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, standardized suspensions
of killed and stained bacteria, agglutinate when mixed with samples containing the homologous antibody.
CLINICAL SIGNIFICANCE
Febrile diseases diagnostic may be assessed either by microorganism isolation in blood, stools or urine, 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 dictates that a serum with high levels of agglutinating antibodies to
O and H 1/100 is indicative of the infection with these microorganism.
PRECAUTIONS
R: EUH208-Contains formaldehyde. May produce an allergic reaction. EUH210-Safety data sheet available on
request Control + / - : H317-May cause an allergic skin reaction. Contain 2-Methylisothiazol-3(2H)-one (Proclin 950).
Follow the precautionary advice given in the SDS and the product label.
REAGENTS COMPOSITION
- Bacterial Antigens: Suspensions of Salmonellas, Brucellas and Proteus in glycine buffer, pH 8.2. Preservative. - Controls: Animal serum. Preservative
CALIBRATION
There is not any International Reference for the sensitivity standardization of these reagents. For this reason,
Spinreact uses an internal control that contains animal serum with antibodies anti-Salmonellas, Brucellas and
Proteus, and titered with commercial reagents of certified quality.
PREPARATION AND STABILITY
Antigen suspensions: Ready to use. Controls: Ready to use. Mix reagents gently before use.
Reagents deterioration: Presence of particles and clumps. All the components of the kit are stable until the expiration date on the label when stored at 2-8ºC, protected from light and contaminations. Do not freeze.
ADDITIONAL EQUIPMENT
- Mechanical rotator adjustable to 80-100 r.p.m. - Heater at 37ºC. - Vortex mixer. - Pippetes 50 µL. - Glass slides.
SAMPLES
Fresh serum. Stable 8 days at 2-8ºC or 3 months at –20ºC.
The samples with presence of fibrin should be centrifuged before testing. Do not use highly hemolized or lipemic samples.
serum titer is defined as the highest dilution showing a positive result.
QUALITY CONTROL
Positive and Negative controls are recommended to monitor the performance of the procedure, as well as a comparative
pattern for a better result interpretation. All result different from the negative control result, will be considered as a positive.
REFERENCE RANGES
Salmonellas: Titers ≥ 1/80 (O antibodies) and ≥ 1/160 (H antibodies) indicates recent infection. Brucellas: Titers ≥ 1/80 indicate
infection. Proteus: 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. 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.
PERFORMANCE CHARECTERISTICS
All the performance characteristics of the Bacterial Antigensmay be found in the corresponding Technical Report and
they are available on request.
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 negativeresults can be obtained in early disease, immune-unresponsiveness, prozone (Brucelosis), and antibiotic
treatment. (somatic).
- Serological cross-reactions with Brucella have been reported in cases of infection or vaccination with some strains of
Vibrio cholerae, Pasteurella, Proteus OX19 and Y. enterocolitica (serotype 9).
NOTES
1. When testing for Brucella antibodies it is recommended to reduce sample volume to 20 µL in order to avoid prozone.
2. In some geographical areas with a high prevalence of febrile antibodies, it is recommended to dilute the sample ¼ en NaCl 9 g/L before to perform the assay.
3. The incubation procedure may be accelerated incubating as follows:
-Somatic (O) and Proteus antigens: 48-50ºC for 4 h. -Flagellar (H) antigens: 48-50ºC for 2 h.
4. 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.
5. 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.
