Immunological and serological tests for diagnosis of Diseases

 

Immunological and serological tests for diagnosis of Diseases

Preface:

Immunological and serological tests are the laboratory methods for diagnosis of various infectious diseases and immunological disorders. These tests are based on the principle of antigen antibody reactions.

Principle:

Any antigen reacts only with its specific homologous antibody. This antigen antibody reaction will result in certain observable effects, which can be demonstrated in the laboratory & thus be used for diagnostic purpose.

Role of serological tests in infectious diseases:

            1. For diagnosis of infectious diseases:

Certain infectious diseases are very difficult to diagnose by microscopic examination or culture methods. For example,

a. Viral infections: These agents are too small to be demonstrated under routine laboratory microscopes and also very difficult to culture. Hence, a diagnostic void is created which is filled up by serology.

b. Occult infections: The infecting agent is present at such a site in the body which cannot be accessed for sample collection; or the local site of infection remains unidentified. In such cases, indirect evidences in the form of serological reactions come in handy.

c. All the infections where the infecting agent cannot be recovered from the clinical samples; and we need laboratory evidence of infection, especially in cases with highly suspicious clinical background, wherein to stamp upon a disease, a diagnostic laboratory report is needed.

            For diagnosis of such diseases, we may take indirect evidence of infections,

- Microbial infection will induce antibody production. Such antibodies are present in patient's serum. To detect these antibodies, patient's serum is allowed to react with laboratory reagent containing commercially (in vitro) prepared antigens of the microorganism belonging to the same species.

- Similarly, microbial antigen present in patient's serum may be detected with the use of its homologous antibodies present in the laboratory reagent, prepared commercially.

 

            2. Detection of a particular antibody titer in patient's serum

Titer: Antibody titer of serum is the highest dilution of the serum which shows observable reaction when reacted in vitro with its specific antigen.

 

Learning objectives:

1. Various serological tests, their principle and clinical applications.

2. The devices used for serological reactions.

3. The parameters of serological tests which measure the efficiency limitations of the test.

 

Parameters of a serological test.

All the serological tests used in the laboratory have certain limitations. So, the results of serological tests should always be interpreted with consideration of patient's clinical background and efficiency of that specific serological test kit with which the test is performed. The efficiency of a test kit is defined with the help of the following parameters.

1. Sensitivity: Sensitivity is the ability of a test to detect the most  minimum amount of antigen or antibody  present in the serum.

            If a test has higher sensitivity,

- We can also diagnose those diseased persons who have a very low antigen concentration or antibody titer.

            - We can get positive results in maximum number of diseased persons.

- If the test is negative, we can be sure that the person would not have had that particular disease, i.e., there are almost no chances of getting a false negative result.

            If a test has lower sensitivity

- It will give false negative results in patients having low antigen concentrations or low antibody titers even if the patient suffers from the disease, i.e., there are chances of getting a false negative result.

2. Specificity: Specificity of a test is its ability to identify the diseased person accurately.

            If a test has higher specificity:

- It will give positive results only for those patients who are actually diseased with that specific infection for which the test is performed.

- When the test is positive, we can be sure that the person would be having that particular disease.

            If a test has lower specificity:

- It may give false positive results, even though the patient may not be having that particular disease.

3. Positive predictive value:

- Positive predictive value signifies the ability of a test to detect true positives.  It will be higher in cases of tests having more specificity and hence, there will be lesser chances of the test giving false positive results.

4. Negative predictive value:

- Negative predictive value signifies the ability of a test to detect true negatives. It will be higher in cases of tests with more sensitivity and hence, there will be lesser chances of the test giving false negative results.

 

Types of serological reactions ( Antigen - antibody reactions )

1. Precipitation:

            When a soluble antigen combines with its specific antibody in the presence of electrolytes, at optimum temperature and pH, it forms an insoluble precipitate, this reaction is called precipitation.

Flocculation:

If in precipitation reaction, the precipitates remain suspended as floccules, the reaction is known as flocculation.

Zone phenomenon in precipitation reaction:

Precipitation reaction in a medium takes place only in the zone of equivalence i.e. where antigens and antibodies are in optimum concentration.

If the antibodies are in excess in a reaction, precipitates will not be formed and test result would be false negative. This is called pro-zone phenomenon.

Different types of precipitation test

            A. Slide precipitation / flocculation test

            e.g. VDRL (Venereal Disease Research Laboratory) test used for diagnosis of syphilis.

                        RPR(Rapid Plasma Reagin) is a modified VDRL test - a slide flocculation test.

            B. Tube precipitation / flocculation test

                        e.g.

                        Kahn test for syphilis.

                        For quantification and standardization of toxins and toxoids.

            C. Gel Precipitation test (Immunodiffusion)s

                        e.g. Elek’s gel precipitation test

                        This test is used to detect the toxigenicity of diphtheria bacilli.

            D. Immuno-elecrophoresis

            E. Counter immuno electrophoresis

 

2. Agglutination test:

             When a particulate (insoluble) antigen reacts with its homologous antibody in a suitable environmental condition in terms of temperature, pH & electrolytes, it  will form large, clumped aggregates visible to the naked eye. This reaction is known as agglutination.

Different types of agglutination tests:

A. Slide agglutination

A drop of suspension containing insoluble antigen(kit reagent) is allowed to react with the patient's serum on a slide. If the serum contains antibodies against that specific antigen, it will form clumps (agglutination). These clumps may be visible to the naked eye or can be demonstrated by a magnifying lens.

This test may also be used to detect antigen; wherein a suspension of antigen is allowed to react with antisera containing known antibodies.

Examples of commonly used slide agglutination tests:

            a. Blood grouping and cross matching

            b. For antigenic typing of bacteria and identification of unknown bacterial cultures:

            e.g.

            i. Lancefield typing of Streptococcus pyogenes

ii. The isolated bacteria can be identified by characterization of their antigenic structures with the help of specific antisera. For example if an isolated colony is suspected to be that of Salmonella typhi or S. paratyphi, we need to use three different antisera, containing antibodies against "H" antigen of S. typhi, S. paratyphi A and S. paratyphi B, respectively.

Test procedure: A drop of saline is placed on three different slides. The isolated bacterial colony  is picked up with a sterile wire loop and emulsified uniformly in the saline. This procedure is done on each slide. One drop each of the three antisera is mixed with this bacterial suspension on respective individual slides and mixed by rotation. The isolated bacterium is identified by formation of agglutination with its homologous antisera.

B. Tube agglutination:

            A serial dilution of the patient’s serum is mixed with the antigen in the tubes. Agglutination will appear if the concentration of antibody is optimum in the mixture. The highest dilution which shows agglutination, will be the titer of the antibody in the patient’s serum.

Examples of tube agglutination tests:

            i. Widal test - For diagnosis of typhoid fever.

            - Test kits have two types of antigens (1) Flagellar - H antigen & (2) Somatic - O antigen.

            - Serial dilution of patient's serum (1:15 to 1: 480) is prepared in two different sets of tube

            1. Conical Dreyer's tube - in this H antigen is mixed with the serial dilutions of the serum.

2. Round bottomed Felix tube - in this O antigen is mixed with the serial dilutions of the serum.

- Highest dilution of the serum which gives agglutination is considered as the titer of the widal test. A titer of more than 1:100 with O antigen and more than 1:200 with H antigen is considered positive for the diagnosis of typhoid fever. 

            ii. Weil-Felix reaction -For diagnosis of various Rickettsial diseases (typhus fever)

- This test is based on the sharing of common antigens by Rickettsiae and Proteus bacilli& hence, sera from the patients of rickettsial diseases will react with the surface antigens of the Proteus spp.

Test kits (commercially available)  have three types of surface antigens of Proteus bacilli: OX 2, OX19 & OX K.

- Patient's serum is allowed to react with each of these three antigens separately, in three different test tubes.

- Sera from the patients suffering from typhus fever and spotted fever  will agglutinate OX 2 & OX 19 antigens; while sera from the patients having scrub typhus will agglutinate OX K antigens.

            iii. Paul-Bunnell test: - For diagnosis of infectious mononucleosis

            Patient's serum is allowed to react with the sheep RBCs in a test tube. The infectious mononucleosis patient's serum contains cold agglutinins of IgM type, which agglutinate sheep RBCs. Hence, if the patient suffers from infectious mononucleosis, agglutination will be visible.

            iv. For diagnosis of Brucellosis:

            Tube agglutination method is used to detect anti brucella antibodies and its titer in patient's serum.

           

C. Passive agglutination test:

When a soluble antigen is allowed to react with its specific antibody precipitation occurs. If this soluble antigen is coated over a carrier particle, and then it is allowed to react with its specific antibody, agglutination will occur, because the carrier molecule will render the soluble antigen insoluble. Thus, by coating soluble antigens over carrier particles, the precipitation reaction can be converted into agglutination reaction. This is known as passive or indirect agglutination test.

Benefits of passive agglutination over precipitation:

            - More convenient to perform.

            - More sensitive for detection of the antibodies.

            - Better visibility of the results of the antigen-antibody reactions.

Reversed passive agglutination:

Instead of antigen, if the soluble antibody (reversed) is coated (adsorbed) over a carrier particle, and the antigen-antibody reaction allowed to occur, it is known as reversed passive agglutination. It is used for detection of antigens.

Commonly used carrier particles

Latex particles, carbon particles, RBCs, etc.

Examples of passive agglutination

            i. Latex agglutination:

a. Anti streptolysin O ( ASO): Patient's serum is mixed with a reagent containing Streptolysin O antigen coated over latex particles. Occurrence of agglutinations suggests that the patient's serum contains ASO antibodies; which in turn indicates that the patient has been infected with Group B beta haemolytic Streptococci. These antibodies cross react with human heart valve. So in suspected case of rheumatic heart disease, ASO is used as a diagnostic marker.

                        b. C- Reactive Protein ( CRP ): It is an acute phase reactant. Its level rises in various inflammatory conditions.

                        c. Rhematoid (RA) factor: It is an anti gamma globulin antibody. Its level rises in autoimmune disorders i.e. Rhematoid arthritis.

            ii. Haem-agglutination

                        a. Treponema Pallidium Haem-agglutination (TPHA) test: The test reagent contains treponemal antigens which are coated over RBCs. This is used for serological diagnosis     of syphilis.

                        b. Rose - Waaler test: Used for detection of RA factor.      

            iii. Co agglutination:

- Cell walls of certain Staphylococcus aureus strains have an affinity to the Fc portion of antibodies. These strains are used as carrier particles for coating antibodies on.

                        - Co-agglutination techniques are used for detection of various capsular antigens of capsulated bacteria from CSF, in suspected cases of pyogenic meningitis. e.g. Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae etc.

 

3. Complement Fixation Test ( CFT )

This  test consists of two systems/ steps  /principles

1. Test system

            System :Reagent 1= antigen + free complement.

Step   :    Patient's serum is allowed to react with the test reagent.(Patient system + Test system)

Principle : When an antigen-antibody reaction occurs, the so formed complex fixes any complement in the system.

Interpretation :If antibodies are present in the patient's serum, they will bind to the antigen and the antigen antibody complex so formed  will further consume(fix) the free complement in the system. This mixture will now contain no more free complement.

If patient's serum does not contain antibodies, there will not be any Ag-Ab reaction and the complement will remain free in the mixture(system).

 

2. Indicator system

            System : Reagent  2 Sheep RBCs coated with anti sheep RBC antibodies.

            Step : Mixture of step 1 + Reagent 2.

            Principle : Free complement in a system lyses RBCs.

Interpretation:

Positive (No haemolysis): If the patient's serum contains antibodies, it will consume complement during first step &complement will not be available during second step. In absence of complement, there will not be analysis of the indicator system.

Negative (haemolysis of sheep RBCs): If the patient's serum does not contain antibodies, the complement would remain free after first step. During second step, the free complement will lyse the indicator system .

 

4. Enzyme - Linked Immuno Sorbent Assay ( ELISA )

ELISA test is used to detect either antigen or antibody in serum or any body fluid.

Principle:

A commercially available ELISA kit contains

            i. A solid phase to which antigens or antibodies are already bound. The most commonly used solid phase device is microtiter plate having 96 wells.

            ii. A reagent enzyme conjugated over antibody.

In the test procedure, this enzyme conjugated reagent is mixed with the the specimen in the well of microtiter plate.

The antigen-antibody reaction is detected by addition of chromogenic substrate, with which the conjugated enzyme will react.

The intensity of colour produced will be in direct proportion to the concentration of antigen or antibody in the specimen.

In the microtiter plate, the intensity of colour change can also be detected by automation.

 

Types of ELISA:

1. Indirect ELISA:

- The microtiter well is already coated with the specific antigen to which antibody in the patient serum is to be detected.

            - The specimen (patient’s serum) is added in the well.

            - The antibody, if present in the specimen will react with the coated antigen in the well.

            - Any free antibodies are washed off with wash buffer available in the ELISA kit.

- The presence of antibodies bound to solid phase antigen is detected by addition of anti-gammaglobulin antibodies(antibody to antibody) conjugated with an enzyme. These will get attached to bound antibodies.

            - Any free labeled antibodies are again washed away.

            - A chromogenic substrate is added to the well.

            - The colour production is measured by specialized spectrophotometer (ELISA reader).

Interpretation:

            If the specimen contains antibody: The colour of substrate will change. Its intensity will be in direct proportion to the concentration of antibody in the specimen.

If the specimen does not contain antibody: No colour change occurs in the microtiter plate at the end of the test.

Uses: For detection of antibodies in cases of infection with

            - Human Immunodeficiency Virus

            - Hepatitis A virus

            - Hepatitis C virus

            - Hepatitis E virus

            - TORCH      etc.

 

2. Competitive ELISA:

            - The microtiter plate is coated with the specific antigen.

            - The ELISA kit contains conjugated antibodies which are idiotypically similar to the antibodies to be detected.

- This reagent is mixed with patient's serum and poured in the well. The sample antibodies and conjugated antibodies (present in reagent), both will compete for binding to the solid phase antigen. More the antibodies in the specimen, the conjugated antibodies will find less  opportunities for binding to the  solid phase antigens.

            - After proper incubation, the remaining antibodies are washed away from the well.

            - The concentration of conjugated antibodies will be detected by addition of the substrate.

-Increased intensity of the colour produced suggests that the conjugated antibodies got more chance to bind with the coated antigens due to less concentration of antibodies in the specimen. So, intensity of colour change will be inversely proportional to the antibody concentration in the specimen.

Use: For detection of antibodies e.g Anti Hepatitis D antibodies

 

3. Sandwich ELISA:

            - The microtiter plate is coated with specific antibodies.

- The test sample is added to the well. If the sample contains that specific antigen, it will react with the coated antibodies and get fixed.

            - The remaining unbound contents  of the system are washed off.

- Another antibody, homologous to the antigen, which is conjugated with an enzyme is added in the reaction well.

            - These conjugated antibodies will get attached to the fixed antigen in the well.

            - In the next step, substrate is added.

- The intensity of colour production will be in direct proportion to the concentration of antigen in the sample.

Use:    For detection of antigens e.g.

            - Hepatitis B surface antigen detection

            - Malarial antigen(HRP2, pLDH) detection

            - HIV viral p24 antigen detection

4.Membrane based ELISA:

In these methods, a specific antigen is immobilized at a specific place over a nitrocellulose membrane in the form of a dot or line. This membrane is fixed over an absorbent membrane, which helps to soak all the free unbound material through the membrane during the  test procedure.

- Patient's serum is pippetted over the membrane in the sample well provided  in the test device.

            - The specific antibody to be detected, if present in serum, will bind with its specific antigen  coated over the membrane.

            - The rest of the unbound material will be soaked off by the absorbent material.

            - The antibody on the membrane is detected by addition of anti Ig antibody conjugated with colloidal gold.

            -  This conjugate binds to the antigen antibody complex and forms red dot or band, which is suggestive of a positive reaction.

Advantage of the test:

Rapid, easy to interpret and it does not require sophisticated equipments.

Use: For detection of variousantibodies in patient serum e.g.

            - Anti HIV antibody

            - Anti HCV antibody

 

5. Immuno-chromatography:

This is nowadays most popularly used immunological method for detection of various antigens and antibodies from the patient serum.

Principle:

For detection of antibody:

The antigen (against which antibody is to be detected) is immobilized in a form of a line(test line) over a nitrocellulose test strip.

The lower half of the strip contains an absorbent pad in which colloidal gold conjugated to similar Ag is loosely impregnated.

The sample is added to the absorbent pad (or absorbent pad is immersed in sample).

Sample moves forwards by capillary action towards the test line.

If the sample contains the specific Ab, it will get attached to the colloidal conjugated Ag and flow forwards withinthe test strip. This Ab will then crosslink with the antigen present over the test line and give a red band there.

Test strip also contains an internal control - anti gamma globulin antibody which is coated (control line) above the test line. Formation of control band suggests validity of the test.

Uses: Detection of Anti HAV antibodies, Detection of Anti HEV antibodies.

 

For detection of antigen:

Monoclonal Ab specific against the Ag to be detected is coated over the test line.

In the absorbent pad, colloidal gold conjugated with a similar monoclonal antibody is loosely impregnated.

The Ag present in the sample will bind to the conjugated Ab and move forward and then then crosslink with the Ab coated over the test line and form the band.

Use: Detection of

            - HBsAg

            - Malarial antigens (pLDH& HRP2)

 

Both antigen and antibody detection system can be incorporated in a single device

e.g. For diagnosis of Dengue infection: Anti Dengue IgM antibody, Anti Dengue IgG antibody and NS1 antigen of Dengue virus-  all these  three parameters  can be detected in a single device.

 

6. Immuno fluorescence test:

Principle:

Fluorescent dyes when exposed to UV light, will absorb it but emit the visible light (wavelength of absorbed and emitted light is different). This emitted light can be visualized by fluorescent microscope. Rhodamine and fluorescein isothiocyanate are the most commonly used fluorescent dyes. These dyes can be conjugated with antigen or antibody to detect their homologous counterparts.

Types:

A. Direct immuno fluorescence test:

This method is used to detect antigen in the specimen.

The kit contains specific antibody conjugated with a fluorescent dye.

A smear is prepared from the specimen over a clean slide.

The fluorescein conjugated Ab is poured over the  slide. If smear contains the Ag, it will absorb the conjugated Ab over it.

After proper exposure time, the remaining material is washed off from the slide.

The smear is now examined under the fluorescent microscope.

The conjugated Ab attached to coated Ag over the slide will be visualized as being fluorescent and indicates a positive test.

B. Indirect immuno fluorescence test:

This method is used to detect antibody in the serum.

A known antigen against which antibody is  to be detected, is  coated over the slide.

Patient's serum is poured over the slide.

After appropriate incubation, the remaining serum is washed away.

On next step, the anti gamma globulin Ab conjugated with fluorescent dye is poured and allowed to react.

If the patient's serum contains the specific Ab, it will get attached to the smeared Ag, which will then absorb the conjugate over it in the next step.

On examination under microscope, this Ag-Ab-Anti Ig Ag- conjugate will fluoresce and indicate a positive result.

Use:

- For diagnosis of Syphilis

- For detection of antibodies against EB virus, influenza and para-influenza viruses

- For detection of rabies virus from corneal smear / smear prepared from post mortem brain specimen

7. Neutralization test:

Used for detection of virus (viral neutralization test); and bacterial exotoxins (toxin neutralization test)

8. Radio-immunoassay (RIA):

A radioactive agent (radioisotope) is used to detect antigen antibody reaction.  Radioactivity is measured with automated reader. This test is used for qualitative as well as quantitative analyses for the antigen in the serum or body fluids.