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").
Antigenic structure of BrucellaA- Surface antigen (OM) Lipopolysaccharide (LPS) It is either S-LPS or R-LPS Constitute 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 ).
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.
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 diagnosticassays : 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 serologicaltests . 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.
Serological assaysIt 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 quantitativeagglutination 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 erythrocytesas 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 HamagglutinationInhibition TestIt measures the ability of antibodiesto inhibit the hem-agglutinating activity of fixed amount of virus
In the absence of anti-virusantibodies Erythrocytes Virus Virus agglutination of erythrocytes
Heamagglutination Inhibition Anti-virusErythrocytes antibodies Virus Viruses unable to bind to the erythrocytes
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 pointThe results are reported as the reciprocal of the maximal dilutionthat gives 50% visible agglutination Serum
Precipitation tests Agar gel immunodiffusion tests “Double immunodiffusion” (AGID). Single radial immunodiffusion (SRID) Immunoelectrophoresis (IE) Countercurrent immunoelectrophoresis (CCIE) Immunochromatography
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.
Principals ofImmunoelectrophoresis 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 (+).
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
Principal steps in indirectimmunofluorescence (IIF)
2 - Principals of Enzyme-LinkedImmuno 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 for Antigen or antibodyMicroplate 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.
Competitive ELISA (cELISA) fordetection 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 FixationTest (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.
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 0.1Tested Ag or 0.1 0.1 0.1 Serum C’ dilution 0.1 0 0.1 (1/10) HS 0.2 0.2 0.2
Each Ag C SC C’ C HS C Ag dil VB 0 0.1 0.2 0.2 0.3Tested Ag 0.1 HIS(1/10) 0.1 0.1C’ (2-4Unit) 0.1 0.1 0.1 0.1(1/10) Incubate at 37 oC for 30 m HS 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 complementfixation obtained with acute and convalescent phase serum specimens,respectively. (2-fold serum dilutions were used) The observed 4-fold increase issignificant and indicates recent infection.
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.
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 No. +ve /TotalVaccine dilution Positive% No. of animals 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 MicroscopyClassical Immune electron microscopy (IEM) - the sample istreated with specific anti-sera before being put up for EM.Viral particles present will be agglutinated and thus congregatetogether by the antibody.Solid phase immune electron microscopy (SPIEM) - the grid iscoated with specific anti-sera. Virus particles present in thesample will be absorbed onto the grid by the antibody.
Uses of Immune Electron MicroscopyAt 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.
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 or 2. Chemiluminescent detection or 3. 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
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 withCPV. Addition of buffer enables the complex to migrate along the test stripwhere gold-conjugated antibodies are captured by the immobilized anti-porcineor 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 thedetection 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 NUCLEICACIDS 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 .
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 lengthpolymorphisms (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 hybridizationassaysPrincipals 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 ofDNA fragments.
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