ELISpot facts for kids

The ELISpot (which stands for enzyme-linked immunosorbent spot) is a special lab test. It helps scientists count how often a single cell releases tiny protein messengers called cytokines. Imagine it like counting how many times a specific cell "talks" by sending out these messages. ELISpot is also a type of immunostaining. This means it uses special antibodies to find and measure a specific protein. The word "analyte" refers to any substance being identified or measured.

The FluoroSpot Assay is a newer version of the ELISpot test. It uses fluorescence (like glowing light) to look at many different proteins at once. This means it can detect more than one type of protein being released by cells.

How ELISpot Started

A scientist named Cecil Czerkinsky first described the ELISpot test in 1983. He found it was a new way to measure how much of a specific antibody was made by certain lab-made cells called hybridoma cells.

In 1988, Czerkinsky improved the test. He used it to measure how much of a protein called a lymphokine was released by T cells. T cells are important immune cells.

Also in 1988, scientists combined the ELISpot test with computers for the first time. This made it easier to count and study the spots that formed during the test. That same year, special plates with membrane bottoms were first used for these tests.

How the ELISpot Test Works

The ELISpot test involves several steps. Different liquids are added to and washed away from small containers called "wells." These wells are found on a lab plate. A plate can have anywhere from 16 to 100 wells.

• Antibody coating: First, special antibodies are added to the wells. These antibodies are designed to stick to the walls of the wells. They are "monoclonal," meaning they come from a single cell type. They can only attach to one specific part of a protein. These antibodies will later grab onto the cytokines.

• Cell incubation: Next, the cells you want to study are added to the wells. Some wells might have things added to them that make the cells release cytokines. Other wells might not. The cells are then left alone for a while. This allows them to react and release cytokines. It's important to handle cells carefully. For example, blood samples should be gently mixed if stored for long. Cells that have been frozen should rest for an hour or more at body temperature (37 degrees Celsius). Also, the incubator where cells are kept needs to be humid. This stops the wells from drying out.

• Cytokine capture: As the cells release cytokines, these cytokines float around. They then stick to the special antibodies that are coating the walls of the wells.

• Detection antibodies: After the cytokines are captured, the wells are rinsed. This washes away the cells and anything else not needed. Only the captured cytokines, stuck to the first antibodies, remain. Then, a second type of antibody is added. These are called "detection antibodies." They are "biotinylated," meaning they have a special tag called biotin. These detection antibodies attach to the cytokines that are already stuck in the wells.

• Streptavidin-enzyme conjugate: Next, a substance called streptavidin-enzyme conjugate is added. This substance loves to bind to biotin. So, it attaches to the biotin tag on the detection antibodies. This creates a chain: well wall -> first antibody -> cytokine -> detection antibody (with biotin) -> streptavidin-enzyme conjugate.

• Addition of substrate: A special liquid called a "substrate" is added to the wells. The enzyme part of the streptavidin-enzyme conjugate then reacts with this substrate. This reaction creates tiny, insoluble spots in the wells. The type of substrate used depends on the enzyme. For example, if the enzyme is ALP, a specific mixture called BCIP/NBT-plus makes clear spots. If the enzyme is HRP, then TMB works better.

• Analysis: Finally, the spots that have formed can be counted. This is usually done with a special machine called an automated ELISpot reader. Sometimes, a microscope is used. Counting these spots helps scientists figure out how many cells are releasing cytokines.

How the FluoroSpot Test Works

The FluoroSpot test is very similar to the ELISpot test. The main difference is that FluoroSpot can look for many different proteins on one plate. ELISpot usually only looks for one. FluoroSpot does this by using fluorescence (glowing light) instead of an enzyme reaction.

• Antibody Coating: Just like ELISpot, special capture antibodies are added to the wells. For FluoroSpot, a mix of different types of antibodies is used. This allows them to detect many different proteins. Before adding antibodies, the plates are treated with ethanol. This helps prevent contamination. The way you treat the plates with ethanol depends on the plate type. After ethanol, the wells are washed many times with sterile water. Then, the antibodies are added.

• Cell Incubation: Cells are added to the wells. They are left to react, with or without things that make them release proteins.

• Cytokine Capture: The proteins released by the cells stick to the capture antibodies on the wells.

• Detection Antibodies: The wells are rinsed to remove cells and other unwanted stuff. Then, different types of detection antibodies are added. One type might have a biotin tag. Other types might have different "tag-labels." Each type of detection antibody is specific for a different protein you want to measure.

• Fluorophore-labeled Conjugates: Instead of an enzyme, FluoroSpot uses "fluorophore-labeled conjugates." These are substances that glow. They attach to the detection antibodies. A special solution is also added to make the glowing signals stronger. This glowing light is what allows FluoroSpot to measure many different proteins at once.

• Analysis: Since FluoroSpot uses glowing light, there's no need to add a substrate like in ELISpot. The final step is to analyze the glowing spots. This is done with an automated fluorescence reader. This machine has special filters to see the different colors of light. These filters must match the specific colors of the glowing substances for accurate results.

Sometimes, when you measure many proteins at once, one protein might affect how much of another protein is released. This is called "capture effects." It can make the amount of a second protein go up or down. To help with this, scientists can add a second antibody. This antibody helps stimulate the production of the affected protein.

What ELISpot and FluoroSpot Are Used For

ELISpot and FluoroSpot tests are used in many areas of research. They help scientists in fields like:

• Developing new vaccines.
• Studying cancer.
• Understanding allergies.
• Learning about different types of immune cells.
• Researching animal health.

With ELISpot, scientists can study how the body's immune system reacts to specific things. They can look at how T cells respond. They can also measure how well vaccines work. They can even map out specific parts of proteins that the immune system recognizes.

The T-cell ELISpot test is especially useful for studying different groups of T cells. It can detect specific cytokines like IFN-y, IL-2, and TNF-alpha (from Th1 cells). It can also detect IL-4, IL-5, and IL-13 (from Th2 cells). Measuring these cytokines helps scientists understand how well vaccines protect against diseases.

With T-cell FluoroSpot, scientists can monitor immune cells that go into tumors. They can also look at IFN-y and granzyme B to study how T cells fight cancer. Both of these are important for cancer research.

B-cell FluoroSpot can also help check how well vaccines work. It measures different types of antibodies like IgG, IgA, and IgM. It can do this before and after a vaccination. Being able to measure multiple immunoglobulins (antibodies) at once is a big advantage of the FluoroSpot test.

See also

In Spanish: ELISPOT para niños