BIOTECH Project, University of Arizona ELISA Assay

TEACHER GUIDE: Using the ELISA Assay for Disease Detection In this laboratory you will use the ELISA (Enzyme Linked ImmunoSorbent Assay) for detecting the presence of an antigen, such as a disease related agent, from a sample of body fluid! "ou will be gi#en a solution that represents your body fluid! "ou will e$change body fluid with three other indi#iduals! %he ELISA test will be used to detect a simulated disease agent in the mi$ed body fluids! &inally, you will analyze the class data set to determine identity of the original carrier(s)! %his assay is similar to that used for the AI'S test, in which an ELISA is used to detect indi#iduals infected with the #irus that causes AI'S ((I))! Materials: Each student should have the following items before starting the laboratory: * Microcentrifuge tube containing "body fluid" (BF) * Disposable transfer pipet Each group of three students should also have: * ELISA test plate The class should have * Positive and negative control solutions (+ and -) * Paper towels * Antibody solution (Ab) * Washing solution (in wash bottle) * Color reagent solution (TMB) Proced re: E!changing Sharing Sol tions *! At the start, you+ll ha#e a transfer pipet and a tube containing a solution that represents your ,body fluids!, "e s re to choose st dents fro# all o$er the class% and not & st in yo r i##ediate area' (ait for the instr ctor)s signal *efore e!changing fl ids' -! &ind one other student in your class and e$change your solution from your +body fluid+ tube with him or her! .se transfer pipet to swap half of your +body fluid+! +, sharing: "rian /! At the instructor+s signal find a different student to e$change your sharing solution with! +- sharing: S san 0! At the instructor+s signal e$change your sharing solution with one more student! +. sharing: /ennifer The three na#es 0ritten a*o$e are the 1eo1le 0ith 0ho# one indi$id al s0a11ed *ody fl id' "e s re st dents s0a1 only 0ith three 1eo1le and that e$eryone does their first s0a1 si# ltaneo sly% then their second s0a1 si# ltaneo sly% then their third' That 0ay% things don)t get conf sing 0ith one 1air on their third s0a1 0hile e$eryone else is on their second'

BIOTECH Project, University of Arizona ELISA Assay

ELISA Test After all three contacts ha#e been completed, you will do an ELISA test on your sharing tube fluid! *! &orm a group (usually four people) at your lab station! "our group will run an ELISA assay on your mi$tures of body fluids from each student in your group using an ELISA plate! St dents can 1artner 1 for the ELISA 0ith 0ho#e$er they)d li2e% not necessarily 0ith their fl id3 s0a11ing 1artners' 4or e!a#1le% I s0a11ed 0ith "rian% S san% and /ennifer% * t #y la* gro 1 for analysis is #yself% To#% Dic2% and Harry' St dents sho ld record the na#es of their la* gro 1 on the ta*le *elo0 for analysis 5not their s0a11ing 1artners% 0ho sho ld *e recorded on the 1re$io s 1age so they don)t forget6' Ma2e s re e$eryone in a gro 1 has their ta*les la*eled the sa#e 0ay 50ith the sa#e 1erson in the first ro0% the sa#e 1erson in the -nd ro0% etc'6' Sa#1les Loaded Res lts

1yself

222222222222222222

%om

222222222222222222

'ick

222222222222222222

(arry

222222222222222222

3ositi#e (4)

222222222222222222

5egati#e ( )

222222222222222222

222222222222222222 222222222222222222

222222222222222222 222222222222222222 The last t0o ro0s are e#1ty% 0hich is fine% or the la* gro 1s co ld *e larger to se those last ro0s' -! .sing your transfer pipet, add three drops of your sharing tube fluid into each of three wells! "e s re to record which wells contain your fluid to a#oid confusing your wells with another person+s! 3. Each group will also do one positive and one negative control. Using the dropper bottle pipets for each control, add two drops of a positive test solution (+) into three wells and two drops of a negative test solution (-) into a different set of three wells.

BIOTECH Project, University of Arizona ELISA Assay

0! After e#eryone has added their solution, lea#e the plate on the lab table undisturbed for fi#e minutes! (hile the sa#1les are standing% I tal2 a*o t the na#e of the test to get st dents thin2ing a*o t 0hat co ld *e ha11ening' The na#e is ELISA% 0hich stands for En7y#e3Lin2ed I## no3Sor*ent Assay' I as2 the st dents to tell #e 0hat )I## no) #eans to the#' Us ally% they)ll say so#ething a*o t the i## ne syste#' Then I as2 0hat o r i## ne syste#s se to fight disease% and at least one st dent says )anti*odies)' I)ll as2 if anyone 2no0s ho0 anti*odies 0or2 5they stic2 to a foreign o*&ect li2e a *acteri # or $ir s 3 called an antigen% so yo r *ody can recogni7e it as foreign and other cells co#e along and destroy anything that anti*odies are stic2ing to6' Scientists ta2e ad$antage of the fact that 0e #a2e anti*odies that stic2 to al#ost e$ery 2ind of disease3 ca sing agent' If 0e too2 those anti*odies 5for e!a#1le% HI8 anti*odies6 and st c2 the# on the *otto# of a dish 5li2e o r ELISA 1late6 and 1 t *ody fl id infected 0ith HI8 into the dish% 0hat 0o ld ha11en9 St dents s ally 0ill say that the HI8 0ill stic2 to the anti*odies' I)ll also as2: if 0e 1 t *ody fl id infected 0ith the fl $ir s into the dish% 0hat 0o ld ha11en9 :othing 0o ld stic2 *eca se the HI8 anti*ody can only stic2 to HI8' "y the 0ay% )Sor*ent) #eans to stic2% li2e the anti*odies stic2 to the HI8' 6! Shake off the fluid into a nearby sink or designated container, making sure that the fluid has emptied from each well! %ap the plate upside down onto the paper towel to remo#e any e$cess li7uid or bubbles! 8! 9ash your group+s plate / times and shake off! (ith this ste1% 0e 0ash off e$erything that isn)t st c2 5fl $ir s% for e!a#1le6' 7. Using the dropper for the antibody solution add two drops of the antibody solution (Ab) to each well. :! Allow fi#e minutes incubation time on the lab table and then shake off the fluid! (e ha$e to *e a*le to tell 0here there is HI8 st c2 and 0here there isn)t' (hat do 0e already 2no0 stic2s to HI89 The HI8 anti*ody; The A* sol tion is #ade of anti*ody to HI8% 0hat 0ill ha11en 0hen 0e add it9 It 0ill stic2 0here there is HI8 and not stic2 0here there isn)t' This *rings s *ac2 to the na#e of the test3 (hat does )Lin2ed) #ean9 5St c2 together6 (hat does )En7y#e) #ean9 5Molec le that cataly7es a reaction% or does a s1ecific &o*6 The anti*ody 0e & st added is )lin2ed) to an en7y#e that changes the TM" sol tion 50hich 0e ha$en)t sed yet6 fro# colorless to *l e' So 0hat ha11ens if there is HI8 in the *ody fl id9 The en7y#e3lin2ed anti*ody sho ld stic2% and 0hen 0e add the TM" it sho ld t rn *l e' (hat a*o t if there is no HI8 in the *ody fl id9 The en7y#e3 lin2ed anti*ody has nothing to stic2 to and so there 0ill *e no en7y#e aro nd to t rn the TM" *l e' :o0 it)s ti#e to 0ash off the nst c2 anti*ody' ;! 9ash your group+s plate / times and shake off! 10. Using the dropper for the TMB solution add two drops of the color reagent solution (TMB) to each well. **! 9atch your plate for the ne$t 6 minutes! 9hat happens< =ecord your results in the table on page -!

BIOTECH Project, University of Arizona ELISA Assay

(atch to see 0hich sa#1les t rn *l e' This is easiest to $ie0 on a 0hite *ac2gro nd' Record on the data ta*le on 1age - in the )Res lts) col #n 0hich sa#1les t rn *l e and 0hich stay colorless' < estions I as2 in the #eanti#e: (hat do yo thin2 the 1ositi$e control is9 "ody fl id that definitely has HI8% so 0hat sho ld ha11en9 It sho ld t rn *l e' (hat do yo thin2 the negati$e control is9 "ody fl id that definitely doesn)t ha$e HI8% so 0hat sho ld ha11en9 It sho ld stay colorless' 4inally% 0hat a*o t the 0ord )Assay)3 it #eans test' So ELISA is a test to see if anti*odies stic2 to an antigen 5e'g' HI86 in yo r *ody fl id *y lin2ing the anti*ody to an en7y#e that ca ses a $is al change 5li2e TM" t rning *l e6'

La*oratory < estions Each group will record the results of the tests! >e sure to record whether you are infected! "ou will also record your test results and list of partners on the blackboard! *! ?i#en the classroom data, who are the original infected carriers< "ased on 0ho has HI8% 0ho doesn)t% and 0ho 1eo1le s0a11ed 0ith% the st dents #ay *e a*le to trace *ac2 to the original infected 1erson' -! 9hy were positi#e and negati#e controls needed< If the 1ositi$e control doesn)t t rn *l e% then #ay*e so#e 1eo1le that ha$e HI8 also didn)t t rn *l e 5this is a false negati$e% see *elo06' (e ha$e to incl de a 1ositi$e control so that 0e 2no0 that e$eryone that has HI8 definitely tests 1ositi$e' If the negati$e control t rns *l e% then #ay*e so#e 1eo1le 0ho don)t ha$e HI8 also t rned *l e 5this is a false 1ositi$e% see *elo06' (e ha$e to incl de a negati$e control so that 0e 2no0 that e$eryone that doesn)t ha$e HI8 definitely tests negati$e' /! 9hat was the purpose of washing the plates between addition of each reagent< =o ha$e to get rid of nst c2 anti*ody' If yo don)t 0ash off the nst c2 anti*ody% yo #ay test 1ositi$e e$en 0hen yo )re negati$e' 0! E$plain what is meant by a false positi#e test! 5ame one error that would result in a false positi#e test! See a*o$e' A false 1ositi$e co ld res lt fro# #any things% incl ding not thoro ghly 0ashing off the nst c2 anti*ody or ha$ing an anti*ody that stic2s to other things *esides HI8 5this is called non3 s1ecific6% for e!a#1le% the fl $ir s' Then% so#eone 0ho has the fl #ight test 1ositi$e for HI8' 6! E$plain what is meant by a false negati#e test! 5ame one error that would result in a false negati#e test! See a*o$e' A false negati$e co ld res lt fro# #any things% incl ding too # ch 0ashing or not )stic2y) eno gh anti*odies% so that the anti*ody doesn)t stic2 to the HI8 and a 1erson 0ith HI8 tests negati$e' 8! (ow can you protect yourself from transmitted diseases<