serology
laboratory no. 4
serology
serum separation
1. collect blood sample in clean container without anticoagulant and allowing it clot by leaving it at room temperature for 2 h or overnight.
2. centrifuge at 2500 rmp for 5 minutes.
3. two layers formed, remove the serum by pasture pipette and put in clean container.
pregnancy test (pt)
human chorionic gonadortopin (hcg)
hcg is a glycoprotein hormone produced by the developing placenta shortly after fertilization. the appearance and rapid rise in the concentration of hcg in the urine make hcg an excellent pregnancy marker. the concentration of hcg in urine can reach 0.2 iu/ml as early as 2-4 days after the first missed menstrual period. the highest values can be demonstrated later in the first trimester of pregnancy.
detection of hcg in urine
principle
the reagent used in the pregnancy test is a suspension of latex particles of uniform size coated with anti-hcg monoclonal antibodies. latex particles allow visual observation of the antigen-antibody reaction. the visible agglutination is due to presence of hcg in the urine.
samples collection and preparation
· a first morning urine specimen is preferred because it contains the highest concentration of hcg.
· if the urine is very turbid, centrifugation or filtration may be necessary.
· urine samples may be stored at 2-8°c for up to 2 days. if the testing is delayed more than 2 days the samples can be stored at -20°c for up to 3 months.
materials required:
1. latex reagent: suspension of latex particles coated with anti-hcg monoclonal antibodies buffered in sodium azide.
2. positive control: hcg solution buffered in sodium azide.
3. negative control: non reactive diluted human serum.
4. slides.
5. pipette.
6. stirrers.
test procedure
- allow the reagents and urine samples to reach room temperature.
- place 50 ?l of the urine and both positive and negative controls on the test slide.
- shake the regent vial and add one droping or reagent next to the droping of sample.
- mix both dropings with stirrer covering the whole surface of the slide.
- rotate the slide for 2 min.
- observe for the presence or absence of agglutination.
reading the results
positive : large clumping with clear background.
negative: absence of agglutination, uniform suspension.
detection of hcg in serum
principle
the rapid chromatographic immunoassay is a qualitative test to detect hcg in urine or serum. the test utilized a combination of antibodies including a monoclonal antibody to selectively detect hcg. the test uses two lines to indicate results.
· the control line is composed of goat polyclonal antibodies and colloidal gold particles.
· the test line is composed of monoclonal antibodies to hcg with colored conjugate.
the assay conducted by immersing the test strip in the urine and serum samples and observing the formation of colored lines. the sample migrates via capillary action along the membrane to react with the colored conjugates. positive samples react with the specific anti-hcg-colored conjugate to form a colored line at the test region of the membrane. absence of this colored line suggests a negative result. the colored line always appear in the control line region indicating that proper volume of sample has been added and membrane wicking has occurred.
samples collection and preparation
the serum samples should be clear and non-hemolyzed.
assay procedure
1. allow the reagents and urine samples to reach room temperature.
2. with arrows pointing toward the urine or serum sample, immerse the test strip vertically in the urine or serum samples for at least 10-15 second.
3. place the test strip on a non-absorbent flat surface, and wait the color line to appear.
4. read the results at 3 minutes when testing a urine sample, or at 5 minutes when testing a serum samples.
reading the results
1. positive: two distanced red lines appear.
2. negative: one red line appears.
3. invalid: control line fails to appear.
toxoplasma serology
toxoplasmosis
toxoplasmosis is an infectious affecting both animals and humans, which is caused by protozoan parasite toxoplasma gondii. acquired toxoplasmosis is usually asymptomatic and benign. in pregnant women, the parasite may inter the fetal circulation through the placenta and cause congenital toxoplasmosis. the consequences of congenital toxoplasmosis include:
1. abortion
2. prematurity
3. generalize and neurological symptoms
4. ocular complications
toxoplasma latex agglutination test
toxoplasma latex test reagent is a suspension of polystyrene latex particles coated with toxoplasma gondii antigen. the agglutination tack place due to the presence of toxoplasma antibodies in the serum, the latex suspension change its uniform appearance and a clear agglutination became visible.
samples
fresh serum
materials required
1. latex reagent: suspension of polystyrene latex particles coated with toxoplasma gondii soluble antigens in buffer containing bovine serum albumin and contains 1% sodium azide.
2. positive control: diluted human serum containing rabbit igg antitoxoplasma + 1% sodium azide.
3. negative control: non reactive diluted serum + 1% sodium azide.
qualitative test
1. allow the reagent to reach room temperature.
2. place 50 µl of the serum onto a slide.
3. shake the reagent vial and add one droping of reagent next to the droping sample.
4. mix both dropings with a stirrer.
5. rotate the slide for 5 minutes.
6. observe for presence or absence of agglutination.
positive reaction = presence of agglutination
negative reaction = absence of agglutination
semiquantitative test
1. place 50 µl of normal saline onto slide section 2-6
2. place 50 µl of the sample onto slide section 1 and 2
3. take and release the sample and the saline on section 2 several times until they are well mixed
4. take 50 µl of the mixture made on section 2 and transfer it to section 3
5. repeat this operation from 3-6 to obtain a well mixing of reagents, and discarding 50 µl
6. test each dilution for agglutination
the titer: the highest serum dilution that give a positive agglutination
interpretation of results
a negative reaction should be interpretated as the absence of toxoplasma antibodies. a positive reaction should be interpretated as presence of toxoplasma antibodies which may reflect either a past infection or an acute toxoplasma infection.
50 µl serum + 50 µl saline
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