ADRENAL AUTOIMMUNITY
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| Autoimmune destruction of the adrenal cortex is the most common cause of Addison’s disease. The adrenal specific enzyme steroid 21-hydroxylase (21-OH) is a major adrenal autoantigen and autoantibodies to 21-OH are important markers of autoimmune adrenal disease. This is the case whether the disease presents as isolated Addison’s disease or as part of the autoimmune polyglandular syndromes (APS) type I or type II. |
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RSR has developed a highly sensitive and specific 21-OH Ab ELISA. ELISA plate wells are coated with 21-OH and after incubation with test sera, antibodies monovalently bound to the coated wells are detected by addition of 21-OH-biotin. The biotinylated antigen binds to the free second antigen binding sites of bound autoantibodies. Streptavidin peroxidase and the substrate TMB are then used to quantitate bound biotinylated antigen with the final absorbance reading being proportional to the amount of autoantibody in the test sample. |
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ISLET CELL AUTOIMMUNITY
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| Type 1 diabetes mellitus is characterised by islet cell autoimmunity and several different islet cell autoantibodies are important in the diagnosis and management of this disease. These include autoantibodies to glutamic acid decarboxylase (GAD), to IA-2, to ZnT8 and to insulin. A combination of these assays allows assessment of the risk of an individual developing type 1 diabetes. |
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RSR has developed a highly sensitive and specific GADAb ELISA. ELISA plate wells are coated with GAD and after incubation with test sera, antibodies monovalently bound to the coated wells are detected by addition of GAD-biotin. The biotinylated antigen binds to the free second antigen binding sites of bound autoantibodies. Streptavidin peroxidase and the substrate TMB are then used to quantitate bound biotinylated antigen with the final absorbance reading being proportional to the amount of autoantibody in the test sample. |
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RSR has developed a highly sensitive and specific IA-2 Ab ELISA using the same principle as the RSR GADAb ELISA. ELISA plate wells are coated with IA-2 and after incubation with test sera, antibodies monovalently bound to the coated wells are detected by addition of IA-2-biotin. The biotinylated antigen binds to the free second antigen binding sites of bound autoantibodies. Streptavidin peroxidase and the substrate TMB are then used to quantitate bound biotinylated antigen with the final absorbance reading being proportional to the amount of autoantibody in the test sample.
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RSR has also developed the ElisaRSR™ Fast IA-2 Ab kit. This has a shorter assay protocol than the ElisaRSR™ IA-2 Ab Version 2 assay. IA-2 Ab in test sera is allowed to interact with IA-2 coated ELISA plate wells for 2 hours at room temperature. IA-2 Ab bound to coated wells is detected through the subsequent addition of IA-2-biotin, streptavidin peroxidase and substrate TMB. The final absorbance reading is proportional to the amount of IA-2 Ab in the test sera. The assay able to be performed in approximately 4 hours without refrigeration and is particularly suitable for automated ELISA processors.
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RSR has developed in addition a highly sensitive and specific ZnT8 Ab ELISA using the same principle as the RSR GADAb ELISA and IA-2 Ab ELISA. ELISA plate wells are coated with ZnT8 and after incubation with test sera, antibodies monovalently bound to the coated wells are detected by addition of ZnT8-biotin. The biotinylated antigen binds to the free second antigen binding sites of bound autoantibodies. Streptavidin peroxidase and the substrate TMB are then used to quantitate bound biotinylated antigen with the final absorbance reading being proportional to the amount of autoantibody in the test sample.
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The ElisaRSR™ Fast ZnT8 Ab™ kit has also been developed and has a shorter assay protocol than the ElisaRSR™ ZnT8 Ab™. ZnT8 coated ELISA plates are incubated with test sera for 2 hours at room temperature. Detection of ZnT8 Ab bound to the coated wells is achieved through subsequent addition of ZnT8-Biotin, streptavidin peroxide and substrate TMB. The final absorbance is proportional to the amount of ZnT8 Ab present in test sera. The assay is able to be performed in approximately 4 hours without refrigeration and is particularly suitable for automated ELISA processors.
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RSR’s 2 Screen ICA ELISA is an earlier version of the 3 Screen ICA ELISA in which plate wells are coated with GAD and with IA-2 only.
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ElisaRSR™ Fast 2 Screen ICA™ is also available with a shorter assay protocol. The assay able to be performed in approximately 4 hours without refrigeration and is particularly suitable for automated ELISA processors.
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In addition to GADAb, IA-2 Ab and ZnT8 Ab ELISAs RSR has developed a highly sensitive and specific 3 Screen
GAD/IA-2/ZnT8 Ab ICA ELISA which detects GADAb, IA-2 Ab and/or ZnT8 Ab simultaneously. ELISA plate wells are coated with a mixture of GAD, IA-2 and ZnT8 and after incubation with test sera, antibody monovalently bound to the coated wells is detected by addition of GAD/IA-2/ZnT8-biotin. The biotinylated antigens bind to the free second antigen binding sites of the divalent autoantibodies. Streptavidin peroxidase and the substrate TMB are then used to quantitate bound biotinylated antigen with the final absorbance reading being proportional to the amount of autoantibodies in the test sample. |
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In RSR’s Insulin antibody (IAA) assay, test serum samples are incubated first with 125I-(A14)-monoiodinated insulin. This is followed by addition of anti-human IgG to precipitate any labelled Insulin-Insulin antibody complexes which have formed. After centrifugation, the precipitates are counted for 125I and the amount of radioactivity in the precipitate is proportional to the concentration of IAA in the test sample |
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NEUROIMMUNOLOGY |
| Muscle weakness in myasthenia gravis (MG) is due to the presence of autoantibodies to the acetylcholine receptor (AChR) and the presence of acetylcholine receptor autoantibodies (AChR Ab) in patient sera is diagnostic for MG. MG can also be diagnosed in canines by the presence of canine acetylcholine receptor autoantibodies (cAChR Ab). Furthermore, ganglionic acetylcholine receptor autoantibodies (gAChR Ab) have been implicated in the impaired synaptic transmission at autonomic ganglia specifically associated with Autoimmune Autonomic Ganglionopathy. Detection of autoantibodies to Muscle Specific Tyrosine Kinase (MuSK) is of considerable value in the diagnosis and management of AChR Ab seronegative MG. The presence of autoantibodies to aquaporin-4 (AQP4) is diagnostic for Neuromyelitis Optica (NMO), an immune-mediated neurological disease that affects the spinal cord and optic nerves. Lambert-Eaton myasthenic syndrome (LEMS) is a different form of MG (often associated with small cell lung cancer) in which autoantibodies are directed against P-type voltage gated calcium channels (VGCCs). Autoantibodies against N-type voltage gated calcium channels (N-VGCC) have been linked to neuromuscular dysfunction in LEMS and further central nervous system disorders including cerebellar degeneration and in paraneoplastic autoimmunity, particularly associated with small cell lung cancer. Also, measurement of voltage gated potassium channel autoantibodies (VGKC Ab) is useful in the diagnosis and management of potassium channelopathies and related neurological disorders. |
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A carefully balanced mixture of detergent solubilised foetal and adult forms of the AChR is the optimum preparation for AChR Ab assays. Consequently a mixture of these 2 receptors labelled with 125I-labelled alpha bungarotoxin provides the basis for RSR’s AChR Ab assay kit. In the assay, labelled receptors are incubated with test sera and any resulting complex of labelled receptor and receptor antibody immunoprecipitated with anti-human IgG. The higher the concentration of autoantibody, the greater the amount of radioactivity precipitated. The kit is easy to use and provides a specific and sensitive assay, being able to detect AChR Ab in about 80% of patients with myasthenia gravis. |
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RSR’s AChR Ab ELISA depends on the ability of AChR Ab in human serum to bind to similar sites on the AChR as various monoclonal antibodies such as MAb1 (coated on ELISA plate wells) and/or MAb2 and/or MAb3 (which are both labelled with biotin). In the absence of AChR Ab a complex is formed between MAb1 coated on the plate wells, the AChR and MAb2 biotin and MAb3 biotin. MAb2 and MAb3 biotin bound are then detected by addition of streptavidin peroxidase, substrate (TMB) and stop solution. In the presence of AChR Ab the formation of the MAb-1-AChR-MAb2/MAb3 biotin complex is inhibited, resulting in less streptavidin peroxidase being bound and a reduction in final absorbance at 450nm. The higher the concentration of AChR Ab in the test serum, the greater the inhibition of MAb biotin binding.
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The ElisaRSR™ Fast AChR Ab has also been developed with a shorter assay protocol. AChR in human sera interacts with MAb1 on coated wells and biotinylated MAb2 and/MAb3 and inhibits the formation of a MAb1/AChR/MAb2/MAb2 complex. The presence of the complex is quantified through addition of streptavidin peroxidase and substrate TMB. Presence of AChR Ab in test sera inhibits the formation of the complex and so the final absorbance is negatively proportional to the AChR Ab concentration in test sera. The assay is able to be performed in approximately 5.5 hours without refrigeration and is particularly suitable for automated ELISA processors.
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RSR’s AQP4 Ab ELISA depends on the ability of AQP4 antibodies in human serum to bind to AQP4 coated onto ELISA plate wells. Bound antibodies are detected by adding biotinylated AQP4 which, due to the divalent nature of antibodies can interact with bound AQP4 antibodies. The amount of biotinylated AQP4 bound is then determined by addition of streptavidin peroxidase, substrate (TMB) and stop solution. The higher the concentration of antibody in the test serum, the higher the absorbance. The kit is easy to use and provides a specific and sensitive assay, being able to detect AQP4 Ab in about 80% of patients with neuromyelitis optica (NMO) or NMO spectrum disorders. |
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RiaRSR™ Canine AChR Ab (25 Tubes)
RSR’s cAChR Ab RIA depends on the use of recombinant cAChR complexed with 125I-labelled alpha Bungarotoxin. 125I-labelled cAChR are incubated with test canine sera and the resulting complexes of cAChR Ab and radiolabelled cAChR are immunoprecipitated with an anti-IgG antibody. The greater the concentration of autoantibody, the greater the amount of radioactivity precipitated. The kit provides a sensitive assay, being able to detect cAChR Ab in most canine MG patients. |
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RiaRSR™ gAChR Ab (25 Tubes)
RSR’s gAChR Ab RIA depends on the use of recombinant gAChR complexed with 125I-labelled epibatidine. 125I-labelled gAChR are incubated with test sera and the resulting complexes of gAChR Ab and radiolabelled gAChR are immunoprecipitated with an anti-human IgG. The greater the concentration of autoantibody, the greater the amount of radioactivity precipitated. The kit provides a specific and accurate assay, with 100% of healthy donors and 96.7% of patients with autoimmune disease other than autoimmune autonomic ganglionopathy negative for gAChR Ab. |
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RSR’s MuSK Ab RIA depends on the ability of MuSK Ab in human sera to bind to 125I-labelled MuSK. In the assay, labelled MuSK is incubated with test sera and any resulting complexes of MuSK Ab and radiolabelled MuSK are immunoprecipitated with anti-human IgG. The higher the concentration of autoantibody, the greater the amount of radioactivity precipitated. The kit is easy to use and provides a specific and sensitive assay for detecting MuSK Ab. |
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RiaRSR™ N-VGCC Ab (25 Tubes)
In RSR’s N-VGCC Ab RIA, N-VGCC Ab in patient sera and controls are allowed to interact with detergent solubilised N-VGCC extracted from rabbit brain tissue and complexed with 125I-labelled ω-conotoxin GVIA. 125I-labelled N-VGCC are incubated with test sera and the resulting antigen-antibody complexes are immunoprecipitated with an anti-human IgG. The greater the concentration of autoantibody, the greater the amount of radioactivity precipitated. The kit provides a specific and accurate assay, with 98.3% of healthy donors and 100% of patients with autoimmune disease other than Lambert Eaton Myasthenic Syndrome negative for N-VGCC Ab. |
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The assay depends on the use of detergent solubilised P-type VGCCs extracted from rabbit cerebellum and labelled with 125I-labelled w-conotoxin MVIIC. The 125I-labelled P-type VGCCs are then incubated with test sera and the resulting complexes immunoprecipitated with anti-human IgG. The higher the concentration of autoantibody, the greater the amount of radioactivity precipitated. Non-specific binding in the assay is determined using a preparation of VGCCs supplied in the kit which have been labelled with 125I-conotoxin in the presence of an excess of unlabelled conotoxin. |
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The assay depends on the use of detergent solubilised voltage gated potassium channels (VGKC) extracted from rabbit brain and labelled with 125I-labelled dendrotoxin. The 125I-labelled VGKCs are then incubated with test sera and the resulting complexes immunoprecipitated with anti-human IgG. The higher the concentration of autoantibody, the greater the amount of radioactivity precipitated. |
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TSH RECEPTOR AUTOIMMUNITY |
TSH receptor autoantibodies (TRAb) - are responsible for a common form of thyroid overactivity known as Graves’ disease and in this condition the antibodies bind to the receptor for thyroid stimulating hormone (TSH) and mimic the effects of TSH causing hyperthyroidism. Clinically, the measurement of TRAb is important to distinguish Graves’ disease from other forms of thyroid dysfunction such as toxic nodular goitre and to monitor treatment of Graves’ disease. TRAb measurement is also indicated for pregnant patients with a history of thyroid disease in order to assess the risk of the neonate developing thyroid disease.
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TSH receptor autoantibodies in patients’ sera are allowed to interact with TSH receptor coated onto plastic tubes. Bound TRAb are detected by their ability to inhibit the binding of 125I-labelled TSH to the receptor coated tubes and TRAb levels are read off a standard curve or expressed as an inhibition index. |
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TRAb ELISA Kit (96 wells)
TSH receptor autoantibodies in patients’ sera are allowed to interact with TSH receptor coated onto ELISA plate wells. Bound TRAb are detected by their ability to inhibit the binding of TSH (in the form of TSH-biotin) to the receptor coated wells. The amount of TSH-biotin bound is then monitored by addition of streptavidin peroxidase and the peroxidase substrate tetramethyl benzidine. TRAb levels are expressed as an inhibition of TSH binding index or read off a standard curve. A newer version of the TRAb ELISA uses competition between patient sera TRAb and a thyroid stimulating human monoclonal antibody (M22) for TSH receptor coated onto ELISA plate wells. This third generation TRAb ELISA displays excellent sensitivity. Another version of the TRAb ELISA assay is RSR’s Fast TRAb ELISA. In this assay M22 is coupled directly to peroxidase allowing fewer steps and shorter assay time.
ElisaRSR™ TRAb Fast™
Technical information
ElisaRSR™ TRAb 2nd Generation
Technical information
ElisaRSR™ TRAb 3rd Generation
Technical information
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TRAb SPEC™ Test (10 tubes)
TRAb SPEC™ is designed to be used in conjunction with a TRAb assay to check the assay specificity. TRAb in patients’ sera are allowed to interact with a purified, concentrated stable TSHR fragment (TSHR 260) coated onto plastic tubes. The concentration of TRAb in a test sample is reduced after incubation in a TRAb SPEC™ tube confirming the specificity of the TRAb result or otherwise.
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