Enzyme Linked Immunisirbent Assay

In this experiment, the primary antibodies against DAT were generated from the species Rattus norvegicus (the lab rat). The secondary antibodies (Chicken Anti-RatDAT) synthesized against Rat Anti-DAT, were generated from Gallus gallus domesticus (chickens). The Chicken Anti-RatDAT antibodies were synthesized by injecting certain purified rat antibodies into a chicken. As a result, a generation of the monoclonal antibodies Rat Anti-DAT, was essential to detect the DAT segment of the protein. Likewise, the polyclonal antibody Chicken Anti-RatDAT, linkage to the Fc portion of Rat Anti-DAT. Secondary antibodies are known to assist in the sorting, detection, and/or purification of target antigens by attaching to a primary antibody, that directly attaches to the target antigen.

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

Andra Larade Bio 30 B7 11/20/15 Liver Enzyme Lab The Effect of pH and Temperature on the Efficiency of Catalase Abstract: In order to determine how pH and temperature affect the liver enzyme catalase, an experiment was conducted. Catalase breaks apart hydrogen peroxide producing water and oxygen.

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.

The results are organized in sections by order to which the experiments were performed. There are a total of four sections that is represented by a major heading and is shown in bold print that corresponds to the four experimental approached used in this study. Each section also contains either a table or a figure or a combination of both that corresponds to each experiment. The first section was to first figure out the best possible combination of these monoclonal antibody cocktails (ZmAb) and MB-003 by using laboratory animals most specifically guinea pigs and NHP’s (rhesus macaques) to test which combinations of MB-003 and ZmAb are the most effective and can be used for further experiments. To test the components of MB-003 (mAbs c13C6,

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.

Observe how protein biomarkers are assayed by ELISA assays beginning with serum samples, and learn how to perform these assays. (Mice sera)

1) Seroconversion using two serum specimens collected at an interval of at least 14 days.

In addition, it appears that the localization in Figure 2 occurred on the surface of the sample due to the bright fluorescence color. Overall, this lab was successful because we did observe localization in the positive well. The technique that was used in this lab was the direct immunofluorescence method. The reagents/materials used in

The enzyme-linked immunosorbent assay (ELISA) is a common laboratory technique used to measure the concentration of an analyte (usually antibodies or antigens) in solution. In the practical anti-BSA antibodies that had undergone serial dilutions were added to a BSA solution in an ELISA plate with goal of seeing how the concentration of anti-BSA antibodies would affect the colour change of the BSA solution. The results clearly showed a direct correlation as the more diluted the anti-BSA antibody solutions became the lower the Wavelength readings at 405nm, which showed that there was less of a colour change.

A mixture of TBST and milk proteins were added to the nitrocellulose along with the primary antibody for the green fluorescent protein (anti-GFP). The milk protein mixture was added with the primary antibody to reduce non-specific binding to other proteins in the blot. A second antibody was then added with an enzyme attached, in this case goat anti-mouse HRP. The substrates for HRP (horseradish peroxidase) were then added and light produced by the enzyme catalyzed reaction was then picked up on a x-ray film. The results of the western blot on this film are given in Figure

The last part of this procedure, antibodies will be used to detect one specific protein from others on the membrane. Incubate the membrane with 10 ml of primary antibody for 10-20 minutes and then place on a rocking platform. Rinse the membrane quickly after with wash buffer on a rocking platform. After three minutes is up, discard the wash and incubate the membrane with 10 ml of secondary antibody for 5-15 minutes on the platform. Rinse the membrane again shortly after and wash the membrane for three minutes. Next, discard the wash and add 10 mL of HRP color detection reagent. Incubation will occur after this for 10-30 minutes. Once it is done, rinse the membrane twice with distilled water and blot dry.

To confirm the blood is human blood, I would use the ELISA test and the Kastle-Meyer test. Once the tests are complete and confirm human blood, I will analyze the splatters on the wall. Stain B looks as though someone attempted to wipe it off, as it seems to be smeared. Stain A appears to be moving left. Stain D and C look like stains from high impact. I would guess a gun was used at this scene, and that is what created the splatters on the

Overall, ELISA is a good serological method in immunodiagnostics, especially for less developed communities due to its cost and efficiency. However, it does come with limitations, mainly due to the economic status of the geographical areas of infection and the infections the individuals are commonly exposed to. As well as the effect that multiple infections can have on the sensitivity and specificity of

While Immunoassay testing is the most popular and cost effective way to drug test, it is not the most accurate. Cone and Menchen (1987) conducted a study involving the KDI Quik Test Drug Screen, which is a drug screen that relies on immunoassay testing alone, and has the ability to detect the use of cocaine, morphine, phencyclidine, amphetamines and other metabolites associated with illicit substances (p. 276). The study focused solely on whether the test indicated a presence of benzoylecogonine, which is the metabolite of cocaine. Overall, the results concluded that the KDI Quik Test was not an accurate test by itself and that positive or negative results would still need to be validate with further testing in a lab. Additional results from the study indicated that immunoassay testing could lead too false positives due to ill trained staff administering the test. In my professional experience, as a Certified Drug and Alcohol Counselor in the State of Delaware, this is true; there were many times when I conducted a urinalysis utilizing the immunoassay method of testing only to decipher that the results were inconclusive, which were generally as a result of the line being too light on the “dip stick”. Therefore, the urine screen would have to be sent to a lab for further testing.