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's assay for 21-OH autoantibodies is based on
highly purified, 125I-labelled, recombinant human 21-OH.
In the assay, test serum samples are first incubated with 125I-labelled
21-OH. This is followed by the addition of solid phase protein A
to precipitate labelled 21-OH-21-OH antibody complexes. After centrifugation,
the precipitates are counted for 125I and the amount
of radioactivity in the precipitates is proportional to the concentration
of 21-OH antibody 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 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 new 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 also developed a new 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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In addition to GADAb and IA-2 Ab ELISAs RSR has developed
a highly sensitive and specific 2 Screen GAD/IA-2 Ab ICA ELISA which
detects both GADAb and IA-2 Ab simultaneously. ELISA plate wells
are coated with a mixture of GAD and IA-2 and after incubation with
test sera, antibody monovalently bound to the coated wells is detected
by addition of GAD/IA-2-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 GAD autoantibody assay, test serum samples
are incubated, first with 125I-labelled human recombinant
GAD 65. This is followed by addition of solid phase protein A to
precipitate the labelled GAD-GAD antibody complexes. After centrifugation,
the precipitates are counted for 125I and the amount
of radioactivity in the precipitate is proportional to the concentration
of GAD antibody in the test sample. |
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In RSR's IA-2 Ab assay, test serum samples are incubated
first with 125I-labelled human recombinant IA-2. This
is followed by addition of solid phase protein A to precipitate
the labelled IA-2-IA-2 antibody complexes. After centrifugation,
the precipitates are counted for 125I and the amount
of radioactivity in the precipitate is proportional to the concentration
of IA-2 antibody 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 autoantibodies to the
acetylcholine receptor (AChR) and the presence of acetylcholine receptor
antibodies (AChR Ab) in patient sera is diagnostic for 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 voltage
gated calcium channels (VGCCs). |
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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 80-90%
of patients with myasthenia gravis. |
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The 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 (SAPOD),
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 SAPOD 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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AQUAPORIN-4 (AQP4) Ab ELISA KIT (96 wells) Technical information
The 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 (SA-POD), 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 90% of
patients with Neuromyelitis optica. |
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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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THYROID AUTOIMMUNITY |
There
are 3 different autoantibodies important in the diagnosis and management
of autoimmune thyroid disease:
a) TSH receptor autoantibodies (TRAb) - These autoantibodies
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 in an uncontrolled way. 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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TRAb Coated Tube RIA Kit (60 or 100 tubes) Technical information
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 inhibit the binding of 125I-labelled
TSH to detergent solubilised TSH receptors in a dose dependent way
and bound and free labelled TSH separated by PEG precipitation.
TRAb levels are read off a standard curve or expressed as an inhibition
index. |
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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 new 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. Furthermore another version of the TRAb ELISA assay
is now available, RSR’s Fast TRAb ELISA. In this assay M22 is coupled
directly to peroxidase allowing fewer steps and shorter assay time.
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| (b
& c) Autoantibodies to thyroid peroxidase (TPO) and to thyroglobulin
(Tg) - Graves' disease is only one
form of autoimmune thyroid disease and a similar proportion (about
1%) of the world's population suffers from thyroid under-activity
associated with thyroid autoimmunity. In this disease (a common form
of which is Hashimoto's disease), thyroid destruction is associated
with the presence of autoantibodies to two other thyroid proteins
- thyroid peroxidase (TPO) and thyroglobulin (Tg). RSR produces several
different isotopic and non-isotopic kits for measuring autoantibodies
to TPO and to Tg; they all show high sensitivity, good precision and
no cross-reactivity with other autoantibodies. |
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The TgAbCT kit uses tubes coated with Tg antibody
and the autoantibodies in patient serum samples inhibit the binding
of 125I-labelled Tg to the coated tube. The assay procedure
is simple and involves only one incubation of undiluted serum and
tracer in the coated tubes. |
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TgAb Direct RIA Kit (50, 100 or 500 tubes) Technical information
This method is based on the direct interaction between
the autoantibodies and 125I-labelled Tg and is one of
a generation of quantitative assays which have the following features:
high sensitivity, flexible design, high precision, easy handling,
high specificity, no interference from other antibodies. Studies
with different groups of patients show that the direct assays are
of greater diagnostic value than less sensitive methods.
|
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This method is based on the direct interaction between
the autoantibodies and 125I-labelled Tg, with separation
of the labelled complex using protein A coupled to a magnetic solid
phase. The assay offers a high sensitivity, specificity and precision
along with simple use and short incubation times. |
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The TPOAbCT kit uses tubes coated with an antibody
to TPO and the autoantibodies in patient serum samples inhibit the
binding of 125I-labelled TPO to the coated tube. The
assay procedure is simple and involves only one incubation of undiluted
serum and tracer in the coated tubes. |
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TPOAb Direct RIA Kit (50, 100 or 500 tubes) Technical information
This method is based on the direct interaction between
the autoantibodies and 125I-labelled TPO and one of a
generation of quantitative assays which have the following features:
high sensitivity, flexible design, high precision, easy handling,
high specificity, no interference from other antibodies. Studies
with different groups of patients show that the direct assays are
of greater diagnostic value than less sensitive methods.
|
| |
This method is based on the direct interaction between
the autoantibodies and 125I-labelled TPO, with separation
of the labelled complex using protein A coupled to a magnetic solid
phase. The assay offers a high sensitivity, specificity and precision
along with simple use and short incubation times. |
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REAGENTS - |
| RSR reagents are available to assist you with your research or manufacturing. |
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HUMAN |
Recombinant Human Thyroid Peroxidase (RSR-TPO -SF9) [freeze-dried or in frozen solution]
|
Specification
|
Thyroid Stimulating Human Monoclonal
Antibody (RSR-M22) [freeze-dried]
|
Specification |
Blocking Type Human Monoclonal
TSH Receptor Autoantibody (RSR-K1-70) [freeze-dried] |
Specification |
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MOUSE |
Mouse Monoclonal TSH Receptor Antibody (RSR-2 -18C5) [liquid]
|
Specification |
Mouse Monoclonal TSH Receptor Antibody (RSR-4 -3C6) [liquid] |
Specification |
Blocking Type Mouse Monoclonal TSH Receptor Antibody (RSR-B2 -9D33) [liquid] |
Specification |
