Enzyme-Linked Immuno-Sorbent Assay (ELISA)

Mechanism: The basic mechanism involved in these test utilizes absorption of antigen to a solid substrate which is placed in contact with a dilution of serum. The reaction which detects and quantifies the binding of antibody uses an antibody labeled with an enzyme followed by the addition of an appropriate substrate on which the enzyme can act to produce a colour reaction. Two distinct test mechanisms are used which we will designate "standard" and "competitive". These are described in detail under "Methods" below.

Reagents: Antigens may be produced using any of the standard techniques. They may be purified by precipitation and ultra-centrifugation or by column chromatography before being absorbed onto the surface. An alternative method which allows the use of relatively impure antigens is to bind specific antibody to the surface and allow it to absorb the antigen from the preparation. Since the latter approach usually adds an extra stage to the test this is not the preferred approach for commercial test kits. In most cases the antigen is delivered pre-absorbed onto plates, though they need to be carefully dried and packed to maintain their stability at refrigerator temperature for a reasonable period. The labeled anti-globulin used in the standard test is included in the test kits as is the appropriate substrates and stop-solutions for use with it. All of these reagents may be purchased separately from a number of sources however their titration to a standard level of activity for a specific purpose is rarely worthwhile for a commercial laboratory carrying out routine serology. Finally, and most importantly, standard negative sera and positive sera of known potency are required. These are even more important than those used in other serological tests since it is common practice to express the results of the test sera by comparing them with those of the control sera (serum-to-positive or serum-to-negative ratios). The objective of this is to remove some of the variation due to operator, environment and plate effects.

Methods:A. Standard. The antigen is absorbed on to a suitable physical surface. Usually this will be the well walls of a polystyrene micro-titre plate, although other surfaces have also been used (Rivetz et alii, 1985). A test-serum dilution (usually between 1:100 and 1:500) is made in a special diluent designed to inhibit non-specific binding of globulins to the physical surface but allowing their binding to the absorbed antigen. A range of tests can be carried out using a single serum dilution (Snyder et alii, 1984). The diluted serum is removed and the surface washed before adding an chicken anti-globulin labeled with an enzyme (usually a phosphatase). The anti-globulin will bind to any globulin attached to the antigen. Once again the excess reagent is removed and the surface washed before adding the enzyme substrate. Any attached enzyme-labeled conjugate causes a chemical change in the substrate which results in a colour change. The enzyme is inactivated after a fixed incubation time to stop the reaction, before reading the results using a colourimeter (Figure 2.7).

B. Competitive. The first part of this mechanism is very similar to the standard test in that antigen is absorbed on the solid surface and a serum dilution is applied. Serum dilutions tend to be much lower however (typically 1:2). The difference lies in the use of a standard anti-antigen conjugate to bind to any antigen sites which have not been bound to by the serum antibody as opposed to the anti-globulin in the standard test which binds to attached antibody. The addition of substrate, stop solution and reading is for the standard test. The interpretation is the opposite of the standard test in that a lack of colour development is indicative of a positive reaction.\par Statistical methods have been developed for the conversion of serum-to-positive ratios into serum titres (Chalquest, R.R., 1988, Briggs et alii, 1986)

Comments: An Elisa for Infectious Bursal Disease (IBD) has been found to be up to 25000 times more sensitive than DID/AGP (Marquardt et alii, 1980). Since 2 attachment sites are not required for the globulin to attach to the antigen this test can detect "incomplete" antibody in the same way as the Coombes test.

Figure 2.7. Micro-titre plate with a standard Elisa test seen from below. The first 2 wells at top right are the negative controls. The following 2 are positive controls. The remaining sera are field test sera. Usually at least 2 wells with a known laboratory positive control are included on each plate.

Assays: A large numbers of different assays based on Elisa technology have been developed for the detection and quantification of antibody response of poultry. A somewhat smaller number of assays are available commercially. See Table 3 for a list of assays and references.

Table 3. A list of poultry diseases for which Elisa tests have been developed for antibody detection. Most of the references deal with non-commercial tests. Commercial kit suppliers are shown in the right-hand column. The assay type is designated as Comp. for Competitive, all others are Standard (see discussion under Methods).
Assay/Disease Reference Kit Manufacturer
Chickens    
Avian Encephalomyelitis Garrett et alii, 198 IDEXX, KPL
  Smart & Grix, 198  
Avian Influenza Snyder et alii, 1985  
  Meulemans et alii, 1987  
Avian Leukosis Tsukamoto et alii, 1985 IDEXX
Avian Rhinotracheitis Chettle et alii, 1990 Cambridge Veterinary Sciences
  Eterradossi et alii, 1992 Svanova Biotech (Comp.)
    Guildhay
Bordetella avium Hopkins et alii, 1988  
Egg Drop Syndrome   Guildhay
Fowl Cholera P.multocida Briggs & Skeeles, 1984 IDEXX,KPL
  Avakian and Dick, 1985  
  Briggs et alii, 1985  
  Avakian et alii, 1986  
Fowl Pox Buscaglia, 1985  
Chick Anaemia Virus Todd et alii, 1990 Guildhay
  Goodwin et alii, 1992  
  Lamichane et alii, 1992  
Coccidiosis Carlson, 1984  
  Onaga et alii, 1986  
Infectious Bronchitis Piela et alii, 1984 IDEXX,KPL
  Snyder et alii, 1984  
  Zellen and Thorsen, 1986  
  Thayer et alii, 1987  
  Martins et alii, 1991  
  Monreal et alii, 1985  
Infectious Bursal Disease Snyder et alii, 1984 IDEXX,KPL
  Solano et alii, 1985 Guildhay
  Briggs et alii, 1986  
  Solano et alii, 1986  
  Tsukamoto et alii,1990  
Inf. Laryngotracheitis Adair et alii, 1985 KPL
Leucocytozoonosis Isobe and Suzuki, 1986  
Marek's Disease Cheng et alii, 1984  
Mycoplasma gallisepticum Piela et alii, 1984 IDEXX
  Talkington et alii, 1985 KPL
  Higgins and Whitear, 1986  
  Mohammed et alii, 1986a,  
  Yagihashi et alii, 1986  
  Avakian et alii, 1988  
  Cummins et alii, 1990  
Mycoplasma synoviae Higgins and Whitear, 1986 IDEXX (+ M.g.)
  Mohammed et alii, 1986a,b KPL
Newcastle Disease Piela et alii, 1984 IDEXX
  Snyder et alii, 1984 Guildhay
  Wilson et alii, 1984 Immuno-Comb
  Marquardt et alii, 1985 KPL
  Rivetz et alii, 1985  
  Thayer et alii, 1987a,b  
  Brown et alii, 1990  
Rotavirus Myers et alii, 1989  
Reovirus Islam & Jones, 1988 IDEXX,KPL
Reticuloendotheliosis   IDEXX
Salmonella typhimurium Hassan et alii,1990,1991,1993 Guildhay

Salmonella enteritidis

Keller et alii, 1993

Guildhay
  Cooper et alii, 1989 IDEXX (Comp.)
.......................................................... Hassan et alii, 1990....................... .......................................................
  Dadrast et alii, 1990  
  Timoney et alii, 1990  
  Nicholas & Andrews, 1991  
  Nicholas & Cullen, 1991  
S.gallinarum/pullorum Barrow et alii, 1992  
Escherichia coli Leitner et alii, 1989  
  Melamed et alii, 1991  
Turkeys    
Enterovirus Hayhow and Saif, 1993  
Haemorrhagic enteritis Ionconescu, 1984  
(Adenovirus Type II) Davidson et alii, 1985  
  Ionconescu et alii, 1985b  
  Hurk and V.d.Hurk, 1986  
Paramyxovirus type 3 Ionconescu et alii, 1985a  
Rotavirus Kang et alii, 1985  
Mycoplasma synoviae Ortiz and Kleven, 1992  
Mycoplasma iowae Jordan et alii, 1987  
Newcastle Disease   IDEXX
Pasteurella multocida   IDEXX
Other birds    
Ornithosis (Chlamydia) Ruppanner et alii, 1984  
Pasteurella anatipestifer Hatfield et alii, 1987  

Note: No endorsement is intended by citing specific manufacturers in the above list. For an up-to-date list of ucts please contact the manufacturer direct:

Cambridge Veterinary Sciences
Unit 8, Henry Crabb Road
Littleport, Ely
Cambs\tab CB16 1SE
UK
Tel +44 (0) 353 861911 Fax 860409
KPL Kirkegaard & Perry Laboratories
3 Cessna Court
Gaithersburg
Maryland 20879-4174 USA
Tel +1 301 948 7755 Fax 9480169
Guildhay Ltd
Riverside Business Centre
Walnut Tree Close
Guildford
Surrey GU1 4UG United Kingdom
Tel +44 (0) 483 573727 Fax 574828
Svanova Biotech
S-751 83 Uppsala
Sweden

T. 46 18 557055 Fax 46 18 553398

IDEXX Laboratories Ltd
Milton Court, Churchfield Road
Chalfont St Peter, Nr Gerrards Cross
SL9 9EW United Kingdom
Tel +44 (0) 753 891660 Fax 891520
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