PRACTICE IMMUNOLOGY SYSTEM

Prof. Dr. MOCHAMMAD HATTA M.D, Clin.Micro (Cons), Ph.D

DEPARTMENT MEDICAL MICROBIOLOGY, FACULTY MEDICINE,

HASANUDDIN UNIVERSITY, MAKASSAR, INDONESIA

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 instructure. Small volumes should be pipetted with individually wrapped, sterile, disposables pipettes.

PRACTICE REGULATIONS !!!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

PRACTICE REGULATIONS !!!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 IIN-VITRO REACTION OF

ANTIGEN ANTIBODY(PRECIPITATION TEST)

PURPOSES :

• 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.

INTRODUCTION

• 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 of Salmonella Lateral flow test :

•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

Short protocol of Salmonella Lateral flow test

• Remove assay device from the packaging and place on a bench top with the test window facing upwards.

• Spot 5µl of serum to the sample pad in the round sample port using a micropipet and a disposable pipet tip.

• Immediately add 130µl 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)

MYCOBACTERIUM LEPRAE PARTICLE AGGLUTINATION TEST

(MLPA TES QUALITATIVE)

• Purposes :For detect the immunoglobulin M (IgM) antibodies from sera of leprosy patients or healthy persons who infected by Mycobacterium leprae (M. leprae).

• Introduction : See guidebook/report

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 propionate-bovine 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 :

• Microplate well containing 96 wells with U-shaped and rigid product by Fujirebio Inc. Japan

• Micropipettes with disposable pipetts tips (volume 10, 100 and 1000 µl)

• Dropper ( volume 25 µl )• 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 2 3

Serum Diluent (ul) 75 25 25

Serum specimen (ul) 25 25 25 discard

Serum dilution (ratio) 1:4 1: 8 1:16

Unsensitized particle (ul) 25

Sensitized particle (ul) 25

Final dilution 1:16 1:32

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

Read and interpret

Step of test procedure :• Deposit 3 drops (75 µl) of serum diluent in well 1 and 1 drop (25

µl) each in wells 2 and 3 using a calibrated pipette dropper• Place 25 µl 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 µl from well 1 to well 2 and repeat the transfer from

well 2 to well 3 in the same way. • Discard the excess 25 µl from well 3.• Add one drop (25 µl) of unsensitized particles to well 2 and one

drop (25µl) 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.

Interpretation criteria for agglutination test MLPA

• Negative : If setting patterns of particles is definite compact button with smooth round outer margin or compact ring with smooth round outer margin

• Mild Positive :If setting patterns of particles is significantly large ring with rough outer margin with agglutination in the periphery

• Strong Positive :If filmy mat of homogeneous agglutination covering entering bottom of wells

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 Asia‑Pacific Symposium Typhoid fever and other Salmonellosis, Denpasar, Bali, page 82. 8‑10 December (1997)

5. Mochammad Hatta, et al. International J. Leprosy, vol 66, no. 4, page 95A, December (1998).

6. Mochammad Hatta. et al, Third Asia‑Pacific Symposium Typhoid fever and other Salmonello sis, Denpasar, Bali, page 82. 8‑10 December (1997)

7. Mochammad Hatta, et al. South‑east Asia J. Tropical Medicine and Hygiene, vol 26, no.4, page 631‑635, December (1995).