PRACTICE IMMUNOLOGY

SYSTEM

PRACTICE REGULATIONS !!!

The following special precautions should be taken in the
preparation and handling of the reagents to be use in-vitro
Antigen antibody reaction test :
1. Prepare by student :
- Laboratory coat (use the coat during working at
laboratorium).
Lab coats should be worn at all times. Lab coats should be
dedicated to immunology laboratory area and washes
fequrently
- Color pencils
2. Pipettes : one group of student will be use one pipette together.
Please check the pipette on your table before start the
practice. If not complete, please ask to assistance or

3. Do not eat, drink, smoke or apply cosmetic (including lip

balm) : during practice in laboratory room. Thoroughly wash
hands and other skin surfaces immediately after any
contamination.
4. Do not insert remove contact lenses
5. In accidents : Clean with water tap immediately if you have
contaminated with poisoning reagens or blood specimens.
Please ask to instructure or assistance if you have trouble with
contaminated reagens or blood specimens. Make sure clean
the table by alcohol or lysol if you dropped the blood, sera or
reagents. Take special care to avoid injuries with sharp object
such as needles and scalpels

6. All used tips, tubes must put on in liquid disposal containing lysol
solution or alcohol.
7. After finish practice (before go out from laboratory room) :
Make clean the pipettes and all equipments by cottton alcohol or lysol.
Turn off all water tap, gas and electricity.
Make clean the tubes and put on the used tips in liquid disposal.,
Check again all equipments and give back to instructure or assistance.
Write all result of experiments on practice book and show to instucture
and signed by them
8. Washing your hand after finish the experiments by alcohol solution or
soap. Prohibit to bring out from laboratory room the results plates
and sera or blood.

PRACTICE I
IN-VITRO REACTION OF
ANTIGEN ANTIBODY
(PRECIPITATION TEST)

PURPOSE

Evaluate the simple Lateral flow assay

for detection of Immunoglobulin M
antibodies (IgM) against Salmonella
typhi (S. typhi) specific antigen
(Lipopolysaccharide) from typhoid fever
patients.

The dipstick consists of a strip of nitrocellulose

membrane containing a 2 mm wide line of
immobilised antigen as detection band and a
separate line of immobilised antihuman IgM
antibody as reagent control, that is adhered to
a rigid backing. The antigen was prepared from
a culture of a recent isolate of S. typhi from
Indonesia

PRINCIPLE

Reaction of precipitation between

IgM antibodies with specific antigen
of Salmonella typhi
(Lipopolysaccharide ) and should be
view by colour band

INTRODUCTION
Laboratory testing is essential because signs and symptoms
may resemble those of other major infectious diseases. The
Typhoid F IgM flow assays provide an indirect measure for
infection through the detection of pathogen specific
antibodies. Specific IgM antibodies usually develop early in
the disease.
The Typhoid F IgM flow assay is relatively simple and rapid
assay that may be used as a point-of-care assay in the field
or at the bed-side. The assay neither requires special
training, equipment, electricity nor refrigeration. Results are
obtained in 10 to 15 minutes. The assay devices and the
running fluid may be stored at +2 C to +25 C.

Reagents use in Salmonella

Lateral flow test
- Each kit contains 25 individually
wrapped assay devices together with 1
bottle of running fluid, sufficient for the
analysis of 25 serum samples, and one
plastic reusable Pasteur pipet

PROTOCOL
Remove assay device from the packaging and place on a bench top with the
test window facing upwards.
Spot 5l of serum to the sample pad in the round sample port using a
micropipet and a disposable pipet tip.
Immediately add 130l running fluid to the round sample port. The running
fluid may be added using a micropipet or using the plastic Pasteur pipet
provided. When using the Pasteur pipet just transfer enough running fluid to
completely fill the round sample port. Keep the Pasteur pipet for later use.
You will see a colour moving across test and control zones. This shows that
the test is working.
Read results at 10 to 15 minutes.
Results are stable for a further 10 to 15 minutes; thereafter false results
may occur.

Example the Salmonella Lateral

flow test results (+4 to Negative)

4+

3+

2+

1+

Neg

Neg

PRACTICE II
ANTIGEN-ANTIBODY
REACTION IN-VITRO
(AGGLUTINATION TEST)

PRINCIPLE
The Mycobacterium leprae particle agglutination test
(MLPA ) test is intended for the qualitative and quantitative
determination of antibody to Phenolic Glycolipid I (PGL-I)
based on the agglutination reaction. The antigen used in
this test is semisynthetic, trisaccharide-phenyl propionatebovine serum albumin (NT-P-BSA) which is very stable
hydrophilic substance with much stronger sero- reactivity
than that of natural Phenolic Glycolipid I, PGL-I (specific
epitop for M. leprae). The principle of the test is indirect
agglutination where NT-P-BSA antigens coated on the
surface of spherical gelatin particles react specifically with
anti- PGL-I antibodies in the blood specimen to aggregate
in filmy form.

Materials and equipments

- Kit MLPA
- Microplate well containing 96 wells with Ushaped and rigid product by Fujirebio Inc.
Japan
- Micropipettes with disposable pipetts tips
(volume 10, 100 and 1000 l)
- Dropper ( volume 25 l )
- Reading Mirror (viewer for observation of
agglutination pattern)

Kit MLPA components containing :

- Reconstituting solution with liquid form. This liquid used
for rehydration of gelatin particles, sensitized and
unsensitized, and the positive control.
- Serum diluent with liquid form. To be used for specimen
dilution
- Sensitized particles with lyophilized form. To be used as
gelatin particles sensitized with NT_BSA which are
rehydrated to form a 1% suspension prior to us
- Unsensitized particles with lyophilized form. To be used as
gelatin particles coated with BSA which are rehydrated to
us
- Positive control with lyophilized form. Serum containing
antibodies with the titer of 1:128 when reconstituted

Working procedure
Well No. 1

Serum Diluent (ul)

Serum specimen (ul)
Serum dilution

75

(ratio)

25 25
25
25 25
1:4

Unsensitized particle (ul)

Sensitized particle (ul)
Final dilution

1: 8 1:16
25
25

1:16 1:32

Mix well, cover the plate, and incubate for 3 hour

Read and interpret

discard

Step of test procedure :

Deposit 3 drops (75 l) of serum diluent in well 1 and 1 drop (25 l) each

in wells 2 and 3 using a calibrated pipette dropper

Place 25 l of each serum specimen in well No 1 introducing it on the
surface of deposited serum diluent and mix well.
Prepare serial dilution (2n) of each serum specimen using a microdiluter
or a micropipette.
Transfer 25 l from well 1 to well 2 and repeat the transfer from well 2 to
well 3 in the same way.
Discard the excess 25 l from well 3.
Add one drop (25 l) of unsensitized particles to well 2 and one drop
(25l) of sensitized particle to well 3 using the droppers supplied in the
kit.
Using an Automatic Try Mixer or Automatic Vibrator mix the fluid of the
wells thoroughly.
Then cover plates and allow to stand at room temperature for 2 two
hours.
Upon completion of the reaction, read the setting patterns.

References
1.Mochammad Hatta, et al. American J. Tropical Medicine and Hygiene. vol

66, no. 4, page 416-421 (2002).

2.Mochammad Hatta. Advance Experiment in Medical Biology. vol 531 :
page 269-278, (2003)
3.Mochammad Hatta, et al. Southeast Asian Journal of Tropical Medicine
and Public Health. vol 33, no. 4, page 182-191 (2002)
4.Mochammad Hatta, et al. Third AsiaPacific Symposium Typhoid fever and
other Salmonellosis, Denpasar, Bali, page 82. 810 December (1997)
5.Mochammad Hatta, et al. International J. Leprosy, vol 66, no. 4, page 95A,
December (1998).
6.Mochammad Hatta. et al, Third AsiaPacific Symposium Typhoid fever and
other Salmonellosis, Denpasar, Bali, page 82. 810 December (1997)
7.Mochammad Hatta, et al. Southeast Asia J. Tropical Medicine and
Hygiene, vol 26, no.4, page 631635, December (1995).