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Finding Out If You’re Infected –

The ELISA antibody test

We know what happens when people get infected with different bacteria and viruses. But how do we know which kind of infection they have? There’s a testing procedure called ELISA – Enzyme-Linked Immunosorbent Assay, and we’ll model it today in class. ELISA can be used in a public health crisis like the one occurring in China right now. The issue is Influenza A, strain H7N9. From Wikipedia: “In March 2013, the Chinese government reported three cases of H7N9 in three human patients, two of whom died, with the third patient in critical condition. This strain did not appear to be highly contagious in human to human transmission because the many people these patients had been in contact with did not get this influenza. As of April 19, 2013, there were 91 confirmed H7N9 cases with 17 fatalities. Both a young boy and girl in neighboring homes became sick, so there is some possibility of human-to-human transmission. On April 5, 2013, the World Health Organization reported both that the genes from influenza A H7N9 viruses causing the first human cases are of bird origin and that these genes also show signs of adaption to growth in mammals, which include an ability to bind to mammalian cell receptors and to grow at temperatures close to the normal body temperature of mammals (which is lower than the normal body temperature of birds). Early suspicions that pigs might be the carrier have not been borne out. Pigs were tested soon after the outbreak with no positive results. At a news conference in Beijing on April 24, 2013, Keiji Fukuda of the World Health Organization stated, "This is an unusually dangerous virus for humans." Researchers have seen a "dramatic slowdown" in human cases in Shanghai after the city's live poultry market was shut down on April 6, also describing the finding as "very encouraging.” Today we have 12 patients to test for Influenza A, strain H7N9. Patient 1 – You are a sick chicken from the Shanghai poultry market. Patient 2 - You are a sick chicken from the Shanghai poultry market. Patient 3 – You are an employee at the poultry market who handles chickens. Patient 4 – You are an employee at the poultry market who handles chickens. Patient 5 – You are the partner of Patient 3. Patient 6 – You are the partner of Patient 4. Patient 7 - You are a European tourist who visited the poultry market and feels sick. Patient 8 - You are an American tourist who visited the poultry market and feels sick. Patient 9 – You are a pig in Shanghai. Patient 10 – You are a pig in Shanghai. Patient 11 – You are a chicken in Beijing. Patient 12 – You are a chicken in Hong Kong.

But how do we test? 2

Assuming that sick people have functioning immune systems, we know they are producing ______against the pathogen that’s causing their illness.

Based on what you saw in the “Holly” video, what do antibodies do the pathogen?

Using that understanding, scientists were able to devise the ELISA test.

1. Protein markers from a virus or bacterium are placed in a plastic well.

2. Plasma from a patient’s blood is added to the well. a. If the person has this pathogen, then their plasma contains antibodies against the pathogen. These antibodies will then ______. b. If the person does NOT have this pathogen, then their plasma does NOT contain antibodies against the pathogen. So what will happen in the well?

3. But we can’t see the reaction in the wells because the molecules are too small. So we must use a special step. In this next step an antibody to the antibody is added. This anti-antibody has been created by injecting existing antibodies into mice or rabbits and extracting their blood plasma. This anti-antibody is added to the well. It will latch on to any antibodies already in the well.

4. Finally, a color indicator that will react to the shape of this “antibody sandwich” is added to the well. If the “antibody sandwich” is present, a colorful reaction will occur.

Patient 1 2 3 4 5 6 7 8 9 10 11 12 3

A B C D E F G H + + + - - -

RULES

 Always add 3 drops!

 Never touch the pipet to liquid already in the wells!

1. Lab Tech 1 adds the pathogen to rows A, B and C to all 12 columns.

2. Lab Tech 2 adds plasma with KNOWN H7N9 antibodies to wells H 1, 2, and 3. This is a positive control.

3. Lab Tech 3 adds plasma with NO H7N9 antibodies to wells H 10, 11 and 12. This is a negative control.

4. 12 patients add their plasma samples to rows A, B and C of their column.

5. Lab Techs 4 and 5 add SECONDARY antibody to every well already in use.

6. Lab Techs 6 and 7 add color indicator to every well already in use.

7. Wait five to ten minutes.

Who is infected?

And what should we do?