected using specific primary antibody and secondary enzyme labeled antibody and substrate.
A protein sample is subjected to polyacrylamide gel electrophoresis. After this the gel is placed over a sheet of nitrocellulose and the protein in the gel is electrophoretically transfered to the nitrocellulose. The nitrocellulose is then soaked in blocking buffer (3% skimmed milh solution) to "block" the non-specific binding of proteins. The nitrocellulose is then incubated with the specific antibody for the protein of interest. The nitrocellulose is then incubated with a second antibody, which is specific for the first antibody. For example, if the first antibody was raised in mouse, the second antibody might be termed "goat anti-mouse immunoglobulin". What this means is that mouse immunoglobulins were used to elicit an antibody response in goats. The second antibody will typically have a covalently attached enzyme which, when provided with a chromogenic substrate, will cause a color reaction. Thus the molecular weight and amount of the desired protein can be characterized from a complex mixture (e.g. crude cell extract) of other proteins by westernblotting. Wear gloves while performing electroblotting.

Western-bloting Procedure
1.Extraction of total protein from tissues/cells.
2.Constructions of western blots using total protein by BG or customers.
3.Immunostaining blots with antibodies provided by customers using ultrasensitive chemiluminescent ECL detection.
4.Quantitative analysis of immunostaining.
5.Sending high quality digital images of X-ray films.

Customer’s resource
Tissue samples 250-500mg;cell samples≥1×106

The principle of ELISA
ANTIGEN A ELISA kit applies the quantitative sandwich enzyme immunoassay technique. The microtiter plate has been pre-coated with a monoclonal antibody specific for IL-1. Standards or samples are then added to the microtiter plate wells and ANTIGEN A if present, will bind to the antibody pre-coated wells. In order to quantitatively determine the amount of ANTIGEN A present in the sample, a standardized preparation of horseradish peroxidase (HRP)-conjugated polyclonal antibody, specific for ANTIGEN A are added to each well to “sandwich” the ANTIGEN A immobilized on the plate. The microtiter plate undergoes incubation, and then the wells are thoroughly washed to remove all unbound components. Next, substrate solutions are added to each well. The enzyme (HRP) and substrate are allowed to react over a short incubation period. Only those wells that contain ANTIGEN A and enzyme-conjugated antibody will exhibit a change in color. The enzyme-substrate reaction is terminated by addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. A standard curve is plotted relating the intensity of the color (O.D.) to the concentration of standards. The ANTIGEN A concentration in each sample is interpolated from this standard curve.

Assay procedure
1)Secure the desired numbers of coated wells in the holder then add 50 uL of Standards or Samples to the appropriate well in the antibody pre-coated Microtiter Plate. Add 100 uL of PBS (pH 7.0-7.2) in the blank control well.
2)Add 100 uL of Conjugate to each well (NOT blank control well). Mix well. Mixing well in this step is important. Cover and incubate the plate for 1 hour at 37°C.
3)Wash the microtiter plate manually or automated. 
4)Add 50 uL Substrate A and 50 uL Substrate B to each well including blank control well, subsequently. Cover and incubate for 10-15 minutes at 20-25°C. (Avoid sunlight).
5)Add 50 uL of Stop Solution to each well including blank control well. Mix well.
6)Determine the Optical Density (O.D.) at 450 nm using a microplate reader immediately.

Customer’s resource
Serum - Use a serum separator tube and allow samples to clot for 2 hours at room temperature or overnight at 2-8°C. Centrifuge at approximately 1000 × g (or 3000 rpm) for 15 minutes. Remove serum and assay immediately or aliquot and store samples at -20°C or -80°C.

Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g (or 3000 rpm) at 2 - 8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C or -80°C.

Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues were rinsed in ice-cold PBS (0.02mol/L, pH 7.0-7.2) to remove excess blood thoroughly and weighed before homogenization. Minced the tissues to small pieces and homogenized them in a certain amount of PBS with a glass homogenizer on ice. The resulting suspension was subjected to ultrasonication or to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates were centrifugated for 15 minutes at 1500×g (or 5000 rpm). Remove the supernate and assay immediately or aliquot and store samples at -20°C or -80°C.

Cell lysates - Cells should be lysed according to the following directions. 
1)Adherent cells should be detached with trypsin and then collected by centrifugation. Suspension cells can be collected by centrifugation directly.
2)Wash cells three times in PBS.
3)Cells were resuspended in PBS and subjected to ultrasonication for 3 times. Alternatively, freeze cells at -20 °C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times.
4)Centrifuge at 1000×g (or 3000 rpm) for 15 minutes at 2-8 °C to remove cellular debris.
5)Assay immediately or store samples at -20°C or -80°C.

Cell culture supernatants and other body fluids - Centrifuge cell culture media at 1000 × g (or 3000 rpm) for 15 minutes to remove debris. Assay immediately or store samples at -20°C or -80°C.

Principle of IHC
Immunohistochemistry is a method of detecting the presence of specific proteins in cells or tissues and consists of the following steps: 1)primary antibody binds to specific antigen; 2) antibody-antigen complex is bound by a secondary, enzyme-conjugated, antibody; 3) in the presence of substrate and chromogen, the enzyme forms a colored deposit at the sites of antibody-antigen binding.
Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Specific molecular markers are characteristic of particular cellular events such as proliferation or cell death (apoptosis). IHC is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue. Visualising an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction (see immunoperoxidase staining). Alternatively, the antibody can also be tagged to a fluorophore, such as fluorescein or rhodamine

Protocol for IHC
1.Roast slides for an hour.
2.Deparaffinization: soak tissue slides with xylene twice, each time for 15 minutes
3.Rehydration: incubate slides in the following graded series of ethanol: 100%I, 95%, 90%, 80%, and 70%. 5 minutes for each solution. Then incubate in the water for 5 minutes.
4.Wash the slides gently with PBS buffer for 3times each time for 5minutes.
5.Antigen retrieval
Bring the slides to boil with citrate acid buffer for 15 minutes to make the antigen retrieval and then let it cool at room temperature.
6.Immerse the slides in 3% H2O2 (in distilled water) for 20 minutes at room temperature. Rinse the slides with 1x PBS (pH7.4) 3 times, circle the tissue section with a Pap Pen (or Para-Pen, ImmEdge Pen)
7.Incubate the slide with 1% Normal goat serum for 20 minutes at 37℃
8.Add the primary antibody to the slides and incubate overnight at 4 ℃
9.Bring the slides to room temperature for 20 minutes then rinse them with PBS buffer 3 times, each time for 5 minutes.
10.Add the secondary antibody (Biotin- labeled goat anti-rabbit IgG) to the slides, incubate at 37℃ for 20minutes then rinse them with PBS buffer 3 times, each time for 5 minutes.
11.Add the third antibody (Streptavidin marked with Horseradish peroxidase) to the slides, incubate at 37℃ for 20minutes then rinse them with PBS buffer 3 times, each time for 5 minutes.
12.Develop the color with DAB, check the result with a microscope and terminate the reaction.
13.Dye the slides again with hematoxylin for 5 minutes, add 75% Hydrochloric acid alcohol solution for 30 seconds then rinse the slides with running water for 5 minutes. 
14.Dehydrate by soaked in the graded series of alcohol: 70%-80%-90%-95%-100%, 5 minutes for each solution; then incubated in Xylene twice, 15 minutes each. 
15.Mount the slide with neutral gum.
Customer’ resource
Tissue slice

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