Up dating on different Serological Assays

Dr.Tariq Mustafa Mohamed AliAl Ain Veterinary Laboratory

Animal Health section,Agriculture sectorDepartment of Municipalities and Agriculture

Serological assays

Antigen (Ag) + Antibody(AB)

Antigen (Ag):

A molecule which elicits a specific immune response when introduced into an animal.

1. Generally large molecules (>10,000 daltons in molecular weight).

2. Structurally complex (proteins are usually very antigenic).

3. Accessible to the immune system .4. Foreign (not recognizable as "self").

Gram-Negative Cell Wall

Examples of bacterial Antigen

Antigenic structure of BrucellaA- Surface antigen (OM)

Lipopolysaccharide (LPS) It is either S-LPS or R-LPSConstitute the major Ag interact in SAT, CFT, RB , MRT

and represents the A &M epitops OMP consists of 2 Ag

1. NH 25-27 K ( Not existed in the Rough strain)2. Poly B 36 – 37 K ( Not existed in the Rough strain)3. NA 31 K ( Existed in B. melitensis )

B- Internal Antigens free from LPS More than 20 protenious Ag(NH and Poly B and A2 ( one of internal Ags) used for

differentiation between Vaccinated and infected animals ).

Antigenic structure of Brucella

Examples for virus antigens

Foot and mouth disease viral antigens

146 S (VP1,2,3 & 4). 75 S ( VP1, 2 &VP0) 12 S ( Vp 1, 2 &3)

Antibody (Ab): A glycoprotein produced in response to an

antigen that is specific for the antigen and binds to it via non-covalent interactions.

The term “Immunoglobulin" is often used interchangeably with "antibody".

Immunoglobulins (Ig) come in different forms (IgA, IgD, IgE, IgG, IgM) that reflect their structure.

Antibody molecule

Antibody molecule

Main classes of antibodies ( IgG, IgA, IgD, IgE, and IgM).

Monoclonal antibodies Typically made by fusing myeloma cells with

the spleen cells from a mouse that has been immunized with the desired antigen to produce what is called Hybridoma cell

Mono-clonal antibody (Mab)

Antigen / Antibody Reactions (A) The hinge region of an

antibody molecule opens and closes to allow better binding between the antibody and antigenic determinants on the surface of an antigen. (B) Hinge flexibility also facilitates the cross-linking of antigens into large antigen-antibody complexes.

Requirements of valid diagnostic assays :

Selection of optimal reagent concentration and protocols parameters

Use the test of optimal Sensitivity and Specificity to the target disease.

Use the test of good Repeatability and Reproducibility

Applied in the Reference animal population Use the test of high prediction or efficiency

Sensitivity of Diagnostic methods

It is the proportion of True positive (TP) that are detected by this method

Increase sensitivity accompanied with a proportion of false positive (FP) .

A test with 90% sensitivity implies 10% will be false negative.

Sensitivity = TP/ (TP+FN) X 100

Specificity of Diagnostic methods

It is the proportion of true negative (TN) that are detected by this method

Increase specificity accompanied with a proportion of false negative ( FN ).

A test with 90% specificity implies 10% will be false positive .

Specificity = TN/ (TN+FP) X 100

Factors yielded false results

Cross reaction with similar epitops Presence of non specific inhibitors in the tested

serum. Some animals show natural or induced tolerance

(BVD). Non specific inhibitors (Anti complementary serum). Kind of AB ( Excess IgG 1 block IgG 2 in brucella

testing. Using unsuitable test (Incomplete antibody). Improper timing for testing the animals (cows for

brucellosis by CFT before abortion might give FN)

Diagnostic objectives of serological tests . Detection of Target Antigen. Study the relation between the field

isolate and reference isolates. Detection of rising antibody titers

between acute and convalescent stage of infection.

Antibody response to infection and reinfection

Serological assays

It is either Qualitative Quantitative test ( Dilution takes place to the unknown

object )

Agglutination tests Card or plate Agglutination Rose Bengal test (RB). Serum Agglutination test (SAT). Micro-agglutination (MAT) Latex Agglutination (LA)

Principals of Agglutination tests The antigen is reacted with the antibody

inducing (clumping) of the antigen.

Application of agglutination tests

Typing of different isolates as in case of E.coli ,salmonella, Pasteurella spp.etc..

Diagnosis of Brucellosis : Rose Bengal test . Milk ring test. SAT Micro agglutination test

Principals of Latex Agglutination (LA) LA tests are similar in principle to bacterial

agglutination . Latex particles could be coated with antibody

or antigen and will agglutinate when mixed with the corresponding antigen or antibody.

Used for Identification of many (Streptococcus ,salmonella typing and toxoplasmosis)

Disadvantages of quantitative agglutination tests Prozone Phenomena :

Lack of agglutination at high concentrations of antibodies due to excess antibody yielded a very small complexes that do not clump to form visible agglutination.

Agglutination but use erythrocytes as targets or carrier

Indirect Hemagglutination (IHA)( Coomb test).

Hemagglutination inhibition tests (HI).

Passive hemagglutination (PHA)

Principals of Hemagglutination

Detect antibody in the mare colostrum in Equine infectious anemia by Coomb’s test (Antiglobulin test)

Principals of Hamagglutination Inhibition Test

It measures the ability of antibodiesto inhibit the hem-agglutinating

activity of fixed amount of virus

In the absence of anti-virus antibodies

Erythrocytes

VirusVirus agglutination of erythrocytes

Heamagglutination Inhibition

Erythrocytes

Virus

Anti-virus antibodies

Viruses unable to bind to

the erythrocytes

Pattern of HI test

Criteria of HI

Diagnosis of haemagglutining viruses such as Influenza ,Newcastle disease etc .

Evaluating the immune response of vaccinated animals or birds.

It is serotype specific

Principals of Passive Hemagglutination (PHA) Passive hemagglutination is a classical

immunological test in which antigen is linked chemically to preserved red blood cells (RBC) using tannic acid, glutraldehyde or CRCl3 .

The labeled cells are then used to detect the appropriate antibody in a simple hemagglutination test.

End point

Serum

The results are reported as the reciprocal of the maximal dilution that gives 50% visible agglutination

Precipitation tests

Agar gel immunodiffusion tests “Double immunodiffusion” (AGID).

Single radial immunodiffusion (SRID) Immunoelectrophoresis (IE) Countercurrent immunoelectrophoresis

(CCIE) Immunochromatography

Double AGID

Principals of Precipitation tests

The serum antibody and the antigen preparations are placed in holes punched into into agar or agarose .

The antigen as well as Antibodies diffuses into the gel, and at the equivalence zone they form a precipitation line.

Principals of Radial Immunodiffusion(Mancini) (SRID) In SRID assay the antibody is incorporated

into the agar gel as it is poured and different dilutions of the antigen are placed in holes punched into the agar to generate a standard curve.

As the antigen diffuses into the gel, it reacts with the antibody and at the equivalence point a ring of precipitation is formed.

Single radial immunodiffusion (SRID)

Principals of Immunoelectrophoresis In immunoelectrophoresis, both antigen

preparation and antibody are placed in a well punched out of an agar gel and exposed to weak electric current for a period of time .

As the Ag and Ab diffuse into the agar a precipitin lines are produced at the equivalence zone when an antigen/antibody reaction occurs

Countercurrent electrophoresis CIE

The antigen preparation into the wells situated on the cathode side of each pair Antigens flow towards the anode (+).

Countercurrent electrophoresis CIE

Using labeled antibodies

A.Radio immune assayB.ImmunofluorescenceC.Enzyme linked immunosorbent assays (ELISA)

Principals of RADIOIMMUNOASSAY (RIA) test Radioactively labeled antibody competes with the

animal’s unlabeled antibody for binding sites on a known amount of antigen.

A reduction in radioactivity of the antigen-animal antibody complex compared with the radioactive counts measured in the control test with no anti- body is used to quantitate the amount of serum antibody bound to the antigen.

Application of RADIOIMMUNOASSAY (RIA) Radioimmunoassay (RIA) is primarily used to

measure antigens (notably certain hormones or proteins) in serum samples

This automated method of detecting antigens is usually performed in a chemistry laboratory.

Of limited application in Veterinary diagnostic laboratory

Principals of Immunofluorescence (IF) test

Immunofluorescence (IF) depends on the coupling of intracellular viral or bacterial antigens with fluorescein isothiocyanate (FITC) conjugated specific antibodies .

The reaction is detected by expose the reaction site to ultraviolet (UV) or blue light. Yielding apple green fluorescence

Immunofluorescense

Principal steps in direct immunofluorescence

Principal steps in indirect immunofluorescence (IIF)

2 - Principals of Enzyme-Linked Immuno Sorbent Assay (ELISA). In a simple terms, antigen is fixed to the solid

plate surface, and then the serum in question is added .

The mixture is washed away followed by addition of anti-species Ab conjugated with an enzyme .

In the final step a substance is added that the enzyme can convert to some detectable signal.

ELISA steps

Direct ELISA

ELISA for Antigen or antibody

Microplate ELISA for HIV antibody: coloured wells indicate reactivity

Indirect ELISA (i ELISA) :

Plate is coated with captured antibody Sample is added, and any antigen present binds to the

captured antibody then washed GP HIS is added, and binds to antigen if exists . Conjugated Rabbit anti GP Ig is followed and binds to

detecting antibody The substrate is added and is converted by enzyme to

detectable form.

i ELISA

Competitive ELISA (cELISA) for detection of RP antibodies. ELISA plates coated with RP Ag. Add tested serum samples and RP Mab in blocking

buffer then Incubate plates with continuous shaking for 1 H at 37oC

Add HRPO conjugated Anti mouse Ab followed by incubation for 1 H at 37oC.

Add the substrate/chromogen and allow colour to develop for ten minutes.

Stop colour development Read plates on ELISA reader at an absorbance of

492 nm.

Standard tests

Complement Fixation test (CFT). Serum neutralization tests (SNT). Protective tests (PD50)

Principals of Complement Fixation Test (CFT) The test consists of 2 Ag/AB reactions. One of them

is the target Ag or serum against the corresponding refrence Serum or Ag .

The 2 nd Ag/Ab called hemolytic system (SRBCs + Rabbit antisheep RbCs “hemolysine”) .

The complement is activated by Ag-Ab complex. If C’ consumed in the first reaction . The HS is not

affected and the HS not been lysed. And vise is versa.

Principals of Complement Fixation Test (CFT)

Criteria of CFT Tested serum should be inactivated at 56°C for 30

minutes. Complement and haemolysin should be titrated first. All Reagents used in the test should be tested for

QC(C’C, RBCs C, Haemolysin C, Serum C) 100 % and 0% lysis) should be included in the test. Complement and haemolysin titration should precede

the test proper. CFT could be done slow or rapid test ICFT could be applied.

Complement (C’) titration

Each C’ dil 0% 100%

VB 0.1 0.2 0.1

Tested Ag or Serum 0.1 0.1 0.1

C’ dilution 0.1 0 0.1 (1/10)

HS 0.2 0.2 0.2

Each Ag dil Ag C S C C’ C HS C

VB 0 0.1 0.2 0.2 0.3Tested

Ag 0.1

HIS (1/10) 0.1 0.1

C’ (2-4Unit) 0.1 0.1 0.1 0.1(1/10)

Incubate at 37 oC for 30 mHS

0.2 0.2 0.2 0.2 0.2

Incubate at 37 oC for 15 m

What does a CFT look like?

Complement Fixation Test in Microtiter Plate. Rows 1 and 2 exhibit complement fixation obtained with acute and convalescent phase serum specimens, respectively. (2-fold serum dilutions were used) The observed 4-fold increase is significant and indicates recent infection.

CFT

Principle of Serum Neutralization (SNT) It demonstrate the amount of antibodies

necessary to inhibit the activity of known amount of virus particles(10 2) .

If it done in tissue culture (TC) tube , the cytopathic effect (CPE) was recorded.

If it done in embryonated eggs death is recorded .

SNT assay The test is done using test tubes, microtiter plate ,

2 oz bottle, embryonated eggs , Mice etc… The target serum is exposed to 4-fold dilutions

followed by the addition of an equal volume of virus suspension diluted to contain approximately 100 ID50 .Following an incubation of serum + virus 1 - 2 hours at room temperature .Inoculate the mixture of each dilution into odd number of target host .Incubated at 37°C, and observed daily for development of viral CPE.

Cytopathic effect on TC

Criteria of SNT The end point is the reciprocal serum dilution

yielded 50% CPE in case of tissue culture. This test is considered the most reliable of all

serologic procedures, being less prone to variation and less subjective in its interpretation.

SNT tests are almost always performed using cell cultures.

Principle of Protection Tests It has the same principal of SNT but in vivo. The target host is immunized with several

Vaccine dilution followed by challenge with constant virus concentration at 3 weeks post vaccination.

Protection tests are used for Vaccine Evaluation An example of a protection test once used is that

for FMD vaccine evaluation.

Calculation of PD50

Vaccine dilution No. +ve /Total No. of animals Positive%

Undiluted 1/5 20%1/4 2/5 40%1/16 3/5 60%1/64 4/5 80%

1/256 5/5 100%m =xk +1/2d- d (-Spi /100)

Immune Electron Microscopy

Classical Immune electron microscopy (IEM) - the sample is treated with specific anti-sera before being put up for EM. Viral particles present will be agglutinated and thus congregate together by the antibody.

Solid phase immune electron microscopy (SPIEM) - the grid is coated with specific anti-sera. Virus particles present in the sample will be absorbed onto the grid by the antibody.

Uses of Immune Electron Microscopy

At least 106 virus particles per ml required for visualization,The magnification power should be 50,000 - 60,000 XViruses may be detected in the following specimens.

Feces Rotavirus, and corona virus & Adenovirus, Calicivirus , Parvoviruses

Skin scrapings Papillomavirus, ORF , Pox

Problems with Electron Microscopy Expensive equipment .

Expensive maintenance .

Require experienced observer .

Sensitivity is often low .

Fluorescence polarization assay (FPA) The assay works on the principle that

molecules in solution randomly rotate at a rate inversely proportional to their size.

A small molecule labeled with a fluorochrome will depolarize plane polarized light at a more rapid rate than a large molecule such as Ag-Ab complex.

Fluorescence polarization assay (FPA) Fluorescence polarization provides a direct readout of the

extent of FITC conjugated Ag binding to serum AB. In positive cases ; the FITC conjugated Ag bind to the Ab

molecules leads to formation of large , slowly rotating molecules and high fluorescence polarization.

In negative cases ; When no reaction takes place with FITC conjugated antigen, the FITC conjugated Ag remain as small, rapidly rotating molecules resulting in low fluorescence polarization.

Fluorescence polarization assay (FPA)

Western blotting

Western blotting is based on the principles of immunochromatography where proteins were separated into poly acrylamide gel according to the isoelectric point and molecular weight.

Immunoblotting is performed chiefly in diagnostic laboratories to identify the desirable protein antigens in complex mixtures.

Criteria of Western blotting

The separated protein bands were detected by :1. Colorimetric detection or2. Chemiluminescent detection or3. Radioactive detection or 4. Fluorescent detection

Western Blot

Western Blot Lane1: Positive Control Lane 2: Negative Control Sample A: Negative Sample B: Indeterminate Sample C: Positive

Immunoblotting

Viral antigens are detected with a polyclonal or a MAb onto nitrocellulose paper.

After incubation, the protein bands (immune complexes) are visualised with peroxidase-conjugated protein and a colour reagent.

A colour develops in the bands where antibody binds to the antigen.

Immunochromatography Test principle.

Antibodies were attached to two different zones on a nitrocellulose membrane .

Purified monoclonal antibody against nucleoprotein (anti-N Mab) that had been identified as IgG2a subtype by using Mab isotyping kit was attached to the test zone at the concentration of 0.75 μg/strip, and purified goat anti-mouse IgG (was attached to the control zone at the concentration of 0.70 μg/strip.

The 30 nm colloidal gold conjugated antibodies were dried on the glass fiber

The test strip was assembled in the following order (Sample pad , gold pad, nitrocellulose paper, and absorption pad (cellulose paper).

All pads are overlapped to enable migration of sample

Immunochromatography

Diagram of the test strip for the detection of anti-canine parvovirus antibody. Serum is added to the sample pad where serum antibodies can interact with CPV. Addition of buffer enables the complex to migrate along the test strip where gold-conjugated antibodies are captured by the immobilized anti-porcine or anti-canine IgG.

Quantitative application of the test

The intensity of the color developed at the T line (T) correlated with the HI assay-determined titer of the reference serum sample shown on each test strip

Diagram of the test strip for the detection of Rabies virus

Saliva is added to the sample pad.

sample pad► gold pad Mab against nucleoprotein ► go through nitrocellulose paper to test line (Rabies Mab) ► Control line ( goat anti-mouse) and absorption pad (cellulose paper).

All pads overlapped to enable migration of sample

DETECTION OF NUCLEIC ACIDS GENOME Polymerase Chain Reaction (PCR) Reverse transcryptase Polymerase Chain Reaction

(RT-PCR). Restriction Fragment length polymorphism

(RFLP). Nucleic acid hybridization (DNA probe) . Microarray Analysis DNA sequencing.

Polymerase Chain Reaction (PCR) The amplification of DNA by the PCR is

accomplished via a succession of incubation steps at different temperatures.

PCR can amplify copies of a small region of 100-400 or more base pairs into millions of copies.

Basic steps of PCR Separation of DNA strands (Denaturation) By

heating to 95 oC . Activate primer’s links (Annealing) By heating

to 55 oC . Activate polymerase activity in 3’ to 5’

direction (Extension) By heating to 72 oC, to synthesis new strand .

Chemical structure of nucleotide

Base bare system

NA Polymerization

Identification of PCR products

Identification of PCR products

Criteria of PCR PCR is a highly sensitive procedure for detecting

infectious agents in host tissues and vectors, even when a small number of host cells are infected

PCR is very useful in the diagnosis of chronic-persistent infections such as :

Bovine leukemia virus Caprine arthritis/encephalitis virus, etc.)

Latent infections by IBR PCR could be used for testing vaccines contamination. It does not differentiate between viable and nonviable

organisms.

Criteria of PCR Extremely liable to contamination Require high degree of operator skill Not easy to set up a quantitative assay. A positive result may be difficult to interpret,

especially with latent viruses such as CMV, where any seropositive person will have virus present in their blood irrespective whether they have disease or not.

Restriction fragment length polymorphisms (RFLP) Detect differences in the genomes of closely related

microbial species DNA is extracted and clipped into fragments of specific

nucleotide sequences with restriction endonucleases. The resultant DNA fragments are then separated in

agarose gel by electrophoresis and visualised with ethidium bromide.

The fragments can then be hybridised with complementary DNA (cDNA) tagged with 32P to determine the differences or similarities in the genomes.

Criteria of FRLP

Uses : Used to detect differences in the genomes of

closely related microbial species or compare field isolates of a given virus.

A distinct limitation of the method is that the presence of a mutation cannot be detected unless that mutation happens to fall within the recognition sequence of the restriction endonuclease being used for digestion of the DNA.

Solid-phase of NA hybridization assays

Principals of the test The double-stranded nucleic acid of a virus is

denatured with alkali to separate strands. The single strands of nucleic acid are attached to

a solid support, usually a nylon or nitrocellulose membrane, to prevent the strands from reannealing.

The nucleic acid attaches to the membrane by its sugar-phosphate backbone; the nitrogenous bases are thus projecting outward.

Nucleic Acid probe A probe (single-stranded DNA or RNA molecule of

known origin - containing the nucleotide sequence specific to that of the target virus - labeled with a radioactive atom or enzyme) is added to the membrane.

Formation of hydrogen bonds occurs between the complementary bases.

Unreacted probe is removed by washing and hybridization is detected by an assay for the probe.

Southern hybridization for detection of DNA fragments.

Northern hybridization for RNA detection

Dot blot hybridization.

The nucleic acid is placed onto nitrocellulose in an apparatus that focuses the individual spots into concentrated areas, similar to a microtiter plate

Application of Microarray Analysis:

The development of microarrays has been fueled by the application of robotic technology to routine molecular biology, rather than by any fundamental breakthrough.

It used for Identification of specific viruses or specific viral sequences as many as included in the test.

DNA microarray

(gene chip, DNA chip or biochip) is a collection of DNA probes attached to a solid surface, such as glass, plastic or silicon chip forming an array. Sample DNA or RNA is extracted, RNA is reverse transcribed to cDNA and the DNA or cDNA is labelled with fluorescent labels. The labelled DNA is denatured and hybridized with the probes on the array. Unbound probes are washed away and the array is visualized using confocal laser microscope scanner

Microarray Analysis