Lab 7

Question 1

How would a lab get a patients antibodies

Answer 1

From their blood, 40ml of blood is centrifuged for 15min, and the supernated containing the sera is kept

Question 2

variables, and problems with the ELIZA assay

Answer 2

Although the procedure is routine and straightforward, it involves a number of variables, such as reagent selection, temperature, volume measurement, and time, which if not adjusted correctly can affect subsequent steps and the test outcome

Question 3

Limitations of the ELISA assay

Answer 3

First, a positive result correctly confirming the presence of antibody does not necessarily mean the patient is sick.

Second, people may be poor producers of antibody or may have some interfering substance in their blood. The amount of antibody, consequently, may be too low to measure accurately or may go undetected. This result is termed a false negative.

Third, a positive result may occur if an unrelated antibody reacts with the antigen nonspecifically

Question 4

What does ELISA measure

Answer 4

whether a specific antibody associated with an illness can be found in a patient’s blood. A positive result indicates that the antibody is there and implies that the person has encountered a particular disease.

Question 5

Why are red and white blood cells removed before an ELISA assay

Answer 5

they can interfere with the test

Question 6

what is the watery, cell free portion of blood called

Answer 6

serum, contains antibodies
= a clear watery fluid obtained after removing blood cells and other components from blood by centrifugation that will contain antibodies

Question 7

ELISA secondary antibody

Answer 7

In ELISAs, the antigen antibody complex is exposed to the second antibody, which binds to the antibody portion of the complex (against which it was formed), creating a sandwich-type structure (Figure 1). The signaling system consists of an enzyme attached to the second antibody. When the appropriate chemical is added, the enzyme converts it to a colored substance that can be measured.

Question 8

how are antibodies quantified in ELISA

Answer 8

This test quantifies how much enzyme is present by the amount of color produced. The more enzyme present, the more secondary antibody must be attached. The amount of secondary antibody present is determined by the amount of target, or first antibody, available. Finally, because the first antibody binds to antigen, the more antigen that is accessible, the more first antibody will be retained. The measure of color, therefore, reflects the amount of antigen initially present.

Question 9

Primary antibody

Answer 9

first antibody used in an immunoassay to detect the foreign particle; in this case, we are testing to see if the serum from the patients contains primary antibodies to SLE.

Question 10

Secondary antibody

Answer 10

he second antibody used in an immunoassay that detects the primary antibody. Note, this antibody must be made in a different species (rabbit, donkey, horse) than the primary antibody, in order to recognize the primary antibody as “foreign”. In this case, we are using HRP-tagged rabbit anti-human antibodies as our secondary antibody.

Question 11

Antibody-enzyme conjugate

Answer 11

antibodies (usually secondary antibodies) are “tagged” or joined to enzymes through a chemical process. Now the antibody is carrying an enzyme, so wherever the secondary antibody binds, the enzyme is present also.

Question 12

Antigenic site

Answer 12

the part of the antigen that is recognized by the antibody

Question 13

ELISA

Answer 13

Enzyme-Linked Immunosorbent Assay; this is an assay that uses an enzyme linked to an antibody. In this experiment, a colorless substrate is turned into a colored product by the bound enzyme. The amount of activity of this enzyme (as determined by detection of the amount of colored product) is used as a measurement of the amount of bound antibody.

Question 14

HRP

Answer 14

horseradish peroxidase; an enzyme used to stimulate the conversion of the colorless substrate into a colored product in this exercise

Question 15

Systemic Lupus Erythematosus (SLE)

Answer 15

Lupus is believed to be an autoimmune disease.Instead of fighting antigens, the antibodies mistakenly fight the body’s own cells. A systemic disease is one in which several different parts of the body may be affected. In systemic lupus, these include the skin, kidneys, nervous system, lungs, heart and/ or blood-forming organs.

Question 16

Titer

Answer 16

the concentration of a substance in a solution. For instance, the amount of a specific antibody in the serum.

Question 17

STEP 2 of Elisa what are you doing

why are there three different solutions

Answer 17

We are preparing serial dilutions to determine the concentration of antibodies in the blood samples

we are using PBS, a common laboratory solution that contains table salt. The amount of salt in the buffer is about the same as that normally found in blood. The phosphate buffer keeps the solution from becoming too acidic or basic.

three solutions for the three serial dilutions (1:2) (1:10) (1:100)

Question 18

What has the ELISA plate been pre-treated with

Answer 18

SLE antigen

Proteins such as antigens and other biological materials can, under proper conditions, physically bind to the plastic material composing the wells of the ELISA plate.

The coating procedure must be done carefully. If too little antigen is used, bare spots will permit antibody or other protein to stick, leading to a false-positive reaction. If too much antigen is used, the excess will be able to bind SLE antibody from patient sera but then will be washed away, creating a false-negative reaction.

The addition of antigen is the crucial first step in the chain of recognition events between antigen and antibody that will end with the formation of color from the enzyme bound to the second antibody.

Question 19

Why is it important to have a postive and negative control

Answer 19

Positive: Primary antibody

Any serum from a patient that contains the antibody for SLE will recognize the antigen in the well and bind to it. Each serum sample contains many different types of antibodies, but because they are so specific in how they react, usually no antibody will recognize the SLE antigen except the SLE antibody.

Negative: only buffer

One control should always produce a positive response if the reagents and conditions are correct. The second control should never produce a positive response. If either control sample fails to react as expected, then the results for the patients’ samples cannot be trusted and the assay must be repeated.

Question 20

WHY does an ELISA plate need to be incubated

Answer 20

Incubating serum samples in antigen-coated wells helps ensure that the antibody present in the sample will interact correctly with the antigen. Because SLE is a disease of humans, the reaction usually occurs at the temperature of the human body, which is 37 degrees C. Time is important: the reaction must proceed long enough for adequate binding to occur or the measurement will be artificially low.

Problem: If the timer is set to less than 15 minutes, the proper reaction will not occur and no color will be evident at the end of the assay. The observer will record the results incorrectly as false negatives.

Problem: If the temperature is set too low, the reaction will not be completed in the allowed time. If the temperature is set too high, protein (antigen and antibody) will be adversely affected via a process known as denaturation, which diminishes its ability to interact. The results will again be recorded as false negatives

Question 21

Why wasH an ELISA plate after incubation

Answer 21

Washing helps remove any antibody that did not react with the SLE antigen in the well. When the fluid is removed from the well, antibody that has reacted with antigen remains attached to the well surface. Unreacted (unbound) antibody may also remain in the well in the small amount of fluid that is left behind. This unbound antibody must be removed, because the anti-human antibody added in the next step will recognize and react with any antibody remaining in the well, regardless of whether that antibody is specific for the SLE antigen. A reaction with non-SLE antibody will produce a false-positive result.

Question 22

HRP substrate

Answer 22

HRP (the enzyme) will interact with a substrate called ABTS (2,2’-azinobis-3-ethylbenzothiazoleine-6-sulfonic acid) to produce a yellow solution.

can be estimated by eye or quantitatively measured in a spectrometer at 414 nanometers.

Question 23

How can the lupus ELISA test be used to test for the presence of HIV

if the reuslts for HIV were the same as this exercise, what would they indicate about the three patients

Answer 23

use a primary antibody that targets HIV

Question 24

Agglutination reactions (LAB)

Answer 24

A positive result is when antibides bind an avalible antigen, causing a clump

Question 25

Direct aggultination

Answer 25

antigen comes incontact with antibodies

add purifed antibodies, or purified antigen depending on what you are wanting to test for

Question 26

Indirect agglutination

Answer 26

artifical constructs like coating latex micobeads with antibodies or antigen

helps to increase the visability of any binding and clumping that occurs

Question 27

What is in the PathoDx strep kit

Answer 27

reg 1 reg 2

both are combined to lyse cells, and releaase the antigen

reg 3inactivated and neutralized the acidic mixture produced by 1 and 2

if this was not donethe antibodies woud be denatured from the latex beads preventing agglutination

Question 28

Antibiotic synergy

Answer 28

more than one antibiotic acts in a synergenic way

if there is no BOOSTED effect, the effect is said to be additive

when the two antibiotics diminish the effects of one another this is said to be antagonistic

Question 29

Tetracycline and vanco

Answer 29

sysergestic effect when used together agaisnt pseudomonas aeruginosa

Question 30

What bacterial structure is the beta-lactam ring similar to in bacteria

Answer 30

d-ala-d-ala of peptidoglycan