Question

fill in the tables and SO question for activity 3 &4.（all questions from physioex 9.1 lab simulation in physiology）

Serological Testing 181 of the wells will appear. The develop te pper soels to remove for each sample (the samples are in the first row, from top to 27. An enlargement to remove ment will progress over time. To determ ELISA, you for each sample (the samples are i bottom, of the microtiter plate): the you steps to reduce any nonspe- e this simulation vells. In a typical . Click the well and the optical density will appear in the i window of the microtiter plate reader Click Record Data to display your resalts in the grid (and record your results in Chart 3) to the dip dispenser to place a tijp to the lest tube containing the Indirect ELISA Results ndaw the sample into the tip. CHART 3 to the micrositer plate to dis- 1Pprells of the plate The tip will auto density HIV test result nd ásposed of in the biohazardous Sample Optical Patient A Patiens B Patient C de rentaining samples will automat e hour by clicking the + button beside nubation time allows the antigens had so the antsbodies present in the od a sart the timer. The simulation com- e ine period into 10 seconds of real time. asing buffer squeeze bottle to the microtiter 」anangssambodies andprevent nonspecific Negative rign and antibhody to the sink to dump washing 28. You will now indicate whether the result for each sample rbund antibodies into the sink. is negative, indeterminate, or positive for HIV y te misnweiter plate to the paper towels. The plate . A result of 0.300 is read as negative for HIV1 o the sarface of the paper towels to remove be mubicharnel pipettor to the pipette tip dis- g te multichunnel pipettor to the developing buffer . A result of 0.300-0.499 is read as indeterminate (need sing!pdfrom the wells. to retest) .A result of -0.500 is read as positive for HIV-1 oas the developing buffer into the tips. fer cuntns the conjugated secondary antibody Click the row for the sample in the grid and then click the - button, IND, or the + button above the HIV test result column to indicate whether the result for the sample is positive, indeter minate, or negative for HIV. Repeat this step for all five samples ch pipettor to the microtiter plate to dis- Record your results in Chart 3 olton into the wells. The tips will automatically be i the tiner to 1 hour and then click Start to start the antibody to bind to dised of in the biohazardous waste disposal. Quiz for Activity3. After you complete the Experiment, take the online Post-lab al livw the conjugated secondary ury anikody if it is present in the sample. Activity Questions 1. Describe how you can tell that this test is the indirect asting buffer squeeze bottle to the microtiter cone any bonspecific binding that occurred. ELISA rather than the direct ELISA te microtiter plate to the sink to dump the contents et o he sufice of the puper toweks to re moue ed to the surf 2. Describe what the secondary antihody bindsto in this ac g ligaid from the wells. nt the üips tivity and why. I pipettor to the pipette tip dis- ichannel pipettor to the substrate bottle to rate into the tips. nnel pipettor to the microtiter plate to dis- on into the wells. The tios will automatically be posed of in the biohazardous waste disposal

Answer 1

CHART-3

SAMPLE	OPTICAL DENSITY	HIV TEST RESULT
PATIENT A	2	POSITIVE
PATIENT B	0.7	NEGATIVE
PATIENT C	0.4	INDETERMINATE
POSITIVE CONTROL	1.0	POSITIVE
NEGATIVE CONTROL	0.1	NEGATIVE

ACTIVITY QUESTIONS

1. The membrane is treated with secondary antibodies in this procedure hence it is indirect ELISA.

2. The secondary antibody binds to the primary antibody in the patient's sample because they are anti-antibodies specifically prepared to bind to antibodies for specific HIV antigens.

3.Seroconversion is the period during initial infection when the the HIV antibodies develop and become detectable.

A sample is said to be seroconverted if the results are indeterminate.

CHART-4

SAMPLE	gp160	gp120	p55	p31	p24	HIV TEST RESULT
PATIENT A	PRESENT(STRIPED)	PRESENT	PRESENT	PRESENT	PRESENT	POSITIVE
PATIENT B	ABSENT(NO STRIPE)	ABSENT	ABSENT	ABSENT	ABSENT	NEGATIVE
PATIENT C	PRESENT	ABSENT	ABSENT	ABSENT	ABSENT	INDETERMINATE
POSITIVE CONTROL	PRESENT(STRIPED)	PRESENT	PRESENT	PRESENT	PRESENT	POSITIVE
NEGATIVE CONTROL	ABSENT(NO STRIPE)	ABSENT	ABSENT	ABSENT	ABSENT	NEGATIVE

ACTIVITY

1. In gel electrophoresis the negatively charged molecules migrate toward anode hence proteins are separated by size.

2. In a western blot, antigens and antibodies are present because the reaction between antigen and antibodies are used in the technique to determine the presence of a positive HIV test.

3.The human serum and bovine serum would have epitopes in common but not completely identical. The precipitin line for positive and indeterminate results are spurred confirming the partial identity of antigens.

ACTIVITY 3 (ELISA)

1.In direct ELISA, the antigen is bound to a protein to block all binding sites except a specific antibody binding site. Teh antibody is also bound to an enzyme to block all other binding sites except the antigen binding site. The mixture leaves the bound antibody and antigen along with bound enzyme. The enzyme is then treated with substrate to detect the antigen.

However,in indirect ELISA,an antibody which recognises the antigen is first added to the antigen after the binding sites are treated with protein. This antibody does not have an enzyme bound to it. Then the enzyme-antibody complex is added which recognises the unbound antibody instead of absorbing the antigen directly. Then the enzyme is treated with substrate to identify the antigen.

2. Usually when a person is infected but seroconverting, the test results may show indeterminate.

3. If the negative control gave indeterminate results, it means the value for determining the HIV negative criteria should be lesser than the negative control optical density, in this case for example if OD<0.3 is negative but negative control is indeterminate, then OD value for negative HIV should be lesser than 0.3 .

4. Antibodies are Y shaped. The upper two arms are the variable region. The bind to specific antigen to neutralise their harmful effect on the body. The lower stick region is the constant region which is the same in all antibodies.

ACTIVITY 4(WESTERN BLOTTING TECHNIQUE)

1. The western blot is more specific than ELISA because the western blot detects antibodies for many different types of HIV antigens at the same time while the ELISA only looks for one at a time.

2. If a patient has indeterminate results, the test has to be performed again after few weeks when the phase of seroconversion is over.

3. Nitrocellulose membrane is prepared by treating by cellulose with a nitrating agent such as nitric acid.

4. In the procedure, the antigen or antibody is washed with antibody-enzyme complex to absorb the antigen or antibody. Again the excess enzyme-antibody complex is washed off so that the bound antigens remain for detection.