Brucella Abortus / Meliteness Antibodies Reagents -
Diagnostics Kit Usage Manual For Slide And Tube Test

INTRODUCTION

Human Brucellosis(Diurnal or undulant fever) is a common febrile illness caused by infection with bacteria of some of the Brucella species(abortus, melitensis). This undulant fever is associated with symptoms, which are often variable and non specific withi chills, fever, sweats and anorexia. On exposure the body responds to this antigenic stimulation by producing specific antibodies whose titres rise slowly at early stages and then increases. Specific antibodies to the Brucella species are detectable a few weeks after exposure and are of considerable importance in the diagnosis of Brucellosis. Information regarding the time of antibodies can be btained by using specific Brucella A/M antigen suspensions.

METHOD PRINCIPLE:

The smooth, attenuated stained BRUCELLA antigen suspensions are mixed eith the patients serum. Specific antibodies to Brucella antigens.

Working with Brucella Abortus / Brucella Melitensis reagents preparation and stability

1. Store the reagents at 2-8*C. Do not Freeze.

2. The shelf life of the reagent is as per the expiry date mentioned on the vial labels

Specimen

Store the reagent at 2-8*C. . The shelf life of the reagent is as per the expiry date mentioned on the vial labels. Avoid exposure to elevated temperatures and air, as the reagents is highly sensitive to denuration and drying.

ADDITIONAL MATERIAL REQUIRED:

Slide Test Method : Stop Watch, Positive control, Isotonic saline and Glass slide with clear / White background, appropriate Pipettes / Micropipettes, Mixing Sticks & high density direct light source.

Quantitative Method:

Timbe, Test Tubes(12mm x 75mm), Test tube rack, appropriate pipettes/Micro pipettes, Isotonic Saline/ 0.25% Phenol saline, Incubator(37C).

Notes:

INVITRO diagnostic regent for laboratory and professional use only. Not for medicinal use.

The reagent contains 0.01% thimersol as perspective. Avoid contact with skin and mucosa. On disposal flush with large quantities of water.

Performance of the reagent must be verified with positive and negative controls and it is recommended that controls be run with each test series.

The reagent can be damaged due to microbial contamination or an exposure to extreme temperature.

Shake the reagent vials well before use to dispense the antigen suspension uniformly and improve test readability.

only a clean and dry glass slides / tubes must be used. Clean the glass slide / tubes with distilled water and dry.

It is necessary to use the calibrated dropper provided in the reagents vial to dispense a reagent drop.

Brucella – A/M antigen suspensions are not from human sources, hence combination due to HBs Ag and HIV is practically excluded.

Donot use damaged or leaked reagents.

Sample Collection and Storage:

1. No special preparation of patient is required prior to sample collection by approved techniques. Do not use hemolysed and turbid serum samples.
2. Clean and dry glassware free from detergents must be used for sample collection.
3. Do no heat inactive in the serum.
4. Though freshly collected serum is preferred samples can be stored at 2-8 C, for 24 hrs or Frozen for 8 Days should be a delay testing occur.
5. Brucella antigen suspensions are not from human sources hence contamination due to HBsAg and HIV is practically excluded.

Procedure- Slide Method

Bring reagent and samples to room temperature before testing, Shake and mix the Brucella antigen suspensions well before dispensing. The procedure for Brucella –A and Brucella-M is identical.

Qualitative Method:

Place one drop of the test sample, positive and negative controls onto separate reaction circles of the glass slide using a sample dispensing pipette.

Add one drop of saline onto the next reaction circle of the glass slide.

Add one drop of patient serum to be tested on the next reaction circle of glass slide.

Add one drop of the appropriate Brucella antigen suspensions in each of the above cirlces( controls and patients sample dispensed)

Mix contents of each circle uniformly over the entire circle with separate mixing sticks.

Gently rock the slide back and froth, observe for agglutination macroscopically at 1 minute against the white background.

Qualitative Method:

Using saline prepare serial dilutions of the test sample positive the qualitative methos 1:2, 1:4, !:8, 1:16, 1:32, 1:64, 1:128 and so on.

Perform the qualitative test procedure using each dilution as test specimen.

The titre is reported s the reciprocal of the highest dilution which shows as positive test result.

Procedure:

Take 8 tests tubes and label them 1 to 8.

Pipette 1.9 ml of istonic saline or preferably 0.25% Phenol saline to tube no.1

To each of the remaining tubes(2-7) add 1.0ml of isotonic saline of preferably 0.25% phenol saline.

To the tube no.1, add 0.1 ml of serum sample to be tested. Mix well.

Transfer 1.0 ml of the diluted serum from tube no1 to tube no 2 and mix well.

Transfer 1.0ml of the diluted serum from tube no.2 to tube no.3 and mix well. Continue this serial dilution till tube no.7.

Discard 1.0ml of the diluted serum from tube no.7

Pipette 1.0 ml of isotonic saline in tube no.8 which serves as a negative control.

To all the tubes and incubate at 37C for 24 hrs.

Observe for agglutination macroscopically in each tube of the dilution series.