SECTION 5: RAINBOW ELISAS

Rainbow ELISA's

At Bio-Diagnostics, we have developed a range of ELISA's to aid the diagnosis of the majority of autoimmune conditions by the simple detection of specific antibodies in patient's serum.

All of our quantitative ELISA's are available in the 'Rainbow' format:

Antigen-specific colour-coded microtitre wells

Ready-to-use, colour-coded reagents and colour coded procedures that enable easy identification of each step.

Quantitative or Rapid (Qualitative) Formats:

Quantitative Assay:

5 Standards,

Positive & Negative Controls;

3 x 30 min. room temp. incubations

Results interpreted using standard curve

Rapid (Qualitative) Assay:

Calibrator,

Positive Control;

3 x 10 min. room temp. incubations

Results interpreted by simple calculation

ELISA SUBSTRATES

A number of different enzyme substrates are represented in our ELISA range. The substrate found in each kit is listed in the following sections. Of particular importance is the fact that MPO and PR3 ELISA's are available with either pNPP or BCIP substrate, a choice that has been introduced in response to customer demand. The plate reader filter required with pNPP is 405 nm. With BCIP, any filter between 595 and 650nm may be used. Many of our other ELISA's use TMB substrate, which requires a 450nm filter.

NEPHROLOGY

ANTI-NEUTROPHIL CYTOPLASMIC ANTIBODIES (ANCA)

ANCA have been shown to be good markers for systemic vasculitis. The characteristic C-ANCA and P-ANCA immunofluorescence staining patterns are caused predominantly by antibodies to the enzymes proteinase-3 (PR3) and myeloperoxidase (MPO) respectively. PR3 antibodies are generally associated with systemic vasculitis, especially Wegener's granulomatosis, whilst MPO antibodies are associated more with a localised renal vasculitis. However, the overlap is considerable, and it is therefore necessary to perform both assays when vasculitis is suspected, or needs to be ruled-out from, for example, infection or malignancy.

ANTI-GLOMERULAR

BASEMENT MEMBRANE (GBM)

The GBM ELISA is useful as an aid to the diagnosis of autoimmune renal disorders such as Goodpasture's' syndrome, in which anti-collagen (IV)-(3-chain antibodies) along with glomerulonephritis and pulmonary haemorrhage, are primary diagnostic parameters.

ENA

Antinuclear antibodies (ANA') directed against a variety of macromolecules occur in extraordinarily high frequency in systemic rheumatic diseases. Many rheumatic diseases are characterised by the presence of one or more of these antinuclear antibodies. Therefore, the identification of specific antibody is useful in the detection and the diagnosis of the disease.

SSA/Ro

Antibodies to SSA/Ro have been detected in approximately 25-30% of patients with SLE and 40-70% of patients with Sjögren's Syndrome. In addition, anti SSA/Ro antibodies have been associated with homozygous complement C2 and C4 deficiencies, subacute cutaneous lupus erythematosus (SCLE) and neonatal lupus.

SSB/La

The SSB/La antigen is a phosphorylated polypeptide that binds a large number of small RNA polyerase III transcripts and also small RNA's produced by some viruses, such as the Epstein-Barr virus, adenovirus and vesicular stomatitis virus. Evidence suggests that the SSB/La particle is either a termination factor for RNA polymerase III or is involved in post termination processing events. Anti SSB/La antibodies are nearly always found in association with Anti-SSA/Ro antibodies and occur in 15% of SLE patients. Anti SSB/La antibodies are also detected in approximately 50-60% of Sjögren's Syndrome patients and are considered a serological marker for that disease. Anti SSB/La antibodies have also been detected in patients with systemic sclerosis, neonatal lupus, false negative immunofluorescence ANA are also found in normal individuals.

SM

Antibodies to Sm, a non-histone nuclear antigen, were first described in a patient with SLE. These antibodies have been identified in the sera of approximately 25-40% of patients with SLE. Anti-Sm is both a diagnostic marker and a reliable measure of disease activity in SLE. Anti-Sm is considered to be a highly specific marker for SLE since it has not been detected in normal sera or in the sera of patients with Rheumatoid Arthritis (RA), Sjögren's Syndrome (SS), Progressive Systemic Sclerosis (PSS), Mixed Connective Tissue Disease (MCTD) or Drug Induced Lupus.

SM/RNP

Sm and RNP antigens are part of the Extractable Nuclear Antigen (ENA) profile. They are composed of small nuclear RNA (U-RNA) complexed with proteins that are extractable with physiological saline. Antibodies to Sm, a non-histone nuclear antigen, were first described in a patient with SLE. These antibodies have been identified in the sera of approximately 25-40% of patients with SLE. Anti-Sm is both a diagnostic marker and a reliable measure of disease activity in SLE. Anti-Sm is considered to be a highly specific marker for SLE since it has not been detected in normal sera or in the sera of patients with Rheumatoid Arthritis (RA), Sjögren's Syndrome (SS), Progressive Systemic Sclerosis (PSS), Mixed Connective Tissue Disease (MCTD) or Drug Induced Lupus.

J0-1

Antibodies to Jo-1, also known as histidyl-tRNA synethetase, are commonly found in a subgroup of connective disease patients with polymyositis and dermatomyositis. The Jo-1 antigen is a 50 kD molecular weight nuclear peptide that is the protein moiety of histidyl-tRNA synthetase.

Antibodies to Jo-1 are present in 18-36% of patients with polymyositis. Polymyositis has been defined as a diffuse inflammatory disorder of striated muscle that causes symmetrical weakness. Symptoms include an erythematosus skin rash, mild arthritis and progressive muscular weakness. Anti-Jo1 antibodies have also been found in patients with interstitial lung disease.

SCL-70

Antibodies to Scl-70 also known as topoisomerase 1, are found almost exclusively in patients with scleroderma. The Scl-70 antigen is a basic chromosomal non-histone protein with a molecular weight of 70 kD. Anti-Scl-70 antibodies are highly specific for scleroderma and are found in 20-70% of patients with the disease. Scleroderma has been defined as a generalised disorder of small arteries, microvessels and diffuse connective tissue, characterised by scarring (fibrosis) and vascular obliteration in the skin, gastrointestinal tract, lungs, heart and kidneys. Anti-Scl-70 antibodies have also been found in patients with CREST variant of scleroderma, and with primary Raynaud's phenomenon.

ENA - POOLS & PROFILES

POOL

Principle of the test

This ELISA test kit provides a qualitative assay for human antibodies to six different antigens simultaneously in an individual test well: nRNP, Sm, SSA, SSB, Scl-70 and Jo-1. The test kit contains microplate strips with wells coated with a pool of these six antigens. In the first reaction step, diluted patient samples, calibrators or controls are incubated in the wells. Autoantibodies to any or all of the six antigens present bind to the microplate wells. After washing to remove any unbound serum components, a peroxidase labelled anti-human IgG is added. The conjugate complexes with bound IgG class antibody. After further washing a substrate solution is added resulting in a colour reaction proportional to the concentration of antibodies.

PROFILE

Principle of the test

This ELISA is intended for the determination of human autoantibodies against all six of the ENA antigens. Test wells are incubated with diluted patient samples or with the calibration sera. In the case of positive samples, specific antibodies of the IgG class (also IgA and IgM) bind to the antigens. In a second step test, wells are incubated with a peroxidase-labelled antibody (enzyme conjugate), which is directed against human IgG. The addition of a colourless substrate solution leads to a colour production by an enzymatic reaction. The intensity of the formed colour depends on the concentration of the antibodies.

DNA

dsDNA

Anti-dsDNA is present in 50% to 70% of patients with SLE. Circulating DNA/anti-DNA immune complexes are considered to play a part in the pathogenesis of SLE. The presence of anti-dsDNA is one of the criteria for SLE. IgG antibodies to dsDNA are considered clinically most useful for the diagnosis and management of SLE. Antibodies to single stranded (ssDNA) and IgM antibodies to dsDNA are found in a number of other connective diseases and liver diseases as well as in some normal individuals. Accurate detection of anti-dsDNA is important in the diagnosis and management of SLE. EIA tests for anti-dsDNA have demonstrated greater sensitivity than standard IFA and RIA tests allowing for improved detection of low titre antibodies to dsDNA.

ssDNA

Autoantibodies to ssDNA occur in several rheumatic diseases, in certain types of cancer and in various types of other disease. The highest incidence of anti ssDNA is found in patients with Systemic Lupus Erythematosus (SLE). In the non-SLE patients the tier of anti ssDNA antibodies often predominates over the anti-dsDNA antibodies. In SLE patients found positive for ssDNA autoantibodies, the titre is used to monitor changes in disease activity and in response to drug therapy. The determination of both dsDNA and ssDNA autoantibodies is highly recommended in determining the prognosis and management of patients with various rheumatic diseases. ssDNA antibodies are not specific for SLE. However, ssDNA antibodies may be the only positive serologic finding in some SLE patients failing to demonstrate a significant ANA titre. Many of these patients possess significant photosensitive cutaneous disease. Approximately 25% of discoid lupus patients possess anti ssDNA of the IgM type, and they may be at risk of developing systemic features. In addition, the presence of anti-dsDNA plays a pathological role in the genesis of glomerulonephritis. Such studies indicate that ss-DNA antibody determinations may be an important diagnostic and even prognostic test to be employed in the evaluation of discoid lupus patient or in patients suspected of having lupus but who fail to demonstrate a significant ANA titre. Since the presence of ssDNA in the nucleus is restricted to replicating cells, and its quantity in non-replicating cells is very small, the ANA immunofluorescence assay usually fails to detect anti ssDNA.

THYROID DISORDERS

Most of the thyroid diseases are autoimmune in nature. Immune responses against specific thyroid antigens appear to play an important role in the disease pathogenesis. Antibodies to TPO (the major antigenic determinant of thyroid microsomes) may be seen in up to 90% of patients with autoimmune thyroid disease (e.g. Hashimoto's thyroiditis, Graves' disease), and also in patients with non-immune thyroid disease, including thyroid cancer. Tg antibodies are less prevalent, and may be associated with disorders such as Hashimoto's thyroiditis, Graves' disease and primary myxoedema.

Bio-Diagnostic's TPO and Tg ELISA's use the internationally recognised NIBSC reference preparations 66/387 and 65/93 respectively.

GASTROENTROLOGY

INTRINSIC FACTOR

Microplate wells are coated with highly purified intrinsic factor, purified from porcine gastric mucosa. Intrinsic factor is a glycoprotein with a molecular weight of 70kD secreted by parietal cells in the stomach. It serves as a transport and protection protein for vitamin B12. Autoantibodies against intrinsic factor are of two types. Blocking autoantibodies (type 1) hinder the binding of vitamin B12 to the intrinsic factor molecule and thus prevent the formation of intrinsic factor/vitamin B12 complexes. Binding autoantibodies (type 2) bind to other parts of the intrinsic factor or to intrinsic factor-vitamin B12 complex.

CLINICAL SIGNIFICANCE

Antibodies against intrinsic factor (IFA) are specific for pernicious anaemia (PA) but are not found in the serum of every patient with PA. Type 1 IFA occurs in 70% of PA patients. Type 2 IFA is found in 35% of PA patients but only in combination with type 1 IFA.

PA is characterised by chronic atrophic gastritis and reduced absorption of vitamin B12 in the ileum. Clinical symptoms are megaloblastic anaemia and often neurological disorders. PA is an autoimmune disease. Antibodies against intrinsic factor and against gastric parietal cells are involved in the pathogenesis. Cellular immune mechanisms also play an important role.

GLIADIN

Clinical significance of antibodies against Gliadin and Endomysium - where patients are intolerant to gluten (gluten sensitive enteropathy; Coeliac disease in small children and non-tropical sprue in adults), the ingestion of cereals containing gluten leads to damage of the mucous membranes of the small intestine. This results in atrophy in the villi and functional disturbances. The clinical picture is characterised by diarrhoea and the consequences of malabsorption (particularly weight loss and growth retardation in children). Some patients with gluten-sensitive enteropathy also suffer from Duhring's dermatitis herpetiforms, a chronic skin disease connected with the formation of blisters. This disease can also appear by itself.

The determination of antibodies against endomysium and gluten, particularly gliadin, is suitable for use in monitoring the course of therapy and for control of a gluten-free diet or a gluten-loading test. A characteristic rise or fall in the titre of antibodies against endomysium or gliadin can spare the patient a first, second or third biopsy of the small intestine. Usually both antibodies occur together, however they are not identical and may not correlate with each other in all cases.

During the acute phase of a gluten-sensitive enteropathy, gliadin antibodies detected consist mostly of the immunoglobulin classes IgA and IgG. Antibodies of the IgA class exhibit a higher specificity for gluten sensitive enteropathy than IgG class antibodies. During a course of therapy by means of a gluten-free diet, the concentration of IgA antibodies against gliadin (and against endomysium) falls to a low value within a few months whereas IgG antibodies generally persist for a longer period.

TISSUE TRANSGLUTAMINASE

This assay is for the determination of specific IgA or IgG antibodies against tissue transglutaminase in human serum, thus enabling the distinction between Coeliac patients and healthy subjects.

MITOCHONDRIAL

The determination of antibodies against mitochondria is of particular significance for the diagnosis of primary biliary liver cirrhosis (PBC). A number of AMA types have been detected in the sera of PBC patients, including antibodies against the antigens M2, M4, M8 and M9.

Antibodies against the M2 antigen are specific and sensitive diagnostic markers for primary biliary liver cirrhosis and can be detected in up to 96% of all patients. The prevalence of antibodies against M4, M8 and M9 antigens is considerably less, antibodies against M4 and M8 always occurring in association with antibodies against M2. The diagnostic relevance of antibodies against M4 and against M9 is still to be evaluated. Data obtained so far indicates that antibodies against M4 are a sign of unfavourable progress of the disease. Antibodies against M9 occur mainly in patients with primary biliary cirrhosis in its early stages (82%) in which antibodies against M2 have in some cases not (yet) been produced. In such cases, M9 antibodies are of the IgM class (greater than 90%). If antibodies against M2 can already be detected, the prevalence of M9 antibodies is only 37% of which 50% are exclusively of the IgM class. The presence of M9 antibodies appears to be an indication of a favourable progress of the disease.

CARDIOLIPIN

SCREENING

Cardiolipin ELISA's allow for the semi-quantitative determination of specific anti phospholipid antibodies found in a variety of disorders. Complete screening is possible through the use of a conjugate that recognises the three major immunoglobulin classes IgG, IgM and IgA.

ISOTYPING

Cardiolipin Isotyping allows for the quantitative determination of IgG and IgM anti cardiolipin antibodies in the sera of patients showing recurrent thrombosis, and unexplained spontaneous abortions. This kit complements the cardiolipin screening kit.

RHEUMATOID FACTOR

IgM RHEUMATOID FACTOR

Quantitative determination of rheumatoid factor IgM factors directed against human gammaglobulin. Screening for IgM RF is a basic test for diagnosing rheumatoid arthritis (RA) and reaching a prognosis for extra-articular symptoms of RA.

IgA RHEUMATOID FACTOR

Quantitative determination of IgA rheumatoid factor (IgA RF) directed against human gammaglobulin. The clinical interest of detecting IgA RF relies on an evaluation of the severity of joint destruction in rheumatoid arthritis. IgA RF is a useful marker of disease activity in RA and therefore can be considered as a prognostic indicator of rheumatoid arthritis.

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THE RAINBOW ELISA

Specific antigen coated microtitre wells and colour-coded reagents are all provided in the kit for the quantitative assessment of patient samples. The following schematic diagrams illustrate the four simple and easy to follow stages of the Quantitative Rainbow ELISA.

STAGE 1

Following equilibration of all the kit components at room temperature (18-24ºC) 100µl of each of the colour coded standards and diluted patient samples are pipetted into the antigen-coated wells. During the 30 minute incubation period specific antibodies are captured by the passively adsorbed antigen on the plate. Free, non-specific antibodies are removed by washing and tapping the wells three times after the addition of 200µl of wash buffer.

STAGE 2

The detection of the antigen bound antibodies is achieved by the addition of, and 30 minute incubation with, 100µl of anti-human horse radish peroxidase conjugate in stage 2. The conjugate is provided as a green, ready to use solution with the kit. Unbound conjugate is removed by washing four times with the wash buffer solution.

STAGE 3

The bound conjugate is then visualised by the addition of, and 30 minute incubation with, 100µl of the ready to use TMB substrate solution. During the incubation period the wells containing the antigen-antibody-conjugate complexes will develop a blue colour as the enzyme reaction takes place. The intensity of the colour is proportional to the amount of antibody, which has been captured on the antigen-coated wells. This phenomenon will be very evident in the wells containing the standards where the colour will increase from standard A to E.

STAGE 4

At the end of the incubation period the reaction is terminated by the addition of 100µl of ready to use stop solution and the wells will turn yellow. The colour is then quantified in a spectrophotometer at 450nm.

The data is processed to produce a standard curve from which the results of the patient samples can be easily calculated.