ELISA

ELISA (Enzyme-Linked ImmunoSorbant Assay)

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This is one of the most powerful qualitative and quantitative tools for dealing with complex molecules. Its discriminatory power lies in the fact that it is based on the extremely specific antibody-antigen complexation. What is given here is NOT a protocol for doing ELISA as no times, concentrations, stirring rates, etc., are given. Instead it is a outline giving the reasons for doing each step and what happens in each step. Added into the outline are how some of the "reagents" (antibodies) are made, even though the secondary, conjugated, or enzyme-linked antibody can be purchased commercially.

ELISA

Step Picture Comments
1 You would generally affix the material you want to test to a surface such as a glass slide, or a blotten onto a type of filter paper, such as made of nitrocellulose fibers. It might be a dried droplet, or it might be a tissue slice.
2 This would be a sub-microscopic side-view of the smudge or tissus slice on a glass slide. Several different types of molecules are shown. For illustrative purposes, we are interested in the aqua or turquoise colored molecule. Our question is: "Is there any of that type of molecule on this slide?" Being that it is so small, it cannot even be seen under a microscope.
3 Our intension is to adsorb an antibody specifically to this aqua-"antigen" and to no others. Unfortunately, if we were to have some of that antibody and dump it onto this slide, the antibody would adsorb nonspecifically to the slide just as all the other antigens have done. So what we do first is block all those possible non-specific adsorption sites with another protein. So we dump a solution of albumin, or even more cheaply, we flood skim milk onto the slide, and then rinse. See: all the surface of the slide is now covered or "blocked" by the skim milk "blocking" agent.
4 The question now is how do we produce the highly specific antibody that will only adhere to the "aqua" antigen and to nothing else. You need to make an immune serum. Well, your friends down the hall have isolated and purified a small batch of this "aqua" antigen (in the tube to the left, below), and you inject a little of this into a mouse (uncooperative lab partners produce immune serum also!). After a week, you give the mouse a booster shot, and a few days after that, you withdraw some of the mouse's blood from its tail vein. Then you centrifuge out all the cellular blood components (name them!) and you have is immune serum, because it is full of anti-"aqua" antibodies. (See that the lower ends of the model indicate the reactive or combining sites on the antibody molecule.) Were you to add a few drops of the immune serum to a batch of your friends' original aqua solution, you would see the solution "agglutinate", or precipitate, as the antibodies complexed with the "aqua" antigens to form an insoluble precipitate, which immunologists call a "precipitin lattice," or "precipitin" for short.

(the "primary" antibody)
(Click this for a better way to make primary antibodies)
5 Next you dump a little of this immune serum onto your "blocked" smudge. You can imagine all the antibody molecules flowing about, but one of them, you see here, is stuck - or adsorbed - to the aqua antigen!
6 You gently rinse the slide with buffer to flush away all the excess specific antibody as well as all the other species of antibodies that the mouse might have in its system, such as those acquired in fighting its own childhood diseases such as mouse mumps and colds. We can see in this diagram that our one little specific antibody is still adsorbed to the aqua antibody. However, in real life, we could not possibly hope to see that one little antibody molecule let alone find it on the broad surface of the slide. We need to find a way to locate it, and we do that by an amplification method.
7 Enzymes are wonderful amplifiers as the longer a reaction is allowed to proceed the more product we get. What if that product were insoluble? There are possibilities! Chances for Nobel gold dance in imaginatiions!!! (Usually these reactions involve a substrate that consists of a large organic molecule that is a dye. Covalently bonded to the dye molecule is a highly charged phosphate or sulfate to make the overall molecule water soluble. However, if the enzyme were able to snip off the charged piece, the remainder would be insoluble and would settle out. Often used enzymes for this are phosphatases and sulfatases - both rather common digestive tract enzymes.

8 You plan is now to make an antibody that reacts to all mouse antibodies; an anti-mouse-antibody antibody, if you will. This is easy: grab your local mouse - any mouse! - and isolate the gamma-globulin fraction of its blood. (Use those lab techniques you've learned - electrophoresis, for example!) Then inject this into a goat; give the goat some booster shots, and soon you have a walking garbage dispos-all that can produce enough anti-mouse antibody to supply the world. Go into business! Yes, there are companies that do this. See the reactive binding sites on the goat antibody. They will bind only to mouse antibodies: just what you need.

9 Your next step is to attach your enzyme "amplifier" onto the goat antibody molecules. There are some neat chemistry tricks for doing this, but just know that you can go to the abovementioned company catalogs to buy these "enzyme-linked goat anti-mouse-IgG antibodies." This you call your enzyme-linked "secondary" antibody.
10 Flood your slide with this enzyme-linked goat antibody, and magically it sticks to wherever it encounters mouse antibody. Of course, rinse away all the excess before going to the next step.
11 Set your slide in a properly buffered solution of the substrate and gently swirl it. As the substrate molecules encounter the enzyme and the phosphates or sulfates are removed, the remaining portion of the dye molecule is insoluble and snows out to stick to nearby things. The longer you allow the reaction to occur, the more the colored "snow" piles up.
12 You "stop" the development of the slide by merely rinsing away the excess substrate solution. You may now look at your slide under the microscope and hopefully the piles of colored precipitate are large enough for you to see.

Step	Picture	Comments
1		You would generally affix the material you want to test to a surface such as a glass slide, or a blotten onto a type of filter paper, such as made of nitrocellulose fibers. It might be a dried droplet, or it might be a tissue slice.
2		This would be a sub-microscopic side-view of the smudge or tissus slice on a glass slide. Several different types of molecules are shown. For illustrative purposes, we are interested in the aqua or turquoise colored molecule. Our question is: "Is there any of that type of molecule on this slide?" Being that it is so small, it cannot even be seen under a microscope.
3		Our intension is to adsorb an antibody specifically to this aqua-"antigen" and to no others. Unfortunately, if we were to have some of that antibody and dump it onto this slide, the antibody would adsorb nonspecifically to the slide just as all the other antigens have done. So what we do first is block all those possible non-specific adsorption sites with another protein. So we dump a solution of albumin, or even more cheaply, we flood skim milk onto the slide, and then rinse. See: all the surface of the slide is now covered or "blocked" by the skim milk "blocking" agent.
4	The question now is how do we produce the highly specific antibody that will only adhere to the "aqua" antigen and to nothing else. You need to make an immune serum. Well, your friends down the hall have isolated and purified a small batch of this "aqua" antigen (in the tube to the left, below), and you inject a little of this into a mouse (uncooperative lab partners produce immune serum also!). After a week, you give the mouse a booster shot, and a few days after that, you withdraw some of the mouse's blood from its tail vein. Then you centrifuge out all the cellular blood components (name them!) and you have is immune serum, because it is full of anti-"aqua" antibodies. (See that the lower ends of the model indicate the reactive or combining sites on the antibody molecule.) Were you to add a few drops of the immune serum to a batch of your friends' original aqua solution, you would see the solution "agglutinate", or precipitate, as the antibodies complexed with the "aqua" antigens to form an insoluble precipitate, which immunologists call a "precipitin lattice," or "precipitin" for short. (the "primary" antibody) (Click this for a better way to make primary antibodies)
5		Next you dump a little of this immune serum onto your "blocked" smudge. You can imagine all the antibody molecules flowing about, but one of them, you see here, is stuck - or adsorbed - to the aqua antigen!
6		You gently rinse the slide with buffer to flush away all the excess specific antibody as well as all the other species of antibodies that the mouse might have in its system, such as those acquired in fighting its own childhood diseases such as mouse mumps and colds. We can see in this diagram that our one little specific antibody is still adsorbed to the aqua antibody. However, in real life, we could not possibly hope to see that one little antibody molecule let alone find it on the broad surface of the slide. We need to find a way to locate it, and we do that by an amplification method.
7	Enzymes are wonderful amplifiers as the longer a reaction is allowed to proceed the more product we get. What if that product were insoluble? There are possibilities! Chances for Nobel gold dance in imaginatiions!!! (Usually these reactions involve a substrate that consists of a large organic molecule that is a dye. Covalently bonded to the dye molecule is a highly charged phosphate or sulfate to make the overall molecule water soluble. However, if the enzyme were able to snip off the charged piece, the remainder would be insoluble and would settle out. Often used enzymes for this are phosphatases and sulfatases - both rather common digestive tract enzymes.
8		You plan is now to make an antibody that reacts to all mouse antibodies; an anti-mouse-antibody antibody, if you will. This is easy: grab your local mouse - any mouse! - and isolate the gamma-globulin fraction of its blood. (Use those lab techniques you've learned - electrophoresis, for example!) Then inject this into a goat; give the goat some booster shots, and soon you have a walking garbage dispos-all that can produce enough anti-mouse antibody to supply the world. Go into business! Yes, there are companies that do this. See the reactive binding sites on the goat antibody. They will bind only to mouse antibodies: just what you need.
9		Your next step is to attach your enzyme "amplifier" onto the goat antibody molecules. There are some neat chemistry tricks for doing this, but just know that you can go to the abovementioned company catalogs to buy these "enzyme-linked goat anti-mouse-IgG antibodies." This you call your enzyme-linked "secondary" antibody.
10		Flood your slide with this enzyme-linked goat antibody, and magically it sticks to wherever it encounters mouse antibody. Of course, rinse away all the excess before going to the next step.
11		Set your slide in a properly buffered solution of the substrate and gently swirl it. As the substrate molecules encounter the enzyme and the phosphates or sulfates are removed, the remaining portion of the dye molecule is insoluble and snows out to stick to nearby things. The longer you allow the reaction to occur, the more the colored "snow" piles up.
12		You "stop" the development of the slide by merely rinsing away the excess substrate solution. You may now look at your slide under the microscope and hopefully the piles of colored precipitate are large enough for you to see.

Betcha can't answers these QUESTIONS!

You are interested in knowing where acid phosphatase is found in cells. You bought some bovine acid phosphatase, and injected a small amount of it into a mouse. After the requisite booster shots, you isolated the serum fraction from the mouse's blood, and that would contain the primary antibody. You then made some thin-section slides of beef liver and ran it through the above steps using your new primary antibody, and later using a secondary antibody that was linked to sulfatase (why not phosphatase?). Get out a picture of an animal cell from your text, and show your instructor what you would see if the picture were your ELISA data and if acid phosphatase was found only in lysosomes.
How could you use ELISA to identify a nasty bacterium that caused diarrhea? Suppose you knew that it had to be one of these three diseases: cholera, dysentery or typhoid. Pretend you are a doctor attending a patient with a severe case of the "runs." Go step by nasty step through the whole procedure. (Don't laugh! Diarrheal diseases are the infectious diseases that kill more humans and livestock than any other. It is very important that analyses be done properly!)
Suppose you had a petri plate in which several hundred colonies had grown. Next suppose that you carefully laid a piece of filter paper over the colonies and blotted them lightly so that a few from each colony would stick to the paper in their respective positions. You know that within that batch were one or two clones that were likely to be producing a new surface antigen, for which you already had a mouse antiserum. How could you use the paper "blot" to tell which colonies, if any, were producing the desired surface antigen?

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