The document describes an experimental workshop protocol to investigate vaccine candidates for malaria. Participants will use ELISA techniques to analyze blood samples from individuals living in malaria-endemic areas and determine if they have developed an immune response to different candidate antigens. This will help identify the most effective vaccine candidates, as antigens that elicit antibodies in immune individuals are most likely to protect against future infection. The protocol involves binding antigens to wells, adding serum and labeled secondary antibodies to form complexes, then detecting the reaction with a colored substrate. Comparing results will reveal the antigen activating the strongest immune response, making it the best potential vaccine candidate.

1. Investigate the
vaccine for malaria
Experimental workshop
PROTOCOL

2. Introduction
Malaria is considered to be the most impor- of different measures that include the use of
tant parasite-caused disease in the world, and mosquito nets impregnated with insecticide,
is responsible for the death of approximately spray insecticides, preventive treatments, the
800,000 people every year, particularly children implementation of educational programmes
under 5 and pregnant women. According to the and environmental intervention, among others.
World Health Organization (WHO), some three
billion persons are at risk of contracting the The scientific community is working hard to de-
infection, while in the year 2010 there were velop a vaccine which, in combination with the
225 million cases of malaria in the world, of current measures, could contribute significantly
which 90% were in Africa. to a better control of malaria. There is already a
vaccine in the clinical study phase, which would
Currently, malaria is endemic in more than be effective in 50% of cases.
100 countries located in sub-Saharan Africa and
regions of South Asia, Latin America and Oceania.
The most recent reports indicate that half the
world's population lives in areas where there is
a risk of contracting the disease, and where in
addition to its consequences for the health of the
population, malaria contributes to further weak-
ening the area’s economic situation.
In this workshop you will do research
To eradicate this disease in areas with a high with different vaccine candidates to
risk of transmission, various interventions are decide which is the most effective.
being conducted that require a combination
Countries or areas where the transmission of malaria occurs. Countries or areas with limited risk of transmission of malaria
Source: World Health Organization (WHO): 2009 data
2

3. How is malaria transmitted? Why is a vaccine against malaria
necessary?
Malaria is an infectious disease which is caught
from the bite of the Anopheles mosquito, which
transmits Plasmodium parasites, thereby act- Historically, vaccines have been one of the most
ing as a vector. In the human body, the parasites efficient measures for the prevention of dis-
multiply in the liver and then infect the red eases and saving lives, especially in the case of
blood cells. Prominent symptoms of malaria are infectious diseases. Obtaining a vaccine that is
fever, headache and vomiting, and they appear partially effective could save hundreds of thou-
from 10 to 15 days after the mosquito bite. sands of lives.
Obtaining a vaccine would be a great step for-
ward that could be added to the current arsenal
of measures used for the prevention of malaria,
such as insecticide-impregnated mosquito nets
and the prompt and appropriate treatment of
persons diagnosed with malaria.
What is being done to Since its short-term effectiveness would be
control malaria? partial, it would be a substitute for these
Basic interventions to control malaria are di- measures, but rather complement them. The
vided into several groups: two together would represent a comprehensive
response to the prevention of malaria.
1 Strategies directed against
the mosquito, or vector,
such as spraying enclosed
spaces with insecticides.
2 Strategies to avoid contact
between the vector and
host, such as the use of
mosquito nets impregnated
with insecticide.
3 Strategies directed
against the parasite.
One of these strategies
consists of treatment
with combinations of
medicines based on a
molecule called artemisinin, which is rapid
and effective. A vaccine would also be a control
strategy directed against the parasite which,
in combination with other strategies, could
contribute significantly to the eradication of
malaria.
3

4. What are we doing at ISGLOBAL, the Barcelona Institute for Global Health?
The Barcelona Institute for Global Health 1. The study of the molecular basis of the
(ISGlobal) is a not-for-profit organisation whose disease as well as the variety of immune
objective is to improve the health and devel- responses.
opment of the most vulnerable populations
through the creation, management, transmis- 2. The development of new medicines and the
sion and application of knowledge. Its vision is assessment of their safety and efficacy.
of a world in which we can all enjoy good health,
and it receives support from the “La Caixa” 3. The assessment of the epidemiological
Foundation, among others. characteristics of malaria in different set-
tings and of the social and cultural factors
One of the essential pillars of ISGlobal is re- that surround it.
search that concentrates on health problems
that affect the most vulnerable populations, 4. The analysis of the effectiveness of various
which is conducted at its Barcelona Centre for prevention tools and the cost-effectiveness
International Health Research (CRESIB). The ratio of these interventions.
research on malaria which is done at CRESIB
focuses on: As part of this role (4) CRESIB carries out clini-
cal studies of the safety and efficacy of vaccines.
It is currently participating in the development
of the RTS,S vaccine against malaria, which is
showing itself to be effective in more than 50%
of infected children. At the same time, CRESIB
investigators are conducting research to identify
new vaccine candidates.
4

5. Workshop objectives 1
In this workshop we invite you to analyse vari-
ous candidates for the vaccine against malaria
along with those that CRESIB is researching
to identify which is the best candidate. The
candidates for the CRESIB vaccine have been
obtained from parasite proteins that have been
purified beforehand.
To analyse them, we have a number of blood
samples from people living in malaria-affected
areas who have had the disease on various oc-
casions and who are now immunised.
To confirm that our vaccine candidates are ef-
fective, we must show that immunised people
have developed a response to these candidates.
If activation of the immune response against
the candidate proteins is found in these people,
it will mean that they could be good vaccine 2
candidates because they could also trigger the
response necessary to protect people against
future infection.
3
5

6. The objective of this workshop is to familiarise that they are capable of activating a good im-
you with one of the techniques most often used mune response, and that they could therefore
in biomedical laboratories, the ELISA (enzyme- be good candidates.
linked immunosorbent assay) technique.
Using this technique, we will specifically
This is an analysis that detects whether anti- discover which of the antigens available in our
bodies are present in blood samples. The pres- laboratory is a good candidate for a vaccine
ence of candidate-specific antibodies indicates against malaria.
Basic principles of the ELISA technique
The basic principle behind this technique is To carry out this identification, the antibodies
based on the interaction of the vaccine candi- used have a molecule attached to them called
date, or antigen (1), with the antibody (2). A spe- an enzyme (3), which has the ability to react
cific antibody will bind to a specific antigen to with a substance called a substrate (4), which
create an exclusive antibody-antigen complex. we will add to produce a colour.
The ELISA technique allows us to identify Therefore, if the sample contains the antibody
whether antibodies were present, and whether, we wish to detect, it will bind to what we have
as a result, antibody-antigen complexes were added, which is bound to an enzyme that will
formed when they came into contact with blood in turn cause the substrate to change colour,
samples containing the vaccine candidates. thereby telling us that the results are positive.
Substrate
Enzyme
Antibody
Antigen
1. Antigen: any foreign substance that binds specifically to the specified antibodies or lymphocytes and activates an immune
response. In general antigens have a high molecular weight; normally they are proteins or polysaccharides.
2. Antibody: proteins (immunoglobulins, Ig) from serum that are formed as a response to the invasion of the body by foreign
molecules, whether due to natural exposure or an antigen introduced by vaccine immunization. They are in the form of
a Y, and are made up of four polypeptide chains that are kept connected by interchain disulfide bonds. Antibodies have a
constant region and a variable region.
3. Enzyme: a protein that facilitates specific reactions of the metabolism.
4. Substrate: a solution that contains a compound acted upon by an enzyme.
6

7. Organisation of the workshop
To analyse which vaccine candidate or antigen against malaria have antibodies against them.
is the most effective, we will test whether the To do so we will divide the experiment into three
blood samples from persons who are immunized main stages.
1. BINDING OF THE CANDIDATE PROTEINS (OR ANTIGENS) TO THE SURFACE OF THE WELLS
The first step will be to fix the candidate proteins that are being studied to a solid support
2. FORMATION OF ANTIBODY-ANTIGEN COMPLEXES
We then add the blood sample, specifically blood serum (blood samples with the cells and clotting
factors removed), and an antibody marked with an enzyme that we will call the secondary antibody.
In the blood samples in which the antibody being studied was present, antibody-antigen complexes
will be formed, which will in turn bind to the antibody marked with the enzyme.
Serum Secondary
from the antibody marked
patients with an enzyme
3. READING THE REACTION
Finally, we will add the enzyme substrate which, if the antibody antigen complex is present, will
change colour. In this way we will learn if the blood samples contain the antibody being studied, and
in what quantity.
Substrate
from the
enzyme
Results and conclusions
Once the results are obtained, we can decide intense immune response and may therefore be
which vaccine candidate activates the most the best candidate.
7

8. Equipment and material
required
Laboratory instruments and utensils
Magnetic stirrer (1) (for Stir bar and Micropipettes
preparing PBS-Tween) “stir bar retriever” of 20 to 200 µl (2)
Test tube, Glass bottle,
Timer Funnel
100 ml 250 ml
Consumables
Strips with 12 wells Graduated plastic Pasteur Tips for the micropipettes
for ELISA, and supports pipettes
Absorbent paper Permanent marker Gloves, goggles and apron
1. If you do not have a magnetic stirrer, the PBS liquid may be purchased
2. If you do not have micropipettes, you may use small-volume Pasteur pipettes
8

9. Reagents and samples
PBS buffer solution (3) Tween-20 detergent (4) Distilled water
Secondary antibody
A B C+ C-
Vaccine candidates Positive controls Secondary antibody
(antigens) and negative controls (5) with enzyme
(peroxidase)
Substrate
1 2 3 4
Substrate, or colouring Serum samples from four people residing in areas in which
solution malaria is endemic, and who are immune to the disease
3. Helps to maintain the solution pH thanks to sodium and potassium phosphates
4. Helps prevent binding of nonspecific antibodies
5. C+: contains a mixture of serum from people residing in areas where malaria is endemic and who
are immune to the disease. C-: mixture of serum from people who have never been exposed to malaria
9

10. Procedures
To identify the presence or absence of specific blood serum) of different patients residing in
antibodies to the vaccine candidate antigens areas where malaria is endemic to see if we
we have in the laboratory, we will use these find specific antibodies against our antigens in
antigens to challenge the blood (actually the their serum.
1 Binding of the antigens to the surface of the wells
The micro-well strips are covered with the vac- The binding of these antigens to the surface of
cine candidates (antigens) that we wish to test the wells is easily brought about since they are
to see if they would be good candidates for a made of a treated plastic that has a great ability
vaccine against malaria. to bind proteins.
PROTOCOL FOR BINDING ANTIGENS TO THE SURFACE OF THE WELLS
1
Note below what you will place in each well (controls, blood samples and the names of the
antigens that you will analyse).
2
Permanently mark the wells
where you place each sample.
10

11. 3
Prepare a washing solution (PBS-Tween 0.05%).
A B
Measure 200 ml of distilled water using the
test tube, adjusting the volume with the Place the stir bar in the bottle, and dilute
Pasteur pipette. Use the funnel to add 200 ml one PBS tablet with water using the
of distilled water to the bottle. magnetic stirrer.
C D
When the tablet has dissolved, extract the
stir bar and add 100 µl of Tween-20 using a Remove it carefully inverting the bottle
plastic Pasteur pipette. several times.
Note: The washing solution contains PBS (phosphate buffer saline), which allows the antibodies to be kept in a stable envi-
ronment that helps to preserve their structure. Tween-20 is a detergent that helps to eliminate the proteins that have been
able to bind in a nonspecific manner, and also adheres to the portions of the well that are not covered by the antigen, thereby
reducing background noise.
11

12. 4 5
Add the two
test antigens to
the respective
wells using the
micropipette
(50 µl per well). It
is important that
you use a clean
tip to dispense the
antigens to avoid
contamination.
Allow it to incubate for 5 minutes at
room temperature.
6 7
Wash it to eliminate the excess antigen
Eliminate the remainder of the antigen not bound to the strip. To do this, fill the
by inverting the strip over the absorbent wells with the washing solution using a
paper. plastic Pasteur pipette.
8 9
Discard the washing solution by inverting
the strip over absorbent paper. Repeat steps 7 and 8.
12

13. 2 Formation of antibody-antigen complexes
In this step we will first condition the serum of called peroxidase. As each primary antibody can
the patients to determine whether they contain bind with more than one secondary antibody,
antibodies against the vaccine candidate. The the amount of colour obtained in step three will
antibodies that we wish to test will be called be enhanced. The sensitivity of the technique is
primary antibodies. We will then add a second- thereby increased.
ary antibody which is marked with an enzyme
Serum Secondary
from the antibody marked
patients with enzyme
PROTOCOL FOR FORMATION OF ANTIBODY-ANTIGEN COMPLEXES
1 2
Add the positive and negative controls Add the different serum samples
to the respective wells using the from 4 residents of the areas
micropipette (50 µl per well). where malaria is endemic to the
The positive control (C+) contains a corresponding wells using the
mixture of serum from people residing micropipette (50 µl per well).
in areas where malaria is endemic and
who are immune to the disease. The
negative control (C-) contains a mixture
of serum from people who have never
been exposed to malaria.
13

14. 3 4
Allow it to incubate for 5 minutes at Eliminate the excess antigen by inverting
room temperature. the strip over absorbent paper.
5 6
Wash all the wells to eliminate the
antibodies that have not reacted with
the antigens and which are therefore not
specific. Fill the wells with the washing Discard the washing solution by inverting
solution using a plastic Pasteur pipette. the strip over absorbent paper.
7 8
Using the micropipette, add the
secondary antibody which is bound to an
Repeat the steps five or six more times. enzyme to all the wells (50 µl per well).
14

15. 9 10
Eliminate the excess secondary
Allow it to incubate for 5 minutes at antibodies by inverting the strip over
room temperature. absorbent paper.
11 12
Wash the wells by filling them with the Discard the washing solution by inverting
washing solution using a plastic Pasteur the strip over absorbent paper.
pipette.
13
Repeat the two previous steps three more times.
15

16. 3 Reading the reaction
After washing to eliminate all the marked
molecules that have not been fixed in the form
of antibody-antigen complexes, the enzyme
substrate solution is added to facilitate the
change of colour.
Substrate
from the
enzyme
PROTOCOL FOR READING THE REACTION
1 2
Add the enzyme substrate to all the Allow it to incubate for 5 minutes. During
wells using the micropipette this time the substrate will bind to the
(50 µl per well). enzyme at room temperature and the
colour will begin to appear.
16

17. 3
Assemble the results in the form of bar graphs.
ANTIGEN 1
Maximum
Intensity
Minimum Samples
C+ C- M1 M2 M3 M4
ANTIGEN 2
Maximum
Intensity
Minimum Samples
C+ C- M1 M2 M3 M4
17

18. Results and conclusions
Interpret and record the results
1. Which of the antigens that you have tested do you believe is the best vaccine candidate?
Do you believe that the antigens that you have tested are good vaccine candidates? Why?
2. When is a reaction positive and when is it negative? Why?
3. Why do you think the controls are used?
18

19. 4. Which part of the primary antibody is recognised by the secondary antibody? The constant
region or the variable region? Give reasons for your answer.
5. What would happen if we did not do the washing before adding the colouring substrate?
6. Could we use blood from your classmates to determine whether our laboratory antigens
are good candidates for a vaccine against malaria? Give reasons for your answer.
7. Do you believe that this experiment has shown that the selected candidate stimulates the
immune response? Would you have to do some other type of experiment to assess whether it
is also capable of activating some other type of response?
19

20. Annex I
OBLIGATORY USE OBLIGATORY USE OBLIGATORY USE
OF GOGGLES OF APRON OF GLOVES
Safety precautions
BE INFORMED or corrosive products. Do not place reagent
Find out where the safety equipment of the containers near a flame. Do not heat inflam-
laboratory or the place where you are experi- mable liquids. Carry bottles holding them from
menting is located (fire extinguishers, show- beneath, never by the neck.
ers or baths, exits, etc.). Read the instructions
carefully before doing an experiment. Do not WASTE DISPOSAL
forget to read the safety labelling for reagents Deposit broken glass, reagents that are toxic,
and equipment. noxious or harmful to the environment and
biological waste in special and appropriately la-
USE PROPER CLOTHING belled receptacles. Never dispose of solid waste
Gloves, apron and goggles. using the sink.
GENERAL STANDARDS In case of accident, advise the instructor im-
Smoking, eating or drinking in the laboratory or mediately. Remember: If you have a question,
area where you are experimenting is prohibited. ask the trainer.
Wash your hands before leaving the labora-
tory. Work in a neat and orderly fashion without SPECIFIC PRECAUTIONS
hurry. If any product should spill, clean it up FOR THIS WORKSHOP
immediately. Always leave materials clean and During this practice session you must follow
orderly. Never use equipment or apparatuses the usual precautions for handling of chemi-
without perfectly understanding how they work. cal products. Below are listed only those that
present the following degrees of hazard:
HANDLING OF GLASS
Protect your hands when handling materials • PBS:
made of glass. Do not use cracked glass items. toxic when ingested, inhaled or in contact with
the skin.
CHEMICAL PRODUCTS
Do not use unlabelled containers of reagents. • Tween 20:
Do not sniff, inhale, taste or touch chemical toxic when ingested, inhaled or in contact with
products. Never pipette by mouth. Wear gloves the skin. Irritant.
and wash your hands frequently if you use toxic
Annex II
Reagent references
NAME: REFERENCES COMMERCIAL MANUFACTURER
PBS P4417-50TAB Sigma
Tween-20 P1379-100ML Sigma
CHK IGY, bagged (= ANTIGEN) 1662406EDU BioRad
RB ANTI-CHK, bagged (= PLASMA) 1662407EDU BioRad
GAR-HRP, bagged (= SECONDARY ANTIBODY) 1662408EDU BioRad
COLOURING SUBSTRATE 1662402EDU BioRad
20

21. Learn more at Xplore Health!
Researchers who have contributed content: Laura Puyol, investigator for CRESIB, ISGlobal.
DEVELOPED BY
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3.0 Unported Creative Commons license. to see a copy of the license, visit
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