Download Bacterial Antistest

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BACTERIAL ANTIGENS
Store at 2 - 8ºC.
vial dropper 5 ml
Bacterial Antigens
Slide and tube agglutination
Bacterial antigens
C. Tube test
CLINICAL SIGNIFICANE
Febrile diseases diagnostic may be assessed either by microorganism
isolation or by titration of specific antibodies, somatic (O) and flagellar (H).
The detection of these antibodies forms the basis for the long established
Widal test. This test dedicates that a serum with high levels of agglutinating
antibodies to O and H >1/100 is indicative of the infection with these
microorganism.
PRINCIPLE OF THE METHOD
The Bacterial Antigens is a slide and tube agglutination test for the qualitative
and semi-quantitative detection of antibodies anti-Salmonella, Brucella and
certain Rickettsias in human serum. The reagents are standardized
suspensions of killed and stained bacteria. When the antibodies are present
in serum, a clear agglutination becomes evident.
REAGENTS
Code
Reagent
5011
5021
5031
5041
5051
5061
5071
5081
5091
5092
5101
5111
5121
5901
5902
Antigen
Salmonella paratyphi A H
a flagelar
Salmonella paratyphi A O
1, 2, 12 somatico
Salmonella paratyphi B H
b flagelar
Salmonella paratyphi B O
1, 4, 5, 12 somatico
Salmonella paratyphi C H
c flagelar
Salmonella paratyphi C O
6, 7 somatico
Salmonella Typhi H
d flagelar
Salmonella Typhi O
1, 9, 12 somatico
Brucella abortus*
somatico
Brucella melitensis
somatico
Proteus OX2 (P. vulgaris)
somatico
Proteus OX19 (P. vulgaris)
somatico
Proteus OXK (P. mirabilis)
somatico
Polyvalent positive control 1 ml
Polyvalent negative control 2,5 ml
1. Prepare a row of test tubes for each
disposition :
Dilution 1/20
1/40
1/80
sample
100 µl
saline
1,9 ml
1 ml
1 ml
1 ml
1 ml
antigen according with the following
1/160
…
1 ml
1 ml
1 ml
1 ml
Discard 1 ml
2. Prepare 2 tubes for Positive and Negative control: 0,1 mL Control + 0,9 mL
NaCl 9 g/L (saline).
3. Add to each tube a drop (50 µl) of antigen suspension, previously shaken.
4. Mix thoroughly and incubate tube test at 37°C for 24 hours.
The incubation period may be accelerated as follows:
- For somatic (O) and Proteus antigens : 4 hours at 48-50°C.
- For flagelar (H) antigens : 2 hours at 48-50°C.
READING AND INTERPRETATION
A single positive result has less significance than the demonstration of a rising
or falling antibodies titer as evidence of infection. A clinical diagnosis should
not be made on findings of a single test result, but should integrate both
clinical and laboratory data.
Slide agglutination method
Examine microscopically the presence or absence of clumps within 1 minute
after removing the slide from the rotator comparing the test results with the
control sera. The reactions obtained in the slide titration method, are roughly
equivalent to those which would occur in tube test with serum dilutions of 1/20,
1/40, 1/80, 1/160 and 1/320 respectively. If a reaction is found it is advisable to
confirm the reaction and establish the titer by a tube test.
(*) also useful for Brucella suis antibodies
Tube agglutination test
REAGENTS COMPOSITION
There is not any international Reference for the sensitivity standardization of
these reagents. For this reason, an internal control is used that contains
animal serum with antibodies anti-Salmonellas, Brucellas and Proteus, and
tittered with commercial reagents of certified quality.
Examine microscopically the pattern of agglutination and compare the results
with those given by all control tubes. A somatic reaction (O) is characterized by
coarse, compact agglutination, which tends to be difficult to disperse, while
flagellar (H) has a characteristic loose, flocculant agglutination.
Positive control should give partial or complete agglutination. Negative control
should not give visible clumping.
Partial or complete clumping with variable degree of clearing of the supernatant
fluid should be recorded as a positive result.
The titer is the highest dilution that shows positive result.
STORAGE AND STABILITY
QUALITY CONTROL
The reagents and controls are stable up to the expiry date when stored at +2
to 8°C. Do not freeze !
The controls and reagents are ready to use. The reagent tends to settle down.
Shake the Reagent before use. After that it must be uniform and without
visible clumping.
The presence of particles and clumps are signs of deterioration.
Positive and Negative controls are recommended to monitor the performance of
the procedure, as well as a comparative pattern for a better result interpretation.
- Bacterial Antigens: Suspensions of Salmonellas, Brucellas and Proteus in
glycine buffer, pH 8.2. Sodium azida 0.95 g/L.
- Controls: Animal serum. Sodium azide 0.95 g/L.
CALIBRATION
ADDITIONAL EQUIPMENT
- Mechanical rotator adjustable to 80 -100 rpm
- Incubator at 37°C
SAMPLES
Use fresh serum obtained by centrifugation of clotted blood. The sample may
be stored 8 days at 2°-8°C or 3 months -20°C.
Highly hemolyzed or lipemic samples must be discarded. The samples with
presence of fibrin should be centrifuged before testing.
PROCEDURE
A. Slide agglutination (qualitative)
1. Bring reagents and specimens to room temperature before use.
2. Gently shake the reagent to disperse the particles until you get a
homogeneous mixture.
3. Place 50 µlnote 1-2 of undiluted serum and 1 drop of Positive and Negative
controls onto separate circles of the slide.
4. Add a drop (50 µl) of the Reagent next to the drop of serum.
5. Mix both drops spreading them over the full surface of the circle
6. Rotate the slide manually or with mechanical shaker (80 - 100 rpm) during 1
minute.
B. Slide agglutination (titration)
1. Using a micropipette, deliver 80, 40, 20, 10 and 5 µL of undiluted serum into
separate circles of the slide test.
2. Place a drop (50 µl) of the Reagent next to the serum.
3. Mix both drops spreading them over the full surface of the circle
4. Rotate the slide manually or with mechanical shaker (80 - 100 rpm) during 1
minute.
REFERENCE RANGES
Salmonellas: Titers ≥ 1/80 (O antibodies) and ≥ 1/160 (H antibodies) indicates
recent infection.
Brucellas: Titers ≥ 1/80 indicate infection.
Proteus: Titers OX19 ≥ 1/80, OX2 ≥ 1/20 and OXK ≥ 1/80 indicate infection.
The level of “normal” agglutinins to these organisms varies in different
countries and different communities. It is recommended that each laboratory
establish its own reference range.
INTERFERENCES
Bilirubin (20 mg/dL), hemoglobin (10 g/L), lipids (10 g/L) and rheumatoid factors
(300 IU/mL), do not interfere.
LIMITATIONS OF PROCEDURE
- False negative results can be obtained in early disease, immuneunresponsiveness, prozone (Brucelosis), and antibiotic treatment. (somatic).
- Serological cross-reactions with Brucella have been reported in cases of
infection/vaccination with some strains of Vibrio cholerae, Pasteurella,
Proteus OX19 and Y. enterocolitica (serotype 9).
- A great number of false positive reactions have been reported in healthy
individuals with Proteus antigens, especially in slide agglutination test. A
titer of less than 1/160 should not be considered significant.
NOTES
1. When testing for Brucella antibodies it is recommended to reduce sample
volume to 20 µl to avoid prozone
2. In some geographical areas with a high prevalence of febrile antibodies, it is
recommended to dilute samples 1/4 in saline before to perform the assay.
BIBLIOGRAPHY
1. Edward J Young. Clinical Infectious diseases 1995; 21: 283-290
2. Coulter JBS. Current Pediatrics 1996; 6: 25-29.
3. David Aet al. Current Opinion in Infectious Diseases 1994; 7: 616-623.
4. David Aet al. Current Opinion in Infectious Diseases 1993; 6: 54-62.
5. Bradley D Jones. Annu Rev Immunol 1996; 14: 533-561.
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