Tuesday, February 3, 2009

ELISA

ELISA
Enzyme-Linked ImmunoSorbent Assay (ELISA) is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light of the appropriate wavelength is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be inferred through the magnitude of the fluorescence.

Types of ELISA
There are 3 types of ELISA. "Indirect", Sandwhich and Competitive ELISA.

"Indirect" ELISA:

The steps of the general, "indirect," ELISA for determining serum antibody concentrations are:
- Apply a sample of known antigen of known concentration to a surface, often the well of a microtiter plate The antigen is fixed to the surface to render it immobile. Simple adsorption of the protein to the plastic surface is usually sufficient.
- A concentrated solution of non-interacting protein, such as bolvine serum albumin(BSA) is added to all plate wells. This step is known as blocking, because the serum proteins block non-specific adsorption of other proteins to the plate.
- The plate wells or other surface are then coated with serum samples of unknown antigen concentration, diluted into the same buffer used for the antigen standards.
- The plate is washed, and a detection antibody specific to the antigen of interest is applied to all plate wells. This antibody will only bind to immobilized antigen on the well surface, not to other serum proteins or the blocking proteins.
- Secondary antibodies, which will bind to any remaining detection antibodies, are added to the wells. These secondary antibodies are conjugated to the substrate-specific enzyme.
- Wash the plate, so that excess unbound enzyme-antibody conjugates are removed.
-Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenic or electrochemical signal.
-View/quantify the result.
The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. A major disadvantage of the indirect ELISA is that the method of antigen immobilization is non-specific; any proteins in the sample will stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding to the well surface.

Sandwhich ELISA:

A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample antigen. The steps are as follows:

- Prepare a surface to which a known quantity of capture antibody is bound.
-Block any non specific binding sites on the surface.
-Apply the antigen-containing sample to the plate.
-Wash the plate, so that unbound antigen is removed.
-Apply primary antibodies that bind specifically to the antigen.
-Apply enzyme-linked secondary antibodies which are specific to the primary antibodies.
-Wash the plate, so that the unbound antibody-enzyme conjugates are removed.
-Apply a chemical which is converted by the enzyme into a color or fluorescent or electrochemical signal.
-Measure the absorbance or fluorescence or electrochemical signal (e.g., current) of the plate wells to determine the presence and quantity of antigen.
The major advantage of a sandwich ELISA is the ability to use impure samples and still selectively bind any antigen that may be present. Without the first layer of "capture" antibody, any proteins in the sample may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized.

Lastly, Competitive ELISA:

A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:
-Unlabeled antibody is incubated in the presence of its antigen.
-These bound antibody/antigen complexes are then added to an antigen coated well.
-The plate is washed, so that unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")
-The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme.
-A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.


A video on the usage of ELISA (:

References:
google
wikipedia

Sunday, February 1, 2009

Picornaviridae

Poliovirus, in the family picornaviridae

Description and Significance
The name "picornavirus" means " little RNA virus" (pico means "very small" in Greek; pico + RNA virus). Picornaviruses are among the oldest viruses, dating back to 1400 B.C., when a temple record tells of poliovirus infections in ancient Egypt. Picornaviruses are also among the most diverse viruses, with over 200 serotypes causing infections such as Polio, Hepatitis A, and the common cold. Foot-and-Mouth Disease Virus (genus: Apthovirus), which causes infections in livestock, was one of the first viruses to be recognized; it was discovered by Loeffler and Frosch in 1898. (sources: Flynn, Bedard)

Genome Structure
The picornavirus genome consists of a single molecule of linear, postitive(+)-sense, single-strand RNA. It is non-segmented. The complete genome is 7000-8500 nucleotides long. The 5'-terminus of the genome has a long untranslated region 600-1200 bases in length, which is important in translation, virulence, and possibly encapsidation. There is a shorter untranslated region (50-100 bases in length) on the 3'-terminus, which is important in (-)strand-synthesis. The 5'-terminus untranslated region also has a "clover leaf" secondary structure known as the Internal Ribosome Entry Site (IRES), which distinguishes picornaviruses from other RNA viruses; this structure is important in translation and replication. The 5'-terminus is modified by a covalently-attached VPg protein (which takes the place of a cap), while the 3'-terminus is modified by polyadenylation. (sources: ICTVdB, Bedard, Flynn)

Virion Structure of a Picornavirus
Picornavirus virions consist of a non-enveloped, icosahedrally symmetric capsid. The capsid consists of 12 capsomers and has a diameter of 27-30 nm, which makes it one of the smallest of all viruses (thus the name "picornavirus"). The genome is tightly packed into the capsid. The capsid has four unique proteins: VP1, 2, 3, and 4. (sources: ICTVdB, Flynn)
Reproductive Cycle of a Picornavirus in a Host Cell
Using different cellular receptors (depending on the picornavirus), a picornavirus virion attaches to a host cell. Uncoating occurs, and the virus' RNA is released into the cytoplasm of the host cell through a membrane channel. Virus replication occurs entirely in the cytoplasm. The host cell's transcription processes are shut off to a degree that varies with different picornaviruses, while the IRES helps to make sure the virus' transcription is left untouched. Replication occurs. RNA is packaged into preformed capsids. Release of the virus occurs when cell lysis occurs (with the exception of Hepatits A, which is non-lytic and thus creates a more persistent infection). (source: Bedard)


Viral Ecology & Pathology
The mode of transmission, ecology, and pathology of picornaviruses vary greatly between the different genera. Interestingly, the genetics of virulence phenotypes of picornaviruses is poorly understood. According to Tracy et al., "Picornaviruses do not have virulence genes per se, but the design of the capsid and how it interacts with the virus receptor expressed on the host cell surface, specific sequences within the nontranslated regions of the viral genome, as well as coding sequences that result in different protein sequences may all have a part in determining the virulence phenotype."

Herpes Simplex Virus (HSV)



























What is Herpes Virus?
Herpes is a virus, named specifically "herpesvirus hominus". Simplex is a sub-category of the herpesvirus family. Herpes Simplex is defined by five categories, types 1, 2, 6, 7, and 8.

Where does it usually infect?
Generally herpes type 1 infects the mouth in humans and type 2 affects the genital area. However, there are recent studies indicating that there are more and more type 1 genital herpes cases are being diagnosed. Type 6 and 7 cause an infection of infancy (i.e. Roseola and Chickenpox) and Type 8 has been associated with Kaposi's sarcoma which is most often seen in HIV related conditions.

Where does the Herpes virus occur?
Herpes simplex virus (HSV) is extremely common in humans. It’s estimated that as many as 80-90% of the entire human population experience oral herpes infections by the age of twelve years old. Some who are infected with oral herpes will have an acute infection that may appear as infected gums and lips which cause high fever. Usually if an infected person has any symptoms, it would be a few only. A substantial portion of the population has recurrent oral herpes infection, showing up as "cold sores" on lips and sides of the mouth. Oral herpes does have a tendency to show up elsewhere on the face thus creating the position for not realizing that the infection actually is “oral herpes.”

Herpes infects the nerve cells of the spinal cord in the pelvic region (as is the case for genital herpes) and of the nerve ganglia in the facial region at the base of the brain (as is the case for oral herpes). Herpes is a DNA-type virus, inserting its DNA directly into the nerve endings of the skin, which then leads along nerve fibers to the nucleus of the nerve cell. Once the viral information is inserted into the cell's nucleus, this blending of viral genetic information with human genetic information is permanent. There is no cure and herpes virus remains in the body forever. The nerve cell then becomes somewhat of a “factory” for making more viral particles which can lead to recurring herpes outbreaks or conditions suitable for active viral shedding.

What herpes is not though it appears to be?
Herpes is not a "skin infection", but rather an infection of nerve cells, by way of the skin. It is not the "skin" that is infected but rather the nerve cell. When the infection becomes active again in the nucleus of the nerve cell, the viral particles flow back out, down the nerve, and out through little blisters that form in the skin. If the quantity of viral particles coming down the nerve cell isn't enough to form a blister, then a number of viral particles can be excreted through the skin without any symptoms at all.


Other facts about HSV
"Genital herpes" and "oral herpes" refer to the location where the herpes infection is found on the individual, not the type of herpes. Most genital herpes is caused by HSV-2, but can be caused by HSV-1 in as many as 30% of new cases. Oral herpes is most often caused by HSV-1, and rarely if ever by HSV-2. Because these locations are often associated with a particular type of herpes (which seem to "take hold" in those particular locations more easily), medical people, websites and literature often equate the location with the herpes type. You might find that people speak of genitally-located herpes infections as HSV-2 and orally-located herpes as HSV-1. However, either virus can occur in either place, and in fact, potentially anywhere on the body depending on the point where infection takes place.
Someone with an HSV-1 lesion on the mouth can transmit the infection to an intimate partner through oro-genital contact with the genitalia of the other (oral sex), causing a recurrent HSV-1 lesion on the genitalia of the partner. Also, other sites of HSV infection can be produced, such as on a finger (often referred to as "herpetic whitlow") or elsewhere.

NOTE: Genital herpes is not always HSV-2 and oral herpes is not always HSV-1.
Genital herpes affects about 20-25% of Americans (that equates to 1 in 5 or even as many as 1 in 4), these numbers likely exceed 50 million Americans. Though most other STDs are seen to be decreasing in their rate of new infections, genital herpes remains one of the fastest growing infectious diseases in the world in absolute numbers of cases. However, Human Papillomavirus (HPV) infections are believed to outnumber herpes infection in both growth rate and likely in absolute numbers.

Generally genital herpes infections are not considered to be extremely contagious. Casual contact on toilet seats, chairs, and similar sorts of common social contact is almost certainly non-contagious, though there is some debate that exists on that issue. Anecdotal case reports of persons acquiring genital herpes through contact in hot tubs have been published though never proven. Obviously such matters would be very difficult to verify. The herpes virus does not survive outside the body for more than about 10 seconds, and although it can survive for slightly longer in warm, damp conditions (i.e. wet towels), it dies very quickly once exposed to the air.


However, genital herpes IS contagious, most oftentimes through direct skin-to-skin contact with an infected area. The method of transmission occurs through an active herpes blister on one person with a challenged or broken area of skin on the other person. For example, a male with an open blister could transmit the virus into the vagina of a female through even the tiniest abrasion in the vaginal mucosa of the female that could occur during intercourse. Similar modes of transmission can occur from female to male, male to male, and even female to female. Oral to oral transmission of either type of virus can also happen much the same way, through infected skin with active viral activity coming in contact with challenged or broken skin.

The virus may be transmitted to the penis, the vagina, the rectum, the mouth, and more rarely, the esophagus, the trachea, and even onto broken areas of skin that may appear anywhere on the body. The New England Journal of Medicine published a photograph of a herpes infection deep down inside the esophagus of a woman in April, 1999. Herpes simplex pneumonias have been reported. And, of course, the Herpes simplex infections of the brain in newborn babies who acquire infection during delivery are well known and may have disastrous consequences. Herpes simplex may also cause wide-spread rashes on the body with redness and swelling in these areas, similar to that of measles.


Once the viral DNA has been transmitted to the receiving person's nerve cells, the infection is permanent.


Viruses are very tiny, far smaller than bacteria and far smaller than the cells of the human body. Viruses are so small that they can even slip through the tiny inter-cellular holes of the "lamb-skin" type of condom which are normally small enough to prevent sperm cells from getting through. Latex-type condoms have been shown to be most protective against viral transmission, considering the condom covers the affected area completely during the sex act. Viral particles from an active lesion can become liquid borne from inside the condom and possibly leak out the base of the condom.

Herpes infections produce a number of different signs and symptoms. Traditional "first episode" herpes, most commonly described in women, are serious illnesses, with high fever, often severe outbreaks, painful urination and even inability to urinate. Hospitalization is sometimes necessary, with catheterization, IV fluids, and intravenous anti-viral medications being required.

Most people diagnosed with a genital herpes infection don't describe having such severe symptoms; however, they might experience a cluster of small blisters surrounded by a red base on or around the genitalia. Often the blisters have already ruptured, leaving behind a cluster of ulcers which scab over and require as much as one to two weeks to heal. Once the blisters have ruptured, it’s likely that a culture cannot be taken to determine if the blister is in fact herpes related. Thus, it is important to seek medical attention upon first notice of blisters to insure that an accurate culture can be taken and proper diagnosis occurs.

During the blister and ulcer phase, herpes lesions contain enormous amounts of viral particles and should be considered highly contagious to any challenged or broken skin it may come in contact with including but not limited to: the eye, mouth, esophagus, trachea, lungs, anus, urethra, penis and vagina.

In most cases herpes symptoms are subtle and often go undetected. Many have reported slight redness in the skin but without obvious lesions. This area, even without textbook symptoms should be considered contagious. Sometimes the skin will form tiny red bumps that don't blister, called "erythematous papules". Sometimes there are no signs on the skin at all but rather a "prodrome" such as urinary urgency, urinary frequency, and/or aching or tingling in the legs. Tingling has been described best as a sensation similar to a rub burn or mild sunburn. Also, itching, burning, tingling, pain or pressure at a previous or potential outbreak site may occur.

Many people with genital herpes and likely in oral herpes cases produce viral particles even when they have no symptoms whatsoever. These people are likely contagious even when they have no symptoms at all. This term is called "asymptomatic shedding."

In cases of women with genital herpes, herpetic lesions inside the vagina may only produce a vaginal discharge as an external symptom, resembling a yeast infection. It may be difficult without examining the patient to know whether the “yeast infection” is actually what it seems or if it is in fact genital herpes. Some women with both chronic yeast infections and genital herpes infections may find themselves confused as to which problem might be causing the symptoms.

These women should be under the care of a trusted doctor and should not attempt to self-medicate until the symptoms have been clearly explained. Self diagnosing and treatment can only make genital herpes symptoms worse.

Viral shedding can occur from people who have acquired the infection asymptomatically. This means that people can acquire a herpes infection and have no symptoms, and later they can be shedding virus and therefore be contagious. In an article that appeared in the New England Journal of Medicine in 2000, Anna Wald concluded that "seropositivity [testing positive in a blood test] for HSV-2 is associated with viral shedding in the genital tract, even in subjects with no reported history of genital herpes." This means that people can be infected and that only their blood tests might be positive, that they may have no symptoms or few symptoms that are recognized as being caused by herpes, and yet that they may still be shedding virus and may be contagious.

HSV Virus Type
1. Herpes simplex virus type 1 or HSV-1 can cause oral and genital herpes and sometimes infect the hands and other parts of the body. About 80% of HSV-1 cases are oral herpes (cold sores, fever blisters).
2. Herpes simplex virus type 2 usually causes genital herpes but like oral herpes can infect other parts of the body. Both stains of herpes simplex viruses tend to be more virulent in their most common places (oral herpes for HSV-1 and genital herpes for HSV-2)
3. Herpes varicella-zoster virus (also called herpes varicella/chickenpox and herpes zoster/shingles). It mostly affects children (varicella) and people over 60 (shingles)
4. Epstein Barr virus (often abbreviated to EBV)
5. Cytomegalovirus (CMV)
6. Human herpes virus 6 (HHV6)
7. Human herpes virus 7 (HHV7)
8. Human herpes virus 8 (HHV8 or it can be called KSHV)

References

http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/dental/tutorials/flora/herpes.html
http://www.best-herpes-treatments.com/information-on-herpes-simplex.html