ELISA

Enzyme-linked Immunosorbent Assays (ELISAs) combine the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily-assayed enzyme. ELISAs can provide a useful measurement of antigen or antibody concentration. There are two main variations on this method: The ELISA can be used to detect the presence of antigens that are recognized by an antibody or it can be used to test for antibodies that recognize an antigen. An ELISA is a five-step procedure: 

1) coat the microtiter plate wells with antigen

2) block all unbound sites to prevent false positive results

3) add antibody to the wells

4) add anti-mouse IgG conjugated to an enzyme 

5) reaction of a substrate with the enzyme to produce a colored product, thus indicating a positive reaction. 

There are many different types of ELISAs. One of the most common types of ELISA is "sandwich ELISA."

Southern Blot

Southern Blotting The first blotting technique to be devised is known as Southern blotting after its originator E. M. Southern.

This technique is capable of detecting a single specific restriction fragment in the highly complex mixture of fragments produced by cleavage of the entire human genome with a restriction enzyme. In such a complex mixture, many fragments will have the same or nearly the same length and thus migrate together during electrophoresis. Even though all the fragments are not separated completely by gel electrophoresis, an individual fragment within one of the bands can be identified by hybridization to a specific DNA probe. To accomplish this, the restriction fragments present in the gel are denatured with alkali and transferred onto a nitrocellulose filter or nylon membrane by blotting.

This procedure preserves the distribution of the fragments in the gel, creating a replica of the gel on the filter, much like the replica filter produced from clones in a library. (The blot is used because probes do not readily diffuse into the original gel.) The filter then is incubated under hybridization conditions with a specific radiolabeled DNA probe, which usually is generated from a cloned restriction fragment. The DNA restriction fragment that is complementary to the probe hybridizes, and its location on the filter can be revealed by autoradiography.

Zebrafish Provide New Hope for Cancer Treatment

ScienceDaily (Dec. 20, 2010) — The imaging of tumour growth in zebrafish has revealed for the first time how newly formed cancer cells have the capacity to co-opt the immune system into spreading the disease, leading the way for investigations into potential therapies for eliminating early-stage cancer in humans. Using different coloured fluorescent tags, scientists at the University of Bristol labelled immune cells and tumour-forming cells in the translucent zebrafish in order to track their behaviour and interactions by live cell imaging.
These dramatic findings, which are the result of a collaboration between academic colleagues in the UK (University of Bristol and University of Manchester) and Italy (Institute of Molecular Oncology, Milan) are published in the online, open access journal PLoS Biology.
Tests showed that cancer cells are less likely to proliferate if white blood cells can be prevented from contacting the precursor cancer cells, suggesting that white blood cells -- the immune cells -- have the ability to promote disease by providing some kind of growth signal. Interestingly, the chemical compound that acts as a draw between the two sets of cells was shown here to be hydrogen peroxide -- commonly used as a disinfectant or antiseptic, but also a natural by-product produced by the body's metabolic process.
Describing the work, Paul Martin, Professor of Cell Biology at the University of Bristol's Schools of Biochemistry and Physiology & Pharmacology, who supervised post-doctoral fellow Yi Feng in the research project, said: "By visualizing the earliest interactions between cancer cells and their host environment, we have shown that even from their earliest stages tumours don't just avoid being destroyed by the immune system. Rather, they appear to court an immune response, co-opting the body's innate immune system to aid and abet their growth."
The team used a method to switch on the human oncogene, HRAS, in specific pigment cells (melanocytes) in the skin of early stage zebrafish embryos. Studies were carried out using zebrafish because they conserve many of the molecular and cellular components of tumour formation seen in mammals and are almost translucent, making it easy to see the cells as they move around and grow. Researchers monitored the first hours and days of development and as the embryo grew, some of the cells were transformed, ie, made cancerous by HRAS. Those transformed cells were found to actively attract the innate immune cells. The researchers got the same results, after inserting HRAS into different, mucous-secreting cells, and again when experimenting with a different oncogene, SRC. They discovered that the tumour cells produced hydrogen peroxide and that immune cells were drawn up the hydrogen peroxide gradient towards the cancer.
The researchers' movies show that the immune cells appear to engulf cancerous cells in a bid to destroy them. However, other cells formed cytoplasmic tethers linking them to cancerous cells and in some cases the cancerous cells appeared to drag the immune cells back when they started to leave the region. In order to see whether the tumour was avoiding destruction or actually co-opting the immune cells, the researchers blocked the immune response in three different ways: they prevented the development of immune cells for the first three days of the zebrafish embryos' life, and separately, they used two different strategies to limit hydrogen peroxide production. In each case, immune cells failed to migrate to the cancer site. And each time, when the immune response was blocked, fewer cancer cells formed.
Professor Martin added: "Yi's movies in Zebrafish larvae give us the first insight into how immune cells sense and then attempt to deal with the earliest stages of cancer. Now we can look closer to discover why it is that immune cells seem to aid growth of these young cancer cells and figure out ways for guiding immune cells how better to search and destroy."

UCSB researchers discover new biotechnology to identify and engineer substrates for proteases

This technology should help solve the puzzles of cancer, Alzheimer’s, atherosclerosis and infectious diseases.

Santa Barbara, California – May 1 , 2006 – Researchers at UC Santa Barbara have developed a new biotechnology that enables scientists to identify and engineer protease substrates, giving them the means of crafting pharmaceuticals to outsmart disease. Their work, authored by Patrick Daugherty, an assistant professor of Chemical Engineering, and Kevin Boulware, a PhD candidate, are published online today in the Proceedings of the National Academy of Sciences.
Proteases (or peptidases) are encoded by about two percent of genes in the human genome and play key roles in nearly all diseases. They act as "molecular scissors" by attaching to specific sequences contained within other proteins, called substrates, and cutting them in specific locations. For example, proteases are responsible for digesting food, for determining the proper time for cells to die, and for removing damaged proteins from the body.

But the substrates for most proteases are unknown, and this has limited researchers' ability to facilitate or thwart protease action. By identifying substrates, scientists gain the ability to regulate protein function, creating the capacity to speed up, slow down or eliminate particular protease actions. Daugherty's approach also makes it easier to measure protease action and thus develop pharmaceuticals that control protease activity.

Daugherty and Boulware developed a general combinatorial approach to identify optimal substrates of proteases, using quantitative kinetic screening of cellular libraries of peptide substrates (CLiPS). The results suggest that CLiPS will be broadly useful for characterizing proteases and developing optimal substrates for therapeutic applications.

Of the roughly 1,000 proteases in the human genome, only about ten percent of the targets have been identified, but Daugherty believes that scientists will identify nearly all of them in the next five to ten years. "This technology will give us a scalable tool that will allow us to effectively tackle this challenge," he says.