8. Since techniques and standardization vary from lab to

lab one tube difference in tube titers can be expected.

9. Do not interchange reagent caps.

Serology/Immunology 627

WIDAL ANTIGEN SET/ANTIGENS FOR SLIDE

AND TUBE TESTS (TYDAL)®

(Courtesy: Tulip Group of Companies)

Reagent

The TYDAL contains ready-to-use concentrated, vitally

stained, smooth antigen suspensions of the bacilli; S

typhi ‘O’, S typhi ‘H’, S paratyphi ‘AO’, S. paratyphi ‘BO’,

S paratyphi ‘AH’, S paratyphi ‘BH’ and/or polyspecific

positive control reactive with these antigens. Each batch

of reagents undergoes rigorous quality control at various

stages of manufacture for its specificity and performance.

Reagent Storage and Stability

1. Store the reagents at 2 to 8°C. Do not freeze.

2. The shelf-life of reagents is as per the expiry date

mentioned on the reagent vial labels.

Principle

When the colored, smooth, attenuated TYDAL antigen

suspensions are mixed/incubated with patient serum,

anti-Salmonella antibodies present in the patient serum

react with the antigen suspensions to give agglutination.

Agglutination is a positive test result, indicating presence

of anti-Salmonella antibodies in the patient serum. No

agglutination is a negative test result indicating absence of

anti-Salmonella antibodies.

Note

1. In vitro diagnostic reagent for laboratory and professional use only. Not for medicinal use.

2. The S typhi ‘O’ reagent contains phenol 0.5%, S. typhi

‘H’, S. paratyphi ‘AH’, S. paratyphi ‘BH’ reagents

contain formal dehyde 0.3% and S paratyphi ‘AO’,

S paratyphi ‘BO’ reagents contain ethanol 0.7% as

preservatives. Avoid contact with skin and mucosa.

On disposal flush with large quantities of water.

3. Only a clean and dry glass slide must be used. Clean

the glass slide with distilled water and wipe dry.

4. Accessories provided with the kit only must be used

for optimum results, (applicable only for TYDAL 4

× 5 mL set and 4 × 10 mL set).

Sample Collection and Storage

1. No special preparation of the patient is required prior

to sample collection by approved techniques. Do not

use hemolyzed samples.

2. Clean and dry glassware free from detergents must be

used for sample collection.

3. Do not heat inactivate the serum.

4. Though freshly collected serum is preferable, store

samples at 2 to 8°C in case of delay in testing, for upto

72 hours.

Material Provided with the Kit

Reagent Pack

The TYDAL 4 × 5 mL set and TYDAL 4 × 10 mL set contain

item Nos 1, 2, 5, 6, 7 and 8 mentioned below. TYDAL 2 ×

5 mL set contains item Nos. 1, 2 and 7 mentioned below:

1. S. typhi ‘O’ Antigen suspension

2. S. typhi ‘H’ Antigen suspension

3. S. paratyphi ‘AO’ Antigen suspension

4. S. paratyphi ‘BO’ Antigen suspension

5. S. paratyphi ‘AH’ Antigen suspension

6. S. paratyphi ‘BH’ Antigen suspension

7. Polyspecific positive control (Goat)

8. Glass slide with six reaction circles, mixing sticks,

disposable sample dispensing pipettes with rubber teats.

Note: Item Nos. 1 to 6 each is available as individual

reagent packs.

Additional Material Required

Slide test method: Stop watch, variable micropipettes.

Note: Item No. 8 from reagent pack is additionally required

for TYDAL 2 × 5 mL set. Item No. 7 and 8 from reagent pack

are additionally required for individual reagent packs of

TYDAL ‘O’, TYDAL ‘H’, TYDAL ‘AO’, TYDAL ‘BO’, TYDAL

‘AH’, TYDAL ‘BH’ antigens.

Quantitative Method

Timer, Kahn tubes/test tubes, pipettes (0.1 mL, 1 mL),

isotonic saline, incubator (37°C), test tube racks.

Procedure

a. Bring reagents to room temperature before testing.

b. Shake and mix antigens well before dispensing.

Slide Screen Method

1. Place one drop of positive control onto a reaction circle

of the glass slide.

2. Place one drop of isotonic saline onto the next reaction

circle of the glass slide.

3. Place one drop of patient serum to be tested onto each

of the required number of reaction circles.

4. Add one drop of appropriate TYDAL antigen

suspensions to the reaction circles containing positive

control and isotonic saline.

628 Concise Book of Medical Laboratory Technology: Methods and Interpretations 5. Add one drop of appropriate TYDAL antigen suspensions

to the reaction circles containing the patient serum.

6. Mix contents of each circle uniformly over the entire

circle with separate mixing sticks.

7. Rock the slide gently back and forth, and observe for

agglutination macroscopically at one minute.

Slide Semiquantitative Method

1. Using a pipette, place 80 µL, 40 µL, 20 µL, 10 µL, and

5 µl of patient serum to be tested on 5 different reaction

circles on the glass slide. The corresponding titers

obtained will be 1:20, 1:40, 1:80. 1:160, and 1:320

respectively.

2. Follow step No. 5 to 7 of slide screen method.

Note: This method is recommended for obtaining quick

approximate titres only.

Quantitative Method

Tube Test Procedure

1. Take appropriate number of sets (as required; one

set for each antigen suspension) of 8 Kahn tubes/test

tubes and label them 1 to 8.

2. Pipette into tube No. 1 of all sets 1.9 mL of isotonic

saline.

3. To each of the remaining tubes (2 to 8), add 1 mL of

isotonic saline.

4. To tube No. 1 of all sets add 0.1 mL of serum sample

to be tested and mix well.

5. Transfer 1 mL of the diluted serum sample from tube

No. 1 to tube No. 2 and mix well.

6. Transfer 1 mL of the diluted serum sample from tube

No. 2 to tube No. 3 and mix well. Continue this serial

dilution till tube No. 7 in each set.

7. Discard 1.0 mL of the diluted serum from tube No. 7

of each set.

8. Now the dilutions of the serum sample achieved

from tube No. 1 to 7 respectively in each set is as

follows 1:20, 1:40, 1:80. 1:160, 1:320, 1:640, 1:1280.

Tube No. 8 in all the sets serves as a saline control.

9. To all the tubes (1 to 8) of each set, add one drop of the

respective well mixed TYDAL antigen suspensions

from the reagent vials and mix well.

10. Cover and incubate at 37°C overnight (approximately

18 hours).

11. Dislodge the sedimented button gently and observe

for agglutination.

Interpretation of Results

Slide Screen Method

Agglutination is a positive test result and indicates presence

of the corresponding antibody in the patient serum.

No agglutination is a negative test result and indicates

absence of the corresponding antibody in the patient

serum.

Slide Semiquantitative Method

Agglutination is a positive test result. The titer of the patient

serum corresponds to the visible agglutination in the test

circle with the smallest amount of serum sample.

Quantitative Method

The titer of the patient serum using TYDAL antigen

suspensions is the highest dilution of the serum sample

that gives a visible agglutination.

Remarks

1. Positive results obtained in the slide test should be

confirmed with the tube test to establish whether the

titers are diagnostically significant or not.

2. TAB vaccinated patients may show a high titer of

antibodies to each of the antigens.

3. ‘O’ being a somatic antigen brings about a coarse,

compact, granular agglutination whereas ‘H’ being a

flagellar antigen brings about larger, loose, flocculant

agglutination.

4. While the ‘O’ antigen is species specific, the ‘H’ antigen

is specific to the serotype.

5. Turbid and contaminated sera should not be used for

testing.

6. Generally antibody titers of 1:80 or more are

considered clinically and diagnostically significant.

However, the significant titer may vary from

population to population and needs to be established

for each area.

7. It is recommended that results of the tests should be

correlated with clinical findings to arrive at the final

diagnosis.

8. Since techniques and standardization vary from

lab to lab one tube difference in tube titers can be

expected.

9. The performance of the reagents should be validated

occasionally using know positive control. Good

physiological saline may be used as a negative

control.

Serology/Immunology 629

Troubleshooting

Problem: False positive results

Possible causes Solutions

1. Past history of immunization inapparent infection or prior disease Positive reaction will be seen in three flagellar antigens: H, AH, BH in

such cases whereas in case of infection, antibodies will be seen only

against the infecting species. Also check the patient’s history

2. Anamnestic response to other vaccines and unrelated fevers in the

case of persons who have had a prior infection or immunization

The anamnestic fever can be differentiated from enteric fever by repetition of the test after a week. The anamnestic response shows only

a transient rise, while enteric fever the rise is sustained

3. Prolonged rocking of the slide causes drying of the test material Agglutination should be observed within one minute.

4. If reagents have been exposed to excessively high temperature,

precipitates could be formed

Ensure that the reagents are not exposed to high temperatures and

are stored properly at 2–8°C

5. Contamination of serum could lead to false positives Ensure that clean and dry glassware free from detergents are used for

sample collection. If serum samples are not test immediately, store at

2–8°C. Turbid serum should not be used for testing

6. Error in interpreting results. Granularity mistaken for clumping The results should be interpreted properly besides by comparing with

the polyspecific positive control provided with the kit

Problem: False negative results

Possible causes Solutions

1. Infection is in very early stages, when antibody titer is very low The agglutinin titer depends on the stage of the disease. Agglutinins

usually appear by the end of the 1st week, so that the blood taken

earlier may give a negative test result. The testing should be repeated

after a week in such cases

Demonstration of a rise in titer of antibodies by testing two or more