An ELISA is a plate-based laboratory technique making use of the binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sample. ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which will passively bind antibodies and proteins.

c-ELISA kits have to be previously validated according to EURL specifications. c-ELISA tests have to be performed according to manufacturer instructions.

 

INTRODUCTION

Antibodies also known as immunoglobulins (Ig) are gamma globulin proteins that are found in blood. These specialized immune proteins are produced following the introduction of an antigen into the body and possess the remarquable ability to combine with the antigen that triggered its production (specific affinity). The antibody recognises and bind to the antigenic determinant region of the antigen.

Enzyme-linked immunosorbent assays rely on specific antibodies to bind the target antigen, and a detection system to indicate the presence and quantity of antigen binding.

 

Brucella antigens (for review, Ducrotoy et al., 2016, DOI: 10.1016/j.vetimm.2016.02.002).

Like other gram negative bacteria, Brucella consist of cytoplasm surrounded by a cell envelope made of an inner membrane, periplasm and an outer membraine. The outer membrane contains two main classes of antigens: lipopolysaccharide (LPS) and detergent-soluble proteins (referred to as outer membrane proteins [Omp]). Structurally, the LPS can be either smooth (S) or rough (R) depending on the Brucella species. These two LPS differ in the presence in the former and absence in the latter of a long polysaccharide section, the O-polysaccharide

 

The antigens relevant to ruminant brucellosis can be classified into 2 categories: S-LPS and the related haptenic polysaccharides (O-polysaccharide linked with most core oligosaccharide sugars or core-O-polysaccharides ; the native haptens (NH) and the polysaccharide B) ; and protein antigens. The O-polysaccharide carries three basic antibody reactivities: A, M and C (Douglas and Palmer, 1988). In diagnosis, the criticial concept is the existence in the serum of an infected animal of a range of antibody reactivities (A>M, A= M and A<M) that in practice correspond to overlapping epitopes highly repeated in the O-polysaccharide (hence their colloective name as Brucella C epitope ; Weynants et al., 1997). Brucella O-polysaccharides cross-react with gram-negative bacteria that contain variable proportions of N-substituted perosamine. Among these bacteria, Yersinia enterocolitica serotype O:9 generates the strongest cross-reactivity.

 

Principle of C-ELISA (source: Laboratoryinfo.com)

This type of ELISA depends on the competitive reaction between the sample antigen and antigen bound to the wells of microtiter plate with the primary antibody.

1-First, the primary antibody is incubated with the sample. This results in the formation of Ag-Ab complex which are then added to the wells that have been coated with the same antigens.

2-After an incubation, unbound antibodies are washed off. The more antigen in the sample, more primary antibody will bind to the sample antigen. Therefore there will be smaller amount of primary antibody available to bind to the antigen coated on well.

3-Secondary antibody conjugated to an enzyme is added, followed by a substrate to elicit a chromogenic signal.

 

Concentration of color is inversely proportional to the amount of antigen present in the sample.

 

Before starting the work, read kit instruction carefully!

 

Results

The ELISA assay yields three different types of data output:

1. Quantitative: ELISA data can be interpreted in comparison to a standard curve (a serial dilution of a known, purified antigen) in order to precisely calculate the concentrations of antigen in various samples.
2. Qualitative: ELISAs can also be used to achieve a yes or no answer indicating whether a particular antigen is present in a sample, as compared to a blank well containing no antigen or an unrelated control antigen.
3. Semi-Quantitative: ELISAs can be used to compare the relative levels of antigen in assay samples, since the intensity of signal will vary directly with antigen concentration.

 

 

Serological tests for Brucellosis [relayed from OIE Terrestrial Manual]:

The buffered Brucella antigen tests (rose bengal test and buffered plate agglutination test), the complement fixation test, the enzyme-linked immunosorbent assays (ELISA) or the fluorescence polarisation assay, are suitable tests for screening of herds/flocks and individual small ruminants, camelids and bovines (cattle and buffaloes). However, no single serological test is appropriate in each animal species and all epidemiological situations, and some of these tests are not adequate for diagnosing brucellosis in pigs. Therefore, the reactivity of samples that are positive in screening tests should be assessed using an established confirmatory or complementary strategy. The indirect ELISA or milk ring test performed on bulk milk samples is effective for screening and monitoring dairy cattle.

 

Several variations of the C-ELISA, using S-LPS or O-PS as antigens, have been described for cattle, small ruminants and pigs employing different antiglobulin-enzume conjugates, substrate or chromogens and antigens prepared from different smooth Brucella strains. Nevertheless, the technique used and the interpretation of results must have been validated in accordance with OIE requirements. It has been shown that the C-ELISA eliminates some but not all False Positive Serological Reactions (FPSR) caused by cross-reacting bacteria in cattle (Muñoz et al., 2005) and swine (Praud et al., 2012). In some cases, in ruminants or pigs, FSPR may be observed in C-ELISA while not in other S-LPS-based tests, including I-ELISA. Moreover, the C-ELISA reduces but not fully eliminates the reactions caused by antibodies produces in response to vaccination.

Some protocols are less sensitive or less specific than others; therefore results obtained from different assays are not always comparable. C-ELISA for diagnosing anti-Brucella antibodies in small ruminants and swine is essentially te same as that described for cattle, but the cut-off should have been properly establhised for these species using the appropriate validation techniqes.

 

Whatever the C-ELISA format used:

i) a positive and a negative control are included in each plate. Optical Density (OD) ranges to be obtained with these two controls must be established to define the criteria for validating each plate results. The OD of the positive control is the one with which the OD of each test serum is compared to establish the final result -negative of positive).

ii) an additional positive serum (internal control) must be included in each plate to validate the repeatability of the test from plate to plate and from day to day (control chart).