Download Generalized ELISA protocol for detecting a target antigen. Enzyme

Names:__________________ (groups of 2)
Score:______/30
DIAGNOSIS OF DISEASE BASED ON IMMUNE RESPONSE
http://www.hhmi.org/biointeractive/vlabs/
Diagnosis
Components of the immune system called ______________________ are found in the liquid portion of blood
and help protect the body from harm. __________________________________________________________
in a laboratory-based assay to help diagnose disease caused by malfunctions of the immune system or by
infections.
Potential Experimental Problems
_____________________________________ is used in many laboratories to determine whether a particular
_________________ is present in a patient's ________________. 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.
Limitations of the Test
First, a positive result correctly confirming ________________________________ does not
necessarily mean the patient is __________________. The body can continue to
_____________________ even though the person may have had the
____________________________________________.
Second, people may be poor _______________________________ or may have some interfering
substance in their ___________________. The amount of __________________________,
consequently, may be too low to measure accurately or may go undetected. This result is termed a
_________________________.
Third, a positive result may occur if ____________________________________________________.
Unlike a ________________________ result where the specific antibody is detected, however, this
___________________. Testing many patients and running tests more than once helps lab workers
distinguish a ___________________ result. To avoid simple experimental mistakes leading to
incorrect results, scientists conduct tests using duplicate (or, sometimes, more than _______)
samples.
How Does This Work?
The interaction of ________________________ outside the body--in the ___________________can
be used to determine if a patient has an ____________________________________. The test
measures whether a __________________________ associated with an _________________ can
be found in a patient's ______________. A positive result indicates that the ___________________
is there and implies that the person has _______________________ a particular disease.
This exercise begins with removing ____________________________________, which can interfere
with the test, from a patient's blood sample. The watery fluid that remains is called ________. A
portion of serum __________________________________ is allowed to react with the target
antigen. A correct match causes the ________________________________ to bind together.
Detection becomes possible when a ________________________. This antibody is prepared from
__________________________________________________________________; the human
antibody in this case serves ___________________________ and the animal thus produces an
______________________________________________. Once isolated, the second antibody can
be ____________________________ to a system that can produce a ________________________.
In ELISAs, the ___________________ is exposed to the second antibody, which binds to the
antibody portion of the complex (against which it was formed), creating a _________________
structure (Figure 1). The signaling system consists of an __________________ attached to the
_________________________. When the appropriate ___________________ is added, the
___________ converts it to a _______________________________ that can be measured.
This test _________________ how much _________________ by the amount of ______________.
The more _________________, the more __________________ must be attached. The amount of
secondary antibody present is determined by the amount of target, or ______________, available.
Finally, because the _____________________________, the more _____________ that is
accessible, the more _____________________ will be retained. The measure of __________,
therefore, reflects the _______________________________________________________.
Lab Notebook
1. What disease is the ELISA testing?
2. What specifically is the test detecting?
3. How long does it have to be spun in the centrifuge? Why?
4. What dilutions must be made? Why?
5. Why do you have to discard the tip in between samples?
6. What is the positive control? What is the negative control?
7. Why don’t you have to switch tips in between the control wells?
8. Why must you incubate the plate?
9. Why is the fluid removed from the plate? Why do you have to use different tips between samples?
10. Why must it be washed several times?
11. What does the secondary antibody bind to? What is its function?
12. What is HRP? What is its function?
13. Why do you have to do the wash step after the HRP?
14. Why is this so annoying?
15. Why must you wait 15 minutes in Step 11?
Now print off your results and attach them to this page.
ELISA Activity Notes - http://www.biology.arizona.edu/immunology/activities/elisa/elisa_intro.html
Figure 1: Generalized ELISA protocol for detecting a target antigen. Enzyme (E) is conjugated to
secondary antibody
A Bind sample to support
C Add secondary antibody-enzyme conjugate; wash
b
B Add primary antibody; wash
D Add substrate
An HIV ELISA, sometimes called an HIV enzyme immunoassay (EIA) is the first and most basic test to
determine if an individual is positive for a ____________________, such as HIV. The test is performed in a 8
cm x 12 cm plastic plate which contains an 8 x 12 _______ of 96 wells, each of which are about 1 cm high and
0.7 cm in diameter.
An ELISA plate
ELISA Method
Partially purified _____________________________________________________
__________________________________________________________________
Patient serum which contains ____________________________________________
____________________________________________________________________
Anti-human __________________________________________________________
____________________________________________________________________
Chromogen or ________________________________________________________
____________________________________________________________________.
How is the positive ELISA different from the negative ELISA?
False positives
It is entirely possible that an individual _________________ with HIV has antibodies which may give
a ________________ in the HIV ELISA. This is called a __________________-. One reason for this
is that people (especially women who have had multiple pregnancies) may possess antibodies
directed against ___________________________________ which are present on the host cells used
to propagate HIV. As HIV buds from the surface of the host cell, it incorporates some of the host cell
HLA into its envelope. _______________________________ can occur during the window between
infection and an antibody response to the virus (seroconversion).
ELISA data from three patients
Positive
Control
1.689
Negative
Control
0.153
Patient A
O.055
Patient B
0.412
Patient C
1.999
Assay
Control
0.123
Above is ELISA data from three patients. Numbers are expressed as ________________________.
The cutoff value indicating a positive result is 0.500. Optical densities of 0.300 to 0.499 are
indeterminate and need to be retested. Values below 0.300 are considered to be negative. In most
cases, a patient will be retested if the serum gives a positive result. If the ELISA retests are
______________, the patient will then be retested by _________________________ analysis.
Review Questions
What is the ELISA test intended to measure?
A. Antibody to HIV only
B. Antigen to HIV only
C. Presence of free, circulating virus in the patient
D. Antibodies directed against HLA molecules
What would happen if serum were omitted from the ELISA, but all other steps remained the same
and were performed properly?
A. Anti-human Ig-conjugate would not bind and be washed away.
B. Anti-human Ig-conjugate would bind non-specifically to the ELISA plate.
C. The O.D. values would be nearly the same as the assay control.
D. Both A and C.
What would happen if the anti-human Ig-conjugate were not washed free of the well before the
substrate was added?
A. The ELISA would not develop when the substrate was added.
B. The ELISA would develop normally.
C. All wells would show uniform over development due to unbound and excess anti-human Ig enzyme
conjugate.
D. Both A and B.
From the ELISA data, which patient is seropositive for HIV?
Positive Control
1.689
A. Patient A
Negative
Control
0.153
B. Patient B
Patient A
Patient B
O.055
0.412
C. Patients B and C
Patient C
1.999
D. Patient C
Assay Control
0.123