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Monday, June 12, 2006

Assays and Reality

When you work in a laboratory in the biomedical world you have to learn a few assays. An assay is a qualitative or quantitative analysis of a substance to determine its components. You put on your white lab coat, grab your pipettes and go do an assay. Are you doing science?

The cargo cult theory says that people will follow the form what they see others do in hopes of acheiving the same results. Scientists run assays so if you want to be a successful scientist someone had better get in that lab and run some assays. Let's say you are an office scientist and you do research on HIV. You will have to hire someone to work in the lab who knows how to test for the HIV virus. There will be an HIV assay. The fastest assay involves testing for antibodies against HIV. This is often the case for assays. You don't test for the component directly but for things associated with the component. The further you get away from the lab however, the less you know about the details of the assay.

The office scientist usually knows about the assay. An ELISA assay involves using an antibody against a protein. The antibody has an enzyme physically linked to it. The enzyme is tested for by adding a substrate that will create a color when cleaved by the enzyme. Remember you are really interested in the protein that is bound by the antibody that is linked to the enzyme that causes the substrate to make the color. How do you assess the significance of the simple development of a color?

The ELISA assay was developed long ago. Someone else did the work to determine that this assay could be used as an assay. Those who come along and choose to use it must determine of the things they are testing for can be used in a similar manner. Here is the cargo cult moment. An office scientist could instruct the young lab worker to run an ELISA assay to determine of protein X is present in some sample. The young lab worker buys a kit, runs the assay and looks for the presence of a color. This is similar to setting up an airport and looking to the skies for airplanes.

You have a sample that you suspect contains your protein. You want to know if it is there and how much of it is there. If you accidentally use too much antibody you will get a strong color. If you use too little you will get a weak color where a strong color should have been observed. You must have controls. If you want to know how much you must have a standard curve. The standard curve must involve y = mx + b and linear regression if it is to be understood quantitatively. Your standard curve must be prepared in the same medium as your sample. Your color must be read at the proper wavelenght. All samples must be stopped at the same time with the acid. If you were to seriously teach about the ELISA there would be more time spent on these issues than on any discussion involving antibodies and substrates. Light absorbance readings for example, have limitations. There is a linear range for reading the absorbance of a color in the machines used in the lab. That means there are limitations that must be known and accounted for. The standard curve is also a test of the limitations.

I bring all of this up because of the limitations of measurements in any science. The machine that measures the abosorbance of a colored solution has limitations. The ELISA assay has an upper and lower limit of detection no matter what you are measuring. That means there is a range where useful and accurate information can be obtained. Do you know what your range is?

Beyond ELISAs there are many assays used in basic biomedical research. They all have limitations that are woven together in a way that can cause great confusion. Even if you have 100% pure samples you will have a margin of error in your assay measurements. Imagine then trying to measure the amount of a protein in a blood sample or in kidney cells. Can you find an internal standard in blood? Are proteins sometimes upregulated and downregulated in times and concentrations that are out of our range to measure? I say hell yes they are yet we constantly read about people claiming to have accurately made the measurements nonetheless. Their measurements conveniently support their assumptions underlying the purpose of the research.

I once worked with RAW cells which are precursors to Osteoclasts. If you add RANK ligand to a RAW cell culture they will fuse together and form large multinucleated osteoclasts in about five days. I observed this process many times. I tested for the presence of tartrate resistant alkaline phosphatase (TRAP) every day. I could see TRAP on the surface of the Raw cells just after adding the RANK ligand. As the cells began to fuse I could see the TRAP on one side of the new ball of cells. By the fourth day the TRAP was nearly gone and the osteoclasts were predominant. These were simple observations. If I wanted to know more about TRAP and it's role in osteoclastogenisis I would have to get more clever than cell staining and microscopic observation. That's when I leave the cargo cult world and figure out what to do. I still have the lab coat and the microscope but I have to do something scientific. I have to know my limitations. I have to think about the location of the TRAP and how the cells pH or polarity might be affecting that location. How might the cells pH or polarity affect my measurements? In the end I would not propose any reasons for the TRAP regulation. I would merely describe what I think is going on. That's all. Next I would follow a lead that came up during the TRAP research. The goal would not be to cure osteoporosis. It's simply to observe nature.

It's like a reality show that only a few can watch. Do you get that channel? If so do you turn it on? If so is the picture fuzzy or clear? If it's clear do you have the ability to tell others about it? If so are you telling the right peope about it?

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