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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.

drink. Do not insert remove contact lenses 5. In accidents : Clean with water tap immediately if you have contaminated with poisoning reagens or blood specimens. Thoroughly wash hands and other skin surfaces immediately after any contamination. 4. Take special care to avoid injuries with sharp object such as needles and scalpels . Please ask to instructure or assistance if you have trouble with contaminated reagens or blood specimens. sera or reagents. Make sure clean the table by alcohol or lysol if you dropped the blood.PRACTICE REGULATIONS !!! 3. Do not eat. smoke or apply cosmetic (including lip balm) : during practice in laboratory room.

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

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

typhi) specific antigen (Lipopolysaccharide) from typhoid fever patients. .PURPOSES : • Evaluate the simple Lateral flow assay for detection of Immunoglobulin M antibodies (IgM) against Salmonella typhi (S.

typhi from Indonesia . The antigen was prepared from a culture of a recent isolate of S. that is adhered to a rigid backing.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.

.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.

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

and one plastic reusable Pasteur pipet .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.

• • • .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. When using the Pasteur pipet just transfer enough running fluid to completely fill the round sample port. Read results at 10 to 15 minutes. The running fluid may be added using a micropipet or using the plastic Pasteur pipet provided. Results are stable for a further 10 to 15 minutes. Immediately add 130µl running fluid to the round sample port. This shows that the test is working. Spot 5µl of serum to the sample pad in the round sample port using a micropipet and a disposable pipet tip. thereafter false results may occur. You will see a colour moving across test and control zones. Keep the Pasteur pipet for later use.

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) .

leprae). • Introduction : See guidebook/report .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.

. trisaccharide-phenyl propionate-bovine serum albumin (NT-P-BSA) which is very stable hydrophilic substance with much stronger sero. PGL-I (specific epitop for M. The antigen used in this test is semisynthetic.reactivity than that of natural Phenolic Glycolipid –I.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. leprae).PGL-I antibodies in the blood specimen to aggregate in filmy form. 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.

Japan Micropipettes with disposable pipetts tips (volume 10. 100 and 1000 µl) Dropper ( volume 25 µl ) Reading Mirror (viewer for observation of agglutination pattern) .Materials and equipments : • • • • Microplate well containing 96 wells with U-shaped and rigid product by Fujirebio Inc.

Kit MLPA components containing : • Reconstituting solution with liquid form. Serum containing antibodies with the titer of 1:128 when reconstituted • • • • . and the positive control. sensitized and unsensitized. To be used for specimen dilution Sensitized particles with lyophilized form. This liquid used for rehydration of gelatin particles. To be used as gelatin particles coated with BSA which are rehydrated to us Positive control with lyophilized form. Serum diluent with liquid 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.

cover the plate. Serum Diluent Serum specimen (ul) (ul) 1 75 25 1:4 2 25 25 1: 8 25 3 25 25 1:16 discard Serum dilution (ratio) Unsensitized particle (ul) Sensitized particle (ul) 25 Final dilution 1:16 1:32 Mix well.Working Procedure : Well No. and incubate for 3 hour Read and interpret .

Then cover plates and allow to stand at room temperature for 2 two hours. Discard the excess 25 µl from well 3. Upon completion of the reaction. Transfer 25 µl from well 1 to well 2 and repeat the transfer from well 2 to well 3 in the same way.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. read the setting patterns. Prepare serial dilution (2n) of each serum specimen using a microdiluter or a micropipette. . 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.

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 .

no. et al. Mochammad Hatta. South-east Asia J. 7. et al. 6. no. .4. vol 66. (2003) Mochammad Hatta. International J. vol 66. et al. Tropical Medicine and Hygiene. December (1995). 4. Denpasar. 3. vol 26. 4. 8-10 December (1997) Mochammad Hatta. Third Asia-Pacific Symposium Typhoid fever and other Salmonellosis. page 82. Tropical Medicine and Hygiene. Bali. Bali. Denpasar. Mochammad Hatta. et al. Third Asia-Pacific Symposium Typhoid fever and other Salmonellosis. page 631-635. Southeast Asian Journal of Tropical Medicine and Public Health. no. 5. vol 531 : page 269-278. 4. page 82. December (1998). page 416-421 (2002). 4. no. et al. page 182-191 (2002) Mochammad Hatta. et al. vol 33. Advance Experiment in Medical Biology.References : 1. 8-10 December (1997) Mochammad Hatta. page 95A. American J. 2. Leprosy. Mochammad Hatta.