You are on page 1of 20

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

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

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

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

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

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

PRINCIPLE 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 assays provide an indirect measure for infection through the detection of pathogen specific antibodies. equipment. Specific IgM antibodies usually develop early in the disease. The assay devices and the running fluid may be stored at +2° C to +25° C.  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.INTRODUCTION  Laboratory testing is essential because signs and symptoms may resemble those of other major infectious diseases. The assay neither requires special training. . electricity nor refrigeration. Results are obtained in 10 to 15 minutes.

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

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

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) .

.

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. PGL-I (specific epitop for M. 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. . 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.

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

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

Working procedure  Well No. 1 2 3  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. and incubate for 3 hour  Read and interpret discard . cover the plate.

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.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. 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. . Discard the excess 25 µl from well 3. Upon completion of the reaction. read the setting patterns.

Tropical Medicine and Hygiene. no. . page 416-421 (2002). December (1995). page 82. Denpasar. et al. vol 66.Mochammad Hatta. no. et al. page 95A. page 182-191 (2002) 4.Mochammad Hatta. Bali. vol 26.Mochammad Hatta. 6. South‑east Asia J. International J. vol 531 : page 269-278. Third Asia‑Pacific Symposium Typhoid fever and other Salmonellosis. Denpasar. 4.4. et al. 8‑10 December (1997) 5. Advance Experiment in Medical Biology. et al. American J. no. page 631‑635. Tropical Medicine and Hygiene. vol 33. vol 66.Mochammad Hatta. Leprosy. 4. (2003) 3.Mochammad Hatta. no. December (1998). et al.References 1. page 82. 2. et al. Third Asia‑Pacific Symposium Typhoid fever and other Salmonellosis. Southeast Asian Journal of Tropical Medicine and Public Health.Mochammad Hatta. 8‑10 December (1997) 7. 4.Mochammad Hatta. Bali.