ImmunoSero Lab 6 - Widal, Tubex, Salmonella typhi antibody

Tubex Test

a semi-quantitative test meaning it can give you a titer of antibodies present

Tubex Test

Only detects Acute typhoid fever that is because it is only capable of detecting the IgM antibody

F

- cannot detect past infections

T/F

Tubex can detect past infections which elicits the IgG antibody

Tubex Test

It detects IgM antibody to Salmonella typhi O9 lipopolysaccharide antigen

Salmonella typhi

Among the three tests, Tubex can only detect

antibodies against ____ and not Salmonella paratyphi

Typhoid IgG/IgM and Widal Test

can detect both antibodies against Salmonella typhi and Salmonella paratyphi

Superparamagentic particles

particles found in the Antigen coated brown reagent

blue dye latex microspheres

particles found in the Antibody coated blue reagent

Antibody coated blue reagent

The reagent in Tubex test that is only capable of detecting IgM antibodies

45 microliter

Tubex test:

volume of the brown reagent

45 microliter

Tubex test:

volume of the patient serum

2 minutes at room temperature

- to allow any antibodies against Salmonella typhi 09 lipopolysaccharide antigen to react with the antigen coated brown reagent.

Tubex test:

Incubation requirements of the brown reagent and the patient's serum

90 microliter

Tubex test:

volume of the blue reagent

2 minutes, back and forth

Tubex test:

After adding the blue reagent, mix for how long?

5 minutes

Tubex test:

Standing time on the magnetic color scale

mix at 90 degrees horizontally

to expose maximum well surface to the mixture

Reading = 0-2

What is the reading?

- Clear pink color

- Negative

Reading = 3

What is the reading?

- borderline

* test at a later date

Reading = 4

What is the reading?

- Weakly positive

Reading = 6-10

What is the reading?

- Positive

* Strong indication of current Typhoid fever

bluish to intense blue solution

Positive result of tubex test?

Pink

Negative result of tubex test?

positive result (blue color)

the patient's antibodies will bind to the antigen coated brown reagent, hence, the antibody coated blue reagent cannot bind with the brown reagent

The patient's serum has antibodies against Salmonella typhi

Interpretation of positive result

negative result (clear pink)

there are no patient antibodies, hence, the antibody coated blue reagent binds with the antigen coated brown reagent

The patient's serum is negative for antibodies against Salmonella typhi

Interpretation of negative result

O9 Lipopolysaccharide antigen

coats the superparamagnetic particles

Antibody monoclonal IgM against O9 LPS antigen

coats the blue dye latex microspheres

pH 8.2

pH of the antigen coated brown reagent and antibody coated blue reagent

Reaction well Strip

contains 6 reaction wells

Sealing tape

seals the mouth of the strips

Magnetic color scale

inside the scale are magnets

Passive agglutination

Principle of Tubex Test

Alpha-D-tyvelose

What makes Salmonella typhi specific?

O antigen

antigen that appears first, disappears first

H antigen

antigen that appears last, disappears last

Typhoid IgG/ IgM

among the three tests, it is purely qualitative

Lateral flow immunochromatographic assay

Principle of Typhoid IgG/ IgM

1 drop / 30-45 microliter

Typhoid IgG/ IgM:

serum volume

1 drop

Typhoid IgG/ IgM:

diluent volume

after 15 minutes

Typhoid IgG/ IgM reading time

Recombinant H and O antigens with colloid gold conjugate

Typhoid IgG/ IgM:

Conjugate pad

Rabbit IgG conjugate

Typhoid IgG/ IgM:

Control line

monoclonal anti-human IgM against Salmonella typhi and Salmonella paratyphi IgM

Typhoid IgG/ IgM:

IgM Band (Nitrocellulose membrane)

monoclonal anti-human IgG against Salmonella typhi and Salmonella paratyphi IgG

Typhoid IgG/ IgM:

IgG Band (Nitrocellulose membrane)

Goat anti-rabbit IgG

Typhoid IgG/ IgM:

Control pad/line (Nitrocellulose membrane)

Colloid gold conjugates

- are nanoparticles that are also carrier particles.

- Similar to latex particles but are smaller that are used to carry the H and O antigens.

antigen-antibody complex

complex formed when the patient's antibody and the H or O antigen are mixed

nitrocellulose membrane

contains Anti-human IgM

and Anti-human IgG

Anti-human IgM

an antibody against the antibody.

early primary infection

Typhoid IgG/ IgM interpretation:

IgM +
C +

active primary infection/ repeat infection

Typhoid IgG/ IgM interpretation:

IgM +
IgG +
C +

Late stage/ latent infection

Typhoid IgG/ IgM interpretation:

IgG +
C +

invalid

Typhoid IgG/ IgM interpretation:

no C line

Widal test

- Semi-quantitative test

- Serological test

Semi-quantitative test

it can give you a titer of antibodies present

serological test

can only detect antibodies

Passive Latex Agglutination

Principle of Widal Test

Standardized suspension of stained bacteria

Reagent of Widal Test

Salmonella typhi O
Salmonella typhi H
Salmonella paratyphi AH
Salmonella paratyphi BH

Enumerate the stained bacteria

F

- it causes cross reaction

T/F

Salmonella paratyphi can use O antigen

H antigens of Salmonella typhi and Salmonella paratyphi

O antigens of Salmonella typhi and Salmonella paratyphi

Widal Test:

Antigen used to coat the latex

Rapid Slide agglutination test

Used as a Screening test

50 microliter

Slide agglutination test:

volume of patient's serum

1 drop

Slide agglutination test:

volume of reagent

1 minute at 100 rpm

Slide agglutination test:

centrifugation

Tube agglutination test

confirmatory test of the slide agglutination test

red-colored

Slide agglutination test:

Color of the reagent that has H antigen

blue-colored

Slide agglutination test:

Color of the reagent that has O antigen

Widal test

Only tube agglutination test is called the ___

1900 microliter

Tube agglutination test:

volume of NSS in the first tube

1000 microliters

Tube agglutination test:

volume of NSS in the succeeding tubes

100 microliter

Tube agglutination test:

volume of serum added to the first tube

2000 microliter

Tube agglutination test:

total volume

1:20

Tube agglutination test:

dilution

Double serial dilution

What kind of dilution is used in tube agglutination test?

1st tube = 1:20
2nd tube = 1:40
3rd tube = 1:80
4th tube = 1:160

Dilution of the:

1st tube
2nd tube
3rd tube
4th tube

1 drop each

Tube agglutination test:

volume of O antigen and H antigen

Tube agglutination test:

24 hours at 37 degrees

Tube agglutination test:

Incubation requirements

48-50 °C

Tube agglutination test:

hasten the results you can accelerate the reaction by incubating it at a higher temperature of ___

2-mercaptoethanol

denature the IgM due to its J-chain.

2-mercaptoethanol

This will differentiate the IgG and IgM in Widal’s test

4

For those mixtures that make use of the O antigen

suspension, you can read results after ___ hours already.

2

For those mixtures that make use of the H antigen
suspension, you can read results after ___ hours.

agglutination

Positive result of the tube agglutination test

somatic reaction

Coarse compact agglutination that is difficult to disperse

flagellar reaction

loose flocculent agglutination

incubate for 24 hours

disadvantage of tube agglutination test

4 hours

Accelerated reading time of O antigen suspension

2 hours

Accelerated reading time of H antigen suspension

Active infection

Tube agglutination test Interpretation:

O antigen titer is greater than 160

Past infection/ Recent immunization/ Vaccination

Tube agglutination test Interpretation:

H antigen titer is greater than 160

6 days to 30 days

Incubation period of the Salmonella

humans

the only reservoir and natural host of Salmonella typhi

Salmonella typhi
Salmonella paratyphi

Typhoidal serotypes

Enteric Fever

typhoid and paratyphoid are collectively called as ___

BASU

1st week = Blood culture

2nd week = Antibody

3rd week = Stool culture

4th week = Urine culture

Lab tests per week

1st week =
2nd week =
3rd week =
4th week =