Posts Tagged ‘ test ’

Diagnostic Tests Used for Viruses

Haemagglutination Assay

Introduction

A simple and rapid method of quantifying the amount of virus in a sample. Haemagglutination is the agglutination of red blood cells. Viruses with envelops or surface proteins are able to bind to the sialic acid, N-Acetylneuraminic acid found in the cell membrane of red blood cells. Because each agglutinating molecule (in this case each viral molecule) can bind to multiple red blood cells, a clump of cells begins to form, this is agglutination. This agglutination forms a lattice structure.

Haemagglutination Inhibition

Haemagglutination inhibition is the addition of an inhibitor of the virus. Antisera (a blood serum which contains antibodies) is used in this case. The antibodies will bind to the virus and thus prevent haemagglutination.

By creating multiple, increasing levels of dilution e.g. 1:2, 1:4, 1:8, etc. of virus to antiserum we can determine a haemagglutination titre, this is the highest value of dilution (i.e. smallest amount of virus) which inhibits agglutination.

A typical haemagglutination inhibition assay looks like this:

You can see that point of agglutination, where the contents of the well go from a homogenous cloudy red, to containing a red spot which is the agglutinated red blood cells. In the above example, sample E (the highlighted row) has a haemagglutination titre of 1:512 as this is the largest dilution which prevents haemagglutination.

By monitoring the haemagglutination titre, we can determine how the virus is progressing over time, for example:

  • 1st assay – an endpoint is reached at a dilution of 1:4
  • 2nd assay – an endpoint is reached at a dilution of 1:16
  • 3rd assay – an endpoint is reached at a dilution of 1:8

From this would we be able to see initially the virus count is low as it is inhibited at a low dilution, however at the next assay the haemagglutination titre is higher (greater dilution of virus reached before it is inhibited) this must mean the virus is replicating as the initial count of virus is higher (i.e. takes greater amount of dilution to reach inhibition). WIth the final assay, we can see that because the haemagglutination titre has decreased, the virus must be reducing in numbers, indicating recovery.

ELISA

Introduction

ELISA is an acronym for Enzyme-linked immunosorbent assay and is used to detect whether or not a certain antibody or antigen is present in a sample. To indicate the presence of an antibody or antigen, reporter molecules are used which are identifiable by a change in colour. Therefore, if an antigen or antibody is present, a colour change will be observed.

Method

A typical ELISA requires the use of a 96-well microtitre plate (as used in the haemagglutination assay), but the wells of the plate are coated with a known or unknown antigen. When diagnosing viruses, the antigen will be unknown as it is the sample which we are testing. To this an antiserum is added, if the virus antigen is present in the sample, then the antibodies will bind. If not, no binding will occur.

To determine whether or not an antibody-antigen complex has formed (i.e. a positive diagnosis of the virus), an antiglobulin is added. This is an antibody which binds to the antibodies used in the initial serum. For example, Goat anti-rabbit IgG is a rabbit antibody (Immunoglobulin G [IgG]) which will bind to goat antibodies. There are many possible variations of antiglobulins.

The antiglobulin will be ‘labelled’ with an enzyme (an enzyme is attached to the antibody which has a negligible effect on its binding capabilities).

Finally, the substrate of the enzyme using to label the antibody is added. This substrate will be broken down if the secondary antibody (antiglobulin) bound to the primary antibody. The break down of the substrate will be coupled with a colour change, for easy identification that this has occurred. The greater the intensity of the colour change, the higher the concentration of the initial antigen.

Sometimes a spectrophotometer (a device used to detect light intensity) will be used to determine the degree of change in colour, as this can be used to calculate reasonably accurate values for initial antigen concentration and thus virus concentration in the host.

Below is an example of the variations in colour which occur during an ELISA test. The coloured wells indicate positive samples:

This type of ELISA is known as an indirect ELISA test, however there are other forms of this test, such as sandwich ELISA, which is where the well is coated in antibody, to which an antigen binds and to this another antibody is added. The antiglobulin tests for the secondary antibody.

There are also competition ELISA tests, where the added sample is an antibody-antigen complex, this is added to antigen coated wells. If the concentration of initial antigen in the sample is high then there will be fewer available antibodies to bind with the antigen in the wells. The wells are washed to remove unbound antibodies and as with the indirect assay, enzyme-coupled antiglobulins are added with substrate that elicit a colour change. However in this case a high initial concentration of antigen in the sample will yield a low change in colour.

Advertisements

Testing Cranial Nerves

Introduction

Cranial nerves arise from the brain directly (unlike spinal nerves which arise from the spinal cord). There are twelve pairs of cranial nerves, varying in length – from supplying nearby structures of the head to the Vagus nerve (X) which is the longest nerve in the body.

Cranial nerves may carry:

  • Sensory information only, i.e. information from an organ to the brain
  • Motor information only, i.e. information from the brain to an organ
  • Both sensory and motor information

Cranial nerves may also be either:

  • Afferent – Meaning to carry sensory information into the central nervous system
  • Efferent – Meaning to carry motor information away from the central nervous system
Cranial Nerve Type of Nerve Fibre Function
Olfactory (I) Sensory Carries sensory information from the olfactory bulb to the brain
Optic (II) Sensory Carries sensory information from the eye to the brain
Oculomotor  (III) Motor Enables the eye to make small, intricate movements
Trochlear (IV) Motor Supplies the extrinsic muscles of the eye
Trigeminal  (V) Both Receives sensory information from the face and supplies motor fibres involved in mastication
Abducens (VI) Motor Supplies the extrinsic muscles of the eye
Facial (VII) Both Supplies motor fibres for facial movements and receives sensory information from ‘anterior taste’
Vestibulocochlear (VIII) Sensory Carries sensory information from the vestibule (balance) and cochlear (hearing) of the inner ear
Glossopharyngeal (IX) Both Carries sensory information from posterior taste (posterior tongue and pharynx) and supplies muscle fibres of the pharynx
Vagus (X) Both Carries sensory information from the pharynx and larynx. Supplies muscle fibres of the larynx as well as; visceral motor fibres to the heart and various thoracic and abdominal organs (including the gastrointestinal tract)
Accessory (XI) Motor Supplies muscle fibres of the neck and shoulders
Hypoglossal (XII) Motor Supplies muscle fibres of the tongue

Testing Cranial Nerves

There are certain tests which can be done to ensure that a cranial nerve is working properly. The tests differ between the nerves due to their different functions. Each test usually has a reflex response which signifies that the cranial nerve is undamaged. The tests have been written primarily with animals in mind, but the majority of these are also observable in humans.

Cranial Nerve Test of Afferent Nerve Test of Efferent Nerve
Olfactory (I) A strong smell is used to test the aversion reflex. If the cranial nerve was undamaged the subject would respond to the smell
Optic (II) Avoiding creating air movement, a finger or hand is thrust towards the eye. If the optic nerve is undamaged, the subject will employ the menace reflex and close the eyelid in response to the finger/hand
Oculomotor (III) Testing eye muscles- Usually tested alongside nerves IV & VI, the movement of the eye and eyelid is observed in response to a stimulus. If this nerve is damaged, the pupils of the eye at rest point down & out

Pupillary reflex- Shining a light into the pupil of one eye should result in the constriction of both pupils

Trochlear (IV) Tested alongside nerve III & VI, if this nerve is damaged a strabismus (abnormal eye alignment) in an up & in direction will be apparent
Trigeminal (V) Touching the skin around the eye will result in the palpebral reflex (closing of the eyelids in response to the touching of the skin). If the nerve is damaged, this will not occur. Also, touching the cornea itself should result in the corneal reflex (closing of the eyelids in response to the touching of the cornea). Again this is absent if the nerve is damaged Should the efferent nerve become damaged, you will be able to observe a drooping jaw in the subject
Abducens (VI) Tested alongside nerve III & IV, if this nerve is damaged a strabismus in a medial, inward direction will be apparent
Facial (VII) The corneal reflex may be tested to check for damage to the nerve. In animals with motile pinna (external ear – not motile in humans), the handclap reflex can be tested. If the nerve is not damaged the pinna will move in response to a loud clap If the efferent nerve is damaged, drooping ears and facial paralysis may be observed. Ptosis (drooping of the eyelid) can also be observed. The menace and palpebral reflexes may be tested to check for nerve damage
Vestibulocochlear (VIII) Testing the Cochlea- The handclap reflex is tested. If the pinna do not respond, this may indicate damage to the nerve.

Testing vestibular responses- In response to altering the orientation of an animal i.e. tilting the body down to face the floor slightly, the neck will self right the head so the head is facing forwards if the nerve is undamaged (tonic neck reflex). If the nerve is damaged, animals may tilt their head with the ear down on the side of lesion/damage. Further observations include nystagmus – spontaneous eye movement, moving slowly in a lateral direction and then returning with a quick eye movement. The direction of the slow movement indicates the side of the lesion/damage

Glossopharyngeal (IX) Bilateral damage to the nerve results in the loss of the gag reflex. Observations that this nerve is damaged include dysphagia – difficulty swallowing
Vagus (X) Similarly to nerve IX, lack of the gag reflex and observing dysphagia can indicate damage. Laryngeal paralysis can be observed with damage, this can cause loss of ability to speak/bark etc. and loud noises when inhaling. Other respiratory and cardiovascular anomalies may arise if damaged.
Accessory (XI)
Hypoglossal (XII) If the nerve is damaged, minor dysphagia and a drooping tongue may be observed – often drooping to the side of damage/lesion if damage is unilateral