Dr. Ibrahim Hassan - Microbiology PhD (C)Dr. Ibrahim Hassan - Microbiology PhD (C)

Detection of Antigen (Ag) or Antibody (Ab) Invitro:Antigen (Ag) is a foreign substances when introduced in the body stimulate the immune system to produce specific Antibody and react specifically in the optimum proportion (zone phenomenon).

Antibody (Ab) is a substance produced by the immune system when a foreign Antigen is introduced in the body, and react specifically with the Ag in the optimum proportion (zone phenomenon or zone equivalence).

Most important types of Ag/Ab reaction:

- Agglutination Test.- Precipitation Test.- Hemolysis Test. - Neutralization Test.

Agglutination Test: Antigen in particulate form;

1- Direct agglutination test: when the Ag & specific Ab are in the optimal proportion react to form visible agglutination without helping factor. - Widal test for Salmonella/Brucella. - ASO test.2- Indirect agglutination test: when the Ag & it’s specific Ab (incomplete or monovalent Ab) react to form invisible agglutination reaction and they become visible after addition of helping factor (i.e. Latex, bovine albumin, ..etc.). - Latex Pregnancy test. - Rapid Plasma Reagin (RPR) - Venereal Disease Research Laboratory (VDRL)

Precipitation Test: Ag in soluble form; when the Ag & specific Ab are in the optimal proportion (zone of equivalence) react to form a visible precipitate (complex) in the presence of electrolytes and suitable temperature and pH (Precipitin line)

Ag

Ab

Precipitin line

Tube Precipitin test:when the Ag and Ab are in soluble form,

and if they are specific result in forming precipitin line; i.e.:- Lance Field grouping.

Gel diffusion Precipitation test:This can be made by different techniques, in plate (with agar) or slide and electric field. 1- Single gel diffusion: Incorporate the Ab and the agar in the plates and add the Ag (soluble) in to the wells and look for precipitin line.

Ag 1

Ag 2Specific

Ab

Precipitin line

2- Double gel diffusion: (Qualitative method) Place the Ag or Ab in a well in the center of plate and add a different Abs or Ags in a different surrounding wells and look for precipitin line.

The same techniques can be made in a gel (or agar) on a test slid in the presence of electric field (Counter-immuno-electrophoresis). it can be Qualitative or Quantitative

Ag

Ab

Precipitin line

Specific Ag

Ag

Ab

Cathode-

Precipitin line

Anode+

The ICT rapid test device is a qualitative, membrane based immunoassay for the detection of Ag or Ab in a patient body fluids (Serum, Plasma, C.S.F, Urine, etc, .....).The Ag(s) is membrane coated.

The Ab From specimen Ag & Ab mixture migrates chromatographically towards the other

end of the membrane and reacts with the Ag(s) in the test line region.

Color line appear in the test line region showing a positive result. the specimen contains specific Ab(s)

CFT can use Ag particulate or soluble it is absorbed during the combination of the Ag(s) to their specific Ab(s). The ability of Ag/Ab complexes to fix complement is made use in the complement fixation test (CFT), it is capable of detecting as little as 0.04 mg of Ab nitrogen and 0.1 mg of Ag.CFT consist of five reagents:- Ag (soluble or particulate).- Ab (heat inactivate at 56 ).℃- Complement Guinea Pig.- Sheep erythrocytes (RBCs).- Hemolysin (Anti-sheep RBCs). Physiological saline (As diluent for the titration).

CFT is two steps:- Step 1: Incubate the heat inactivated patient serum with the Wasserman Ag and guinea pig complement (two units).Ag +Ab = complex +guinea pig complement (utilized) Ag +Ab = complex +guinea pig complement (non-utilized) - Step 2: Add sensitized cells (sheep erythrocytes coated with hemolysin), and incubate at 37 for 30 min, and then ℃centrifuge to check hemolysis.

Result:Lysis of RBCs = complement not fixed = No Ab = -ve result.No Lysis of RBCs = complement fixed = +ve Ab result.

The Ag detection (Direct) test:- the microassay plate coated with Ab.- Add the Ag and incubate 30-60 min.- the specific Ag will be adsorbed to the coated Ab.- Wash thoroughly.- Add the Anti-Ab conjugated with alkaline phosphatase enzyme .- The Ag will conjugate with the complex.- Wash thoroughly.- Add the substrate paranitrophenyl phosphate.- The substrate will split to form yellow colored product.- The result is read by ELIZA reader.- Control positive and negative are included.

Ab Ag Enzyme Substrate

The Ab detection (Indirect) test:- the microassay plate coated with Ag.- Add the Ab (serum) and incubate 30-60 min.- the specific Ab will be adsorbed to the coated Ag.- Wash thoroughly.- Add Anti-human globulin antibody conjugated with horse serum peroxidase enzyme .- The conjugate will attach to the anti-Ab.- Wash thoroughly.- Add the substrate.- The substrate will split to form red colored product.- The result is read by ELIZA reader.- Control positive and negative are included.

Ab Ag Enzyme Substrate

Very sensitive Avoid hazards of radioactivity To measure Ab, use “indirect method” To measure Ag, use “double Ab technique

= direct Ab sandwich method”

Are used to show the combination of Ag with it’s specific Ab (the complex) by using fluorescent dyes (or fluorochromes) e.g. Fluorescein isothiocyanate which illuminate when exposed to ultraviolet (UV) light. This technique is used for rapid identification of Ag (e.g. bacteria) e.g.:- Group A Streptococci in throat swab.- H. influenzae, N. meningitidis. S. pneumoniae in C.S.F.

Direct Immunofluorescence (Ag) test:

Ag +Fluorescent Ab UV light Fluorescence

Indirect Immunofluorescence (Ag) test:

Ag + Ab +Fluorescent Anti- UV light Fluorescence specific Globulin

Direct detection of DNA or RNA Acids in clinical specimens by nucleic acid probes◦ Detects the presence of complementary

sequence of microbial genes in clinical specimens

Polymerase chain reaction (PCR)◦ Amplification of a short sequence of target DNA or RNA

result in several copies of DNA or RNA.

Techniques:

◦ Qualitative : Detection of Ag (+ve or –ve result)

◦ Quantitative: Measure Ag quantity. Monitor the infection. Evaluate the treatment and prognosis

Advanced and very sensitive & specific diagnostic tech.

Automated temperature controlled recycling of 3 simple reactions;1- DNA denaturation (95 C)

Separation of 2 strands of DNA2- Primer annealing (40-60 C)

Each primer anneals to its complementary sequence of the 2 single DNA strands

3- Primer extension Formation of 2 complementary DNA strands