Besides the hit-or-miss applications of various chemicals prior to the discovery of the sulfa antibiotics, perhaps the first true application of biotechnology occurred with the use of variolation in the control of smallpox. This was the transfer of pus from a mildly infected person to a healthy person in hopes another mild case would occur and thereafter the person would be immune. After small-scale use of variolation in both the Eastern and Western worlds, the first large-scale application of variolation was done at Valley Forge upon the American soldiers by Lafayette's French doctor. While there were no American cases of smallpox that winter, approximately a quarter of the Hessians contracted the disease greatly weakening the British side. Of course, Joseph Lister, went one step further into biotech by reasoning that rather than immunizing with a partially attenuated strain of smallpox, he would use a different, but closely related virus - cowpox (vaccinia, from "vacca", cow). So was born vaccination. Look around you. How many people do you know who have been vaccinated against smallpox? Why haven't you?

    Now let us turn to a form of biotech that employs antibodies - the pregnancy test. (Ask your mother or grandmother what was done in their day to check on pregnancy.) Today's do-it-yourself pregnancy test is undoubtedly the most used of any home biomedical kits because it is totally private. Despite the simplicity of its use, the "home pregnancy test" is very high-tech. Yet, if you disassemble the small apparatus, it seems to consist of a nearly blank strip of blotting paper packed within a plastic container. The high-tech invisible parts of the assembly are the three different antibodies that are embedded within portions of that paper wick. After your instructor gives you a mini-lecture on antibodies, you will appreciate the extremely elegant specificity of this test - especially considering that the user puts a highly variable liquid onto the end of the paper wick and comes up with a very reliable result . Urine can vary widely in pH, and in inorganic and organic concentrations - all this is dependent on what and how much of that "what" the user has consumed.

    On the left, below, is a Y-shaped representation of a generalized antibody. On the right is shown an antibody that has "caught" and bonded to its target molecule, which is called an antigen. In this case the antigen is one of the pregnancy hormones that the placenta secretes into the pregnant woman's blood stream to maintain her pregnancy so that her system doesn't slip back into the menstrual cycle. This is a protein hormone called human chorionic gonadotropin, or hCG, for short. (Antibodies are often abbreviated as "Ab", and antigens as "Ag.") In this case hCG is an Ag, and the right side's complex structure is one of anti-hCG combined with hCG, where "anti-hCG" is an "antibody that binds hCG".

    Sometimes other things can be attached to the inactive tails of the Ab's - sometimes dyes or colloidal bits of metals such as gold or platinum, and sometimes even enzymes that produce insoluble colored products from soluble precursors, or even attaching the tails to paper to keep the Ab's imobilized as other fluids flow over them. Sometimes Ab's can even be made that react with other Ab's! Here are some examples:

Immobilized Antibodies
"anti-hCG"	"anti-anti-hCG" (!)

So now we have all the possibilities, and let's put some them together on the paper strip, and then see what happens.

Here's what happens when the woman is pregnant:

1. A few drops of urine or blood are placed in the application window. (Imagine that the plastic casing has three windows in it. This is the first window, which might be merely an open end.)
2. The fluid fraction along with its dissolved components including hCG, move along with the liquid front.
3. When the fluid reaches the unbound Ab#1, which is in great excess, a unique portion of the hCG molecules begin reacting with Ab#1, but the complexes and the excess unreacted Ab#1 are both soluble and continue to flow along with the current.
4. When this reaches the line where Ab#2 is immobilized to the paper, another portion of the hCG is "displayed" by Ab#1 and binds with Ab#2. When this happens, the distorted Ab#2 triggers an enzyme to start making an insoluble dye, which upon accumulating causes the vertical bar on the "plus sign" to become visible.

   

5. The fluid front continues moving along and eventually crosses the "control window," where the excess Ab#1 starts reacting with Ab#3 (aka: anti-Ab#1), and that triggers the formation of a dye that completes the vertical portion of that plus sign.

   The control window shows "+" to indicate that the antibodies in the paper were alright and working. This is to ensure the woman that the device had not become overheated during transit. If it had, she would have two minuses - the second one telling her to take the device back for a refund because it could be a false negative in the pregnancy window.

   

6. Thus a pregnant woman ends up with two plusses. Only in the first 10 days of pregnancy is the concentration of hCG so low that positive tests usually don't appear. Otherwise, the test is about 99.9% accurate. (It might be noted that there are types of cancer that can trigger positive tests in both men and women.)

Now let's consider a woman (or a man, for that matter) who is NOT pregnant.

7. The urine or blood is applied.
8. When the fluid meets Ab#1, there is no hCG to react with it. Only Ab#1 floats along with the current.
9. In the pregnancy window, there is no "displayed" hCG to react with Ab#2, and no color reaction occurs. Only the printed minus continues to be shown.
10. Finally, in the control window, the Ab#1 is bound to Ab#3 and the "plus" is revealed, showing that the antibodies were effective, and that the fluid had flowed that far - far enough.
11. The test result is "minus-plus."

1. In the above scheme, name the two different anti-hCG's. One is Ab#? and the other is Ab#?
2. A 20 year old woman obtained a positive pregnancy test, but months later it was obvious that she was not pregnant. How can this be?
3. A 25 year old husband is convincing his wife that performing a pregnancy test does not cause pain, so he puts some of his own urine on it and gets a positive test. How can this be?
4. A destitute woman who had intercourse three days earlier for the first time, does the test and gets a negative result. She puts the device back into the cabinet to dry out and be ready for use later. She misses her period and starts getting morning sickness. She takes out the used device and runs the test again. She gets what result?

| The Pregnancy Test | The Ovulation Test |

    The home ovulation test has many similarities with the home pregnancy test - both deal with the fluctuating hormones, and both make use of rather sophisticated ELISA technology.

    Of course, the only time a woman can conceive is that very short span of time between her ovaries' release of an ovum (egg) and the time before it passes by the uterus to then be flushed from her body. Only if the ovum is fertilized by spermatozö (sperm) to be come a zygote (fertilized cell - the very beginning of a baby), can it implant itself in the uterine lining to develop into an embryo and onwards to becoming a fetus and then being born as a neonate (infant).

    As a woman's ovaries near the time of ovulation, a number of hormonal changes set in motion within the woman's body, as shown in the graph below:

Figure from ClearBlueEasy.com

In the ovary that is filled with immature ova (plural of ovum), one of these ova is triggered by the woman's release of the hormone FSH (follicle stimulating hormone) to start maturing to be ready for its release. Immediately upon its release for its adventures to proceed down the fillopian tubes to the uterus, another hormone is released in a short, intense pulse that lasts only for a day or two. This one, LH (lutenizing hormone) might be thought of as helping to heal the crater left behind in the ovary by the departing ovum. For the next few days, the ovary wears a "scar" of yellowing cells (the corpus luteum, which means "yellow body") as evidence of the ovum's departure.

    The home ovulation tests all test for the presence of LH in urine. What is so neat about this is that LH is only present in a woman for a day or two a month, and none at all at other times. This makes it nice for testing as a more simple - qualitative - test is all that is needed. (A quantitative test which must be able to differentiate moderate from high levels of stuff is not needed.)

    You can now return to reading about how the pregnancy test works but substitute LH instead of hCG. You will see how antibodies against LH are conjugated to chromogenic (color producing) enzymes and these are used to grab an LH molecules that might be present and then set about making colored compounds on the test-wand's wick.

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