Serological tests

Serological tests
Serum separation:
1. Collect blood sample in clean container without anticoagulant and allowing it clot by leaving it at room temperature for 2h or overnight.
2. Centerifuge at 2500 rpm for 5 minutes.
3. Two layers formed, remove the serum by pasture pipette and put in clean container.

Pregnancy test (PT)

Human Chorionic Gonadotropin (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 Ul/ml as early as 2-4 days after the first missed menstrual period. The highest values can be demonstrate later in the first trimester of pregnancy.

A- Detection of hCG in urine or serum by latex
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.

Sample collection and preparation:
• A first morning urine specimen is preferred because it contain the highest concentration of hCG.
• If the urine is very turbid, centrifugation or filtration may be necessary.
• 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. Automatic pipette.
6. stirrers.

Test procedure
1. Allow the reagents and urine samples to reach room temperature.
2. slide.
3. Shake the regent vial and add one drop or reagent next to the drop of sample.
4. Mix both drops with stirrer covering the whole surface of the slide.
5. Rotate the slide for 2 min.
6. Observe for the presence or absence of agglutination.

Reading the results
Positive : Large clumping with clear background.
Negative: Absence of agglutination, uniform suspension.

B- Detection of hCG in urine or serum by strip
Principle
The rapid chromatographic immunoassay is a qualitative test to detect hCG in urine or serum. The test utilized a combination of antibodies including amonoclonal 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 practices.
• 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 migrate 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.

Sample 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 pointed toward the urine or serum sample, immeres 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 aurine 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.

Toxoplasmosis test

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 congental Toxoplasmosis include:
1. Abortion
2. Prematurity
3. Generalize and neurological symptoms
4. Ocular complications

Toxoplasma latex agglutination test reagent is a suspension of polystyrene latex particales coated with Toxoplasma gondii antigen. The agglutination tack place due to the presence of Toxoplasma antibody in the serum, the latex suspension change its uniform appearance and a clear agglutination became visible.

Samples:
Fresh serum

Materials required:
1. 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% sodum azide.
3. Negative control: non reative diluted serum +1% sodum 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 drop of reagent next to the drop sample.
4. Mix both drops 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

Semiquantitave test:
1. Place 50 µl of normal saline onto a slide section 2-6
2. Place 50 µl of the sample onto slide.section 1&2
3. Take and release the sample and the saline on section 2 several times until they are well mixed.
4. Take 50µl 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 reagent , and discarding 50µl
6. Test each dilution for agglutination.

The titer: the highest serum dilution that give a positive agglutination.







Serologic Diagnosis of typhoid fever
Typhoid fever "Enteric fever"
Typhoid fever is a syndrome caused by Salmonellae Spp. This bacteria are often pathogenic for humans or animals when acquired by the oral route. They are transmitted from animals and animal products to humans where they cause enteritis, systemic infection and enteric fever.

Serologic methods:
Serologic techniques are used to identify unknown cultures with known sera and also be used to determine antibody titers in patients with unknown disease.

The titer: It is the highest dilution that gives a positive (antigen-antibody reaction), for example: the highest dilution at which agglutination occurs is 1:320, the titer is 320 antibody units per milliliter of serum. A higher titer mean there is a greater antibody response of the individual to the disease.

Agglutination test
This test is depend on the agglutination between a suspensions of Salmonellae different antigens and anti- Salmonellae antibodies in patients sera. The presence or absence of visible agglutination is usually related with presence or absence of anti- Salmonellae antibodies in the tested samples.

Samples: fresh, clear serum. After the serum has been separated it must be stored at 2-8°C up to one week, or at -20°C for longer periods.

Materials required:
• Suspension of Salmonellae antigens contain killed bacteria buffered sodium azide:
- Salmonellae typhi H (flagellar antiegn)
- Salmonellae Typhi O (somatic antigen)
- Salmonellae paratyphi H (different serotypes)
- Salmonellae paratyphi O (different serotypes)
• Positive control: contain Salmonellae antiserum buffered in sodium azide.
• Negative control: contain non reactive serum buffered in sodium azide.
• Glass or white test cards.
• Disposable stirrers (woody sticks).
• Test tubes (12 × 100 mm).
• Automatic pipettes.
• Saline solution (NaCl 0.9%)

Test procedure

A- Slide agglutination (Qualitative test)
1. Bring the test reagents and samples to room temperature.
2. Resuspend the antigens in the vial gently.
3. Place 5 ?l of the serum into a row on the card. Place 1 drop of +ev & -ev controls.
4. Add 1 drop of each antigens next to the drops of serum.
5. Mix the antigens and serum sample with stirrer.
6. Rock the slide gently by hand for 1 min.
7. Observe the agglutination under light source.
Reading the results:
- Negative : Smooth suspension with no agglutination.
- Positive: Visible agglutination.

B- Tube agglutination (Widal test)
1. Dilute the patients serum as following:

2. Add 1 drop of Salmonella antigen to each tube.
3. Incubate the tubes in water bath (48-50 °C for 2 hours).
4. Examine the tubes for agglutination.

Reading the results :
Positive: agglutination as clumping sediment, the titer is reported as the highest dilution that show agglutination.
Negative: No clumping visible.

? Note: The serological diagnosis of typhoid fever must perform at least 1 week after the symptoms appears.






Serologic Diagnosis of Brucellosis
Brucellosis (Malta fever)
Brucellosis is a bacterial infection caused by Brucella spp which are a obligate endoparasite of animals and humans. The disease characterized by an acute bacteremic phase followed by chronic stage that may extend over many years and may involve many tissues.

Serologic Diagnosis of Brucellosis (Rose Bengal test)
This test is depends on the ability of antibody in the patient s serum to agglutinate the bacterial antigens. When this occurs the aggregates become clearly visible to the naked eye.

Samples:
Fresh serum. After the serum has been separated it must be stored at 2-8°C up to one week, or at -20°C for longer periods.

Materials required:
1. Brucella bacteria, killed and suspend in lactate buffer.
2. Positive control: serum containing antibodies to Brucella.
3. Micropipettes.
4. Test slide.
5. Stirrers.
6. Saline.

Test procedure
1. Allow reagents and patients serum to come to room temperature.
2. Transfer one drop (50?l) of patient s serum to the slide.
3. Shake the reagent, the using the dropper to add one drop of suspension to the slide next to the serum s drop.
4. Mix the drops.
5. Gently rock the test slide for 4 min.
6. read the results.

Reading the results

Positive: Visible agglutination.
Negative: no change in the suspension on the test slide.