Assay For Quantification Of Anti Rabbit Proteins

Antibodies are involved in the immune response. They're made up of two light polypeptide chains and two heavy polypeptide chains bonded together. Antibodies have a variable region which acts in a similar way to the active site of an enzyme. Each antibody has a variable region that is the complementary shape for a particular antigen and

The ELISA test can also be used to detect antibodies that are produced in response to a specific antigen. Using information about how you completed this ELISA experiment, outline a procedure for testing for antibodies in the blood.

In the gel filtration step, we began with a slurry of Bio-Gel P-100 beads suspended in 20 mM phosphate buffer (Equilibration buffer) and an upright column with a stopcock. We added enough of the Bio-Gel P-100 beads to the column until we reached a height of 4.7 cm, making sure not to allow the column to run dry and that the top of the beads column is flat. We then added the sample, a mixture of Blue Dextran (2 mg/ml, 2 MDa), Hemoglobin (2 mg/ml), Bovine Serum Albumin (2 mg/ml), and Yellow Food Coloring (5 μl/ml, ~500 Da) (Sheffield, 37). This was followed by another ~1 ml of equilibration

Protein Assay: The Pierce BCA Protein Assay (Thermo Scientific) is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein concentration. A series of standard solution of Bovine Serum Albumin (BSA) ranging from 0-2000 µg/ml was prepared from a stock solution of 2 mg/ml BSA. 25ul of diluted crude (1:500, 1:250), desalted (1:100, 1:50), and 6 peak fractions from cibarcon blue column (1:10, 1:5) were loaded in microplate along with 175ul of BCA working reagent. Microplate was incubated for 30min at 370C and then the absorbance was measured at 562nm.

Colorimetric assay is a process of determining a concentration of a solution based on absorbance of light. The purpose of this lab is to determine if the Bradford assay is an accurate way to determine an unknown concentration of two samples of protein. The Bradford assay is done by measuring wavelength of light passing through a cuvette filled with Bradford dye and concentrations of PBS and proteins. After the cuvettes are mixed they are placed into a spectrophotometer to measure wavelength. The wavelength given will be used to plot a standard curve based on concentration (x-axis) and wavelength (y-axis). The standard curve is then used to measure an educated guess on the concentrations of unknown protein concentrations. We hypothesized that if we use the Bradford assay and colorimetric spectrophotometry we can determine an accurate concentration of two unknown concentrations of proteins. The results of this lab failed to reject our hypothesis based on accurate measurements of protein concentrations. The standard curves are drawn with a linear increasing slope. The Bradford assay is an accurate way to demine the concentration of an unknown concentration.

The ending result of this experiment confirms that as five test tubes are lined up with the varying level of absorbance, different results in the level of absorbance will appear as well, this is visible in above table. Thus, this is due to the varying amount of water in the solution. The blank sample had a 0.30 in its level of absorbance.

This, according to the clonal selection theory, the immune system functions on the “ready-made” rather than the “made-to-measure” principle. The question of how an animal makes so many different antibodies there for become a problem of genetics rather than one of protein chemistry” (Bowden 1006). This means that genetics determine how well the immune system works and this the key to figuring out a way to boost the immune system to help fight of infections and diseases before they even begin to weaken and infect the whole body. If someone decided they wanted to be immune to a snake bite or HIV the only thing that would have to be done is find one person just one that is immune and clone that person gene and add it to the other person who also wants to be immune to a certain disease, physical or chemical problem. “Scientist have grown replacement organs for sheep, rats and rabbits using the animal's own cells and alb molds to help the tissue take shape a technique that could be used someday to make spare parts for

IgG – funtions in neutralizing, opsonation, compliment activation, antibody dependent cell-mediated cytocity, neonatal immunity, and feedback inhibition of B-cells and found in the blood.

Eisenthal, R. & M.J. Danson (Eds.) (2002). _Enzyme Assays: A Practical Approach_. United Kingdom: Oxford University Press

The homogenates provided were made by homogenizing tissues in a sucrose phosphate buffer in a 1:20 ratio. The protein concentration in bovine cells was measured by diluting the homogenate with a 1:5 ratio; 50 microliters of homogenate and 200 microliters of water. Then 5 known protein concentration samples which were 0.4, 0.8, 1.2, 1.6, 2.0 mg/ml of bovine serum were used to determine absorbance with a spectrophotometer. Two additional samples were made; one was blank and the other was for the specific homogenate sample. Then 3 microliters of bradford assay reagent, which indicates the amount of protein present

The Enzymes linked immunoabsorbant assay (ELISA) is a commonly used biochemical technique, often used in immunology as a way to detect the presence of an antibody or antigen in a sample.

After the substrate solution was added, five drops of the enzyme were quickly placed in tubes 3, 4 and 5. There were no drops of enzyme added in tubes 1 and 2 and in tube 6 ten drops were added. Once the enzyme solution has been added the tubes were then left to incubate for ten minutes and after five drops of DNSA solution were added to tubes 1 to 6. The tubes were then placed in a hot block at 80-90oC for five minutes. They were then taken out after the five minute period and using a 5 ml pipette, 5 ml of distilled water were added to the 6 tubes and mixed by inversion. Once everything was complete the 6 tubes were then taken to the Milton Roy Company Spectronic 21 and the absorbance of each tube was tested.

The following Diluted Albumin (BSA) Standards have been prepared using Bovine Serum Albumin (BSA) Standard Solution (2.0mg/ml) and the Diluent Phosphate Buffered Saline (PBS) having

Three animal model mice, camel and rhesus masques were vaccinated with a synthetic DNA vaccine encoding the MERS spike protein. The vaccine had induced potent cellular immunity and neutralizing antibodies against Spike protein in all animal models. However, rhesus masques seroconverted rapidly after only single immunization and thery demonstrate high levels of neutralizing antibodies. To assess protective immunity of the MERS-COV vaccine, vaccinated rhesus masques was challenged with MERS-COV infection after 4 weeks of immunization. To compare the control group was vaccinated with pVax1. The control group have demonstrated progressive symptoms of pneumonia with radiographic changes of varying severity, in the other hand vaccinated group had failed to develop neither symptom of pneumonia nor radiographic changes. Concluding that synthetic consensus anti–spike protein DNA vaccine can induce immunity against MERS-COV.(5)

Consistent with the previous reports that IFN-γ is required to induce class switching of IgG2a (29-30), Ara-LAM was successfully able to induce IgG2a antibody in infected re-challenged mice. It is noteworthy to mention that naïve mice treated with Ara-LAM when infected after 14 or 18 weeks, could not rescue from disease progression. Contrary to that, the results described in this study indicated, successful induction of a host-protective effector response is possible by adoptive transfer of Ara-LAM treated immune CD4 or total T cells (but not CD8 T cells). It is obvious that in uninfected mice, Ara-LAM does not stay for such a long time in the body as opposed to what happens in the case of infected mice. Though, SLA immunized mice can show a mixed Th1/Th2 immune responses, but it is insufficient to control L. donovani infection. Altogether, it is obvious that Ara-LAM can generate a pool of immune CD4 T cells in infected mice and that is