Acetylcholine Receptor Antibody

Normally, no acetylcholine receptor (AChR) antibody exists in the bloodstream. Binding antibody is the most common antibody found in myasthenia gravis (MG) patients. As binding and blocking antibody together have high sensitivity and specificity (99.6%) for MG patients and chances to find a positive modulating antibodies in patients negative for above antibodies is less than 0.4%. ^[1] Therefore the reflex panel detects anti-acetylcholine receptor (blocking and binding) antibodies in the serum, if antibody level is greater than 0.4nmol/L, or antibody level is greater than 15% then modulating antibody is added. ^{[1, 2]}

Normal findings ^[3] :

• AChR (muscle) binding antibodies: ≤ 0.02 nmol/L
• AChR (muscle) modulating antibodies: 0-20% (reported as percentage loss of AChR)
• Striational (striated muscle) antibodies: < 1:60 

An image depicting an AChR can be seen below.

AChR antibody levels

Anti-acetylcholine receptor antibody is found in 70-90% of patients with generalized acquired myasthenia gravis (MG). Lambert-Eaton syndrome is a close differential, as less than 13% of patients have clinical presentation similar to MG and antibodies against acetylcholine receptors in high titers. More than 30% of patients with MG are seronegative (ie, anti-acetylcholine receptor antibody is absent in these patients). ^[4] Around 17.4% of this seronegative group converts to seropositive in a span of one year from initial diagnosis. The seronegative group of patients may have other antibodies like anti-MuSk antibody-positive (in < 40% of seronegative MG patients), anti-RyR antibody, titin antibody, and anti-striated muscle antibody. ^[5]

Presence of other antibodies is highly variable, so these antibodies are not confirmatory for MG. Rather, their presence supports the diagnosis of MG. Also seronegative MG patients have a different presentation with presence of these antibodies. For example, patients with anti-MuSk antibody have more facial muscle involvement, whereas anti-striated muscle antibody presents with more severe form of MG. ^[6] Presence of these other antibodies is helpful, as they determine the course of disease and also explain the variable response to universal treatment modalities for MG.

Tindall reported AChR Ab results and mean Ab titers in group of patients with MG. ^[7] The data suggest a trend towards higher Ab titers in more severe disease, though the titer does not predict the severity of disease in individual patient.

Prevalence and titers of Antibody to Acetylcholine Receptor in patients with Myasthenia Gravis ^{[7, 8, 4]}

Table. (Open Table in a new window)

AChR = Acetylcholine receptor , MG Myasthenia gravis

*Osserman classification: R = remission, I = ocular only, IIA = mild generalized, IIB = moderate generalized, III = acute severe, IV = chronic severe. ^{[7, 8]}

Binding antibody

Binding antibody is the most common antibody found in patients with MG. These antibodies bind to the AChR on the muscle end plate and activate endocytosis/complement system leading to degradation of these receptors which results in poor muscle strength. ^[1] Antibodies are positive in 90% of patients, but 10% patients have no detectable levels of binding/blocking/modulating antibodies. Out of these 10%, less than 40% have positive anti-MuSk antibodies. The rest might have anti-smooth muscle/anti-RyR/titin antibodies in the serum.

Sensitivity and specificity with blocking antibody level together is 99.6%, and hence they are the tests of choice.

Test name: Myasthenia Gravis Antibody Reflex Panel/Muscle Weakness Autoimmune Panel.

Method: Quantitative radioimmunoassay

Reference intervals: ^{[1, 7, 2]}

Negative: 0.0-0.4 nmol/L

Positive: 0.5 nmol/L or greater

Blocking antibody

Blocking antibody is the second most common antibody found in MG patients. These antibodies bind to the AChR on the muscle end plate and impairs binding of Ach with the receptor, which results in poor muscle strength.

Sensitivity and specificity with binding antibody level together is 99.6%, and hence they are the tests of choice.

Test name: Myasthenia Gravis Antibody Reflex Panel, Muscle Weakness Autoimmune Panel

Method: Semi-quantitative flow cytometry

Reference intervals: ^{[1, 7, 2]}

Negative: 0-26% blocking

Indeterminate: 26-41% blocking

Positive: 42% or greater blocking

Modulating antibody

Modulating antibody is also found in MG patients. These antibodies bind to the AChR on the muscle end plate and causes endocytosis of the receptor, which results in loss of AChR expression and hence poor muscle strength. ^{[1, 2]} This antibody has the maximum positive correlation with the severity of the disease.

Sensitivity and specificity with blocking antibody level together is 99.6%, and hence they are the tests of choice. ^[4]

Test name: Myasthenia Gravis Antibody Reflex Panel, Muscle Weakness Autoimmune Panel.

Method: Semi-quantitative flow cytometry

Reference intervals: ^{[1, 7]}

Negative: 0-45% modulating

Positive: 46% or greater modulating

Further interpretation and use of results: Binding and blocking antibody together have sensitivity and specificity for MG patients. The likelihood of positive modulating antibodies in patients negative for above antibodies is less than 0.4%. ^[1] Therefore, the reflex panel detects anti-acetylcholine receptor (blocking and binding) antibodies in the serum. If antibody level is greater than 0.4 nmol/L or antibody level is greater than 15%, then modulating antibody is added. ^{[1, 2]} If titers are more than 1:40 on initial dilution then anti-striated muscle (Sm) antibody titers are added to this panel. In seronegative patients with clinical signs in favor of MG, anti-MuSk antibody levels are tested. ^[9]

Anti-acetylcholine receptor antibody can be blocking type (present in 90% of myasthenia gravis [MG] patients), modulating type (positive in 65% of MG patients) and binding form (seen in < 40% patients with MG). The reflex panel detects anti-acetylcholine receptor (blocking and binding) antibodies in the serum. If antibody level is greater than 0.4nmol/L or antibody level is greater than 15%, then modulating antibody is added. If titers are more than 1:40 on initial dilution then anti-striated muscle (Sm) antibody, titers are added to this panel. ^{[1, 10, 11]}

In seronegative patients with clinical signs of MG, the Muscle Weakness Autoimmune Panel is done, which includes anti-MuSk antibody/titin Ab/anti-RyR antibody/ANA/rheumatoid factor. ^{[1, 5, 6]}

Binding Antibody level: ^{[10, 11]}

Test: MG Reflex Panel

Method: Quantitative radioimmunoassay

Pretest preparation: No preparation required. Patient need not be fasting.

Specimen: Freshly drawn blood sample, minimum of 2 mL of serum or 1 mL of blood.

Container: Blood is collected in red top or serum separator tube.

Collection Method: Adequate sterile measures followed by venipuncture. Collect 1-2 mL in red top/serum separator tube. Avoid shaking the tube, rigorous mixing, and other methods that can result in hemolysis or leak of lipid from the cell walls. Avoid contamination of the sample. After collection of the blood sample, centrifugation/serum separation should done as soon as possible or within 2 hours.

Stability: Serum should be stored in a refrigerator. Separated serum can be used up to 2 hours if stored at room temperature, 2 weeks for the sample preserved in refrigerator, and it can be reused within a year if sample is well preserved in freezer section of the refrigerator (avoid repeated thaw/freeze cycles).

Transport: At room temperature. Avoid rigorous shaking or mishandling of sample that can cause damage to RBCs and result in hemolysis. Serum separation in 2 hours of blood collection and adequate storage of serum depending on need for future use of the same sample.

Unacceptable Conditions: plasma, contamination, hemolysis or severely lipemic conditions.

Blocking Antibody level: ^{[10, 11]}

Test: MG Reflex Panel

Method: Semi-quantitative flow cytometry

Collection:

Pretest preparation: No preparation required. Patient need not be fasting.

Specimen: Freshly drawn blood sample, minimum of 2 mL of serum or 1 mL of blood.

Container: Blood is collected in red top or serum separator tube.

Collection Method: Adequate sterile measures followed by venipuncture. Collect 1-2 mL in red top/serum separator tube. Avoid shaking the tube, rigorous mixing, and other methods that can result in hemolysis or leak of lipid from the cell walls. Avoid contamination of the sample. After collection of the blood sample, centrifugation/serum separation should done as soon as possible or within 2 hours.

Stability: Serum should be stored in a refrigerator. Separated serum can be used up to 2 hours if stored at room temperature, 2 weeks for the sample preserved in refrigerator, and it can be reused within a year if sample is well preserved in freezer section of the refrigerator (avoid repeated thaw/freeze cycles).

Transport: At room temperature. Avoid rigorous shaking or mishandling of sample that can cause damage to RBCs and result in hemolysis. Serum separation in 2 hours of blood collection and adequate storage of serum depending on need for future use of the same sample.

Unacceptable Conditions: Plasma, contamination, hemolysis or severely lipemic conditions.

Modulating Antibody level: ^{[10, 11]}

Test: MG Reflex Panel

Method: Semi-quantitative flow cytometry

Collection:

Pretest preparation: No preparation required. Patient needed not be fasting.

Specimen: Freshly drawn blood sample, minimum of 2 mL of serum or 1 mL of blood.

Container: Blood is collected in red top or serum separator tube.

Collection Method: Adequate sterile measures followed by venipuncture. Collect 1-2 mL in red top/serum separator tube. Avoid shaking the tube, rigorous mixing, and other methods that can result in hemolysis or leak of lipid from the cell walls. Avoid contamination of the sample. After collection of the blood sample, centrifugation/serum separation should done as soon as possible or within 2 hours. ^{[10, 11]}

Stability: Serum should be stored in a refrigerator. Separated serum can be used up to 2 hours if stored at room temperature, 2 weeks for the sample preserved in refrigerator, and it can be reused within a year if sample is well preserved in freezer section of the refrigerator (avoid repeated thaw/freeze cycles).

Transport: At room temperature. Avoid rigorous shaking or mishandling of sample that can cause damage to RBCs and result in hemolysis. Serum separation in 2 hours of blood collection and adequate storage of serum depending on need for future use of the same sample.

Unacceptable Conditions: plasma, contamination, hemolysis or severely lipemic conditions.

Related Tests

See the list below:

• Striated muscle antibody

• Anti-MuSK antibody

• Cholinesterase

• Rheumatoid factor

• Antinuclear antibody (ANA)

• Thyroid panel

Anti-acetylcholine receptor antibodies are heterogeneous antibodies that cause loss of muscle function by several mechanisms. Anti-acetylcholine receptor antibodies are present in 80%- 90% of the patients with myasthenia gravis (MG) and 50-60% of patients with ocular MG. Existence of end plate protein was first suggested by Simpson in 1973, as he found globulins that could block binding of alpha bungaratoxin (a polypeptide from venom of Bungarus multicinctus) to acetylcholine receptor in rats. ^[12] Almon et al showed IgG antibodies blocked the binding of alpha bungaratoxin to acetylcholine receptors. ^[12] Bender and colleagues confirmed above with radiolabelled IgG.

The first detailed study was done by Lindstorm et al. It showed no correlation between acetylcholine receptor antibodies and various determinants like age, sex, duration of disease, or ethnicity. ^[13] A positive correlation between antibody levels and severity of disease was established as scientists found low antibody levels in ocular MG. The titers were much lower than those in patients with generalized MG in clinical remission. ^{[12, 13]}

Lefvert et al found a broader peak on elution profiles, which established the polyclonal nature of anti-acetylcholine receptor antibody in patients with MG. ^[12] Further studies found that the most common antibodies present in these patients are IgG3 subclass. ^[5] Researches showed anti-acetylcholine receptor antibodies are affected by different variables, such as site of muscle involvement, function of muscle complex, and denervated muscle.

So far, the only genetic association found is between HLA A1B8DRw3 and young patients (< 40 y) with MG but no thyoma. ^[6] Site of production of these antibodies has been an intriguing question, and based on the exponential decrease in antibodies levels after thymectomy (at a rate of 42.5 fmoles /1,000,000 cells /24 hours) few researchers believe that the main site of origin of these antibodies is the thymus (myoid and epithelial cells). Other sites of production exist as antibodies remain positive in patients in clinical remission after thymectomy. ^[6]

Mechanism of action of anti-acetylcholine receptor antibody

There are three proposed mechanisms of action: binding, blocking, and modulating the end plate acetylcholine receptors. ^[1] Anti-acetylcholine receptor antibody forms a complex with acetylcholine receptors and increases its degradation by either activation of complement pathway (especially C3/C9 mediated destruction of the Ag-Ab complex) or increasing endocytosis of the complex with subsequent degradation of the complex.

Some authors believe that these antibodies may block the binding of acetylcholine to receptor by occupying their space, a theory based on the minimal decrease in the receptor density on end plates of muscle fibers. ^{[13, 14]} A few researchers believe that the variable expression of disease with same amount of antibody burden is due to difference in resynthesis of receptors after degradation. Other researchers believe that difference in expression is due to variability in binding capacity of antibodies to the receptor, which determines further activation of the destruction process. ^{[12, 13, 14]}

Application/indication

Positive antibodies are found in patients with MG with the following thymus disorders: ^[7] thymus hyperplasia in 60% patients, thyoma in 20-25%, atrophica thymus in 9%. Thymus is normal in less than 8% patients. Other diseases with positive anti-acetylcholine receptor antibodies are lupus, rheumatoid arthritis, liver disease, autoimmune hepatitis, Lambert-Eaton syndrome, small cell lung cancer, and Graves disease. Positive anti-acetylcholine receptor antibodies are also seen in patients on immunosuppressive therapy. Antibody levels are independent of variables like age, sex, ethnicity, and duration of disease. ^{[7, 11]}

Antibody levels have a direct correlation with severity of disease, and higher titers are associated with severe forms of disease. The only exception to this is in patients with paraneoplastic syndrome in small cell lung cancer. An inverse relation is described between antibody levels and B cell numbers as they increase the antibody clearance from the serum of the patient.

Anti-acetylcholine receptor antibody is helpful in diagnosing MG and is used as the first test in patients with clinical indicators of the disease. Its high sensitivity and specificity of 99.6% (binding and blocking antibody types) makes it a good screening test, as well as a part of confirmatory test panel. ^[1] Antibody levels also determine the severity of the disease, are used to monitor response to treatment, and are used to assess for relapse in patients in clinical remission after surgical or medical treatment. Levels are also used to differentiate the congenital form of MG from the acquired form (as antibody is negative in former). Antibody positivity also helps in deciding the type of treatment in patients without thymoma. Researchers have seen beneficial outcomes of thymectomy in antibody-positive patients without thymoma and borderline or no improvement in patients with low antibody titers and no thymoma (seronegative MG). ^[4]

Anti-acetylcholine receptor antibody levels are determined by quantitative and semi-quantitative assay, which look for binding type of antibody and modulating/blocking forms respectively in serum of patients with clinical presentation of MG. These tests are part of the reflex panel for diagnosing MG. Other tests that are done to support the diagnosis are anti-striated muscle antibody, anti-MuSk antibody, titin antibody, imaging of the head/neck and chest, and single fiber electrical muscle stimulation (EMS). ^{[1, 7, 4]}

Limitation and other tests

Anti-acetylcholine receptor (AChR) antibody is a sensitive test, with over 90% positive results, so it is used to confirm diagnosis. Negative antibody levels do not rule out MG , and other tests, like single fiber electromyography or repetitive muscle stimulation, can be performed to confirm the diagnosis of MG. ^[15]

AChR can be falsely elevated in other autoimmune diseases or in other etiologies that have presentations similar to MG, such as Lambert-Eaton Syndrome, small cell lung cancer (15% of these patients carry significant titer of anti-AchR antibody), autoimmune hepatitis, systemic lupus erythematosus (SLE), rheumatoid arthritis, and liver disease. Other conditions, like thyoma without MG, can also be associated with high titers of antibody, and antibody can be detected in patients with recent radiation exposure or patients receiving immunosuppressive treatment (especially those receiving D-pencillamine therapy). ^{[12, 13, 7]} Infrequently these antibodies can be found in first-degree relatives of MG patients (perhaps HLA association plays a role in positive antibodies among relatives without activity of disease) and elderly persons with other autoimmune disease, as mentioned previously.

False-negative results can be found in patients with congenital MG or ocular MG (as these patients' hlocalized disease is likely associated with high burden of antibody in their serum) or seronegative MG patients. Seronegative MG patients are those who have the typical symptoms of MG but do not carry detectable levels of anti-AChR antibodies in their serum. ^{[7, 4, 5, 6]} Though these patients represent fewer than 10% of MG patients, diagnosing them is challenging and their response to conventional treatment of MG is variable. These patients are often found to have anti-MuSk antibody and anti-striated muscle antibody in their serum. ^{[5, 6]}

In cases with strong clinical indicators but negative MG Reflex Panel (consisting of binding/blocking anti-AChR antibody detection), a Muscle Weakness Autoimmune Panel is done, which consists of voltage-gated calcium channel antibody, voltage-gated potassium channel antibody, titin antibody, and anti-striated muscle (IgG) antibody. ^{[13, 14, 1]}