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Hall S, Ohrfelt A, Constantinescu R, et al. Accuracy of a panel of 5 cerebrospinal fluid biomarkers in the differential diagnosis of patients with dementia and/or parkinsonian disorders. Arch Neurol 2012;69:1445-1452.
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An Enzyme-Linked Immunosorbent Assay for Detection of Botulinum Toxin-Antibodies Dirk Dressler, MD, PhD,1* Frank Gessler, DVM, PhD,2 Pawel Tacik, MD1 and Hans Bigalke, MD, PhD3 1
Movement Disorders Section, Department of Neurology, Hannover €ttingen, Germany Medical School, Hannover, Germany 2miprolab, Go 3 Department of Toxicology, Hannover Medical School, Hannover, Germany
ABSTRACT Introduction: Antibodies against botulinum neurotoxin (BNT-AB) can be detected by the mouse protection assay (MPA), the hemidiaphragm assay (HDA), and by enzyme-linked immunosorbent assays (ELISA). Both MPA and HDA require sacrifice of experimental animals, and they are technically delicate and labor intensive. We introduce a specially developed ELISA for detection of BNT-A-AB and evaluate it against the HDA. Methods: Thirty serum samples were tested by HDA and by the new ELISA. Results were compared, and receiver operating characteristic analyses were used to optimize ELISA parameter constellation to obtain either maximal overall accuracy, maximal test sensitivity, or maximal test specificity. When the ELISA is optimized for sensitivity, a sensitivity of 100% and a specificity of 55% can be reached. When it is optimized for specificity, a specificity of 100% and a sensitivity of 90% can be obtained. Results: We present an ELISA for BNT-AB detection that can be—for the first time—customized for special purposes. Adjusted for optimal sensitivity, it reaches the best sensitivity of all BNT-AB tests available. Conclusions: Using the new ELISA together with the HDA as a confirmation test allows testing for BNT-AB in large numbers of patients receiving BT drugs in an economical, fast, and more animal-friendly way. C 2014 International Parkinson and Movement Disorder V Society
Movement Disorders, Vol. 29, No. 10, 2014
Key Words: botulinum toxin type A; therapy failure; antibodies; enzyme-linked immunosorbent assay; hemidiaphragm assay
Botulinum toxin (BT) has been used with remarkable success to treat various disorders caused by hyperactivity of muscles or of exocrine glands.1 Its use to treat pain disorders is currently being explored. For therapeutic purposes, BT type A (BT-A) and BT type B (BT-B) are being used. In some patients receiving BT therapy antibodies against BT (BT-AB) are formed. If they are directed against botulinum neurotoxin (BNT, BNT-AB), they can block BT’s therapeutic action so that secondary therapy failure is induced.2 If they are directed against complexing proteins, they are not interfering with BT’s therapeutic action. BNT-AB can be detected by functional tests, including the mouse lethality assay or mouse protection assay (MPA)3 and the mouse diaphragm assay or hemidiaphragm assay (HDA).4 These tests monitor all steps of BT action on the neuromuscular synapse, including binding, translocation, and cleavage. However, they require sacrifice of experimental animals, are technically delicate and are labor intensive. BNT-AB can also be detected by immunological tests, including the radioimmunoprecipitation assay5 and enzyme-linked immunosorbent assays (ELISAs). Immunological tests are easy and quick to perform and are cost effective. However, they may also detect BNT-ABs that are not blocking BNT’s function. The radioimmunoprecipitation assay is not readily available because of involvement of radioactive substances. We wanted to introduce a specially developed ELISA for detection of BNT-A-AB and wanted to evaluate it against the HDA.
Methods Design Thirty serum samples that tested positive or negative by HDA (Toxogen, Hannover, Germany) for BNT-A-AB formation on a routine clinical basis were reevaluated in a blinded fashion by ELISA (miprolab, G€ ottingen, Germany). The ELISA-testing was performed with multiple-parameter constellations for coating antigen, coating dilution, serum dilution, and cutoff values
-----------------------------------------------------------*Correspondence to: Dirk Dressler MD, PhD, Professor of Neurology, Head of Movement Disorders Section, Department of Neurology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany, E-mail: [email protected]
Relevant conflicts of interest/financial disclosures: Nothing to report. Full financial disclosures and author roles may be found in the online version of this article. Received: 28 May 2014; Accepted: 29 May 2014 Published online 29 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/mds.25956
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TABLE 1. Results of botulinum toxin antibody testing with the various ELISA versions based on hemidiaphragm assay (HDA) data as reference Maximizing Sensitivity ELISA Version
#1 #2 #3 #4 #5 #6
BNT-A1 BNT-A1 BNT-A1 BNT-A1 complex BNT-A1 complex BNT-A1 complex
0.5mg 1.0mg 0.5mg 0.5mg 1.0mg 0.5mg
1:100 1:100 1:40 1:100 1:100 1:40
0.87 0.96 0.87 0.78 0.62 0.87
90% 100% 90% 100% 100% 90%
82% 55% 82% 9% 9% 82%
70% 90% 70% 30% 30% 70%
100% 100% 100% 100% 100% 100%
mL21 mL21 mL21 mL21 mL21 mL21
Data were calculated by receiver operating characteristic (ROC) analysis.
(ELISA 1 to ELISA 6). Mathematical modeling was used to optimize parameter constellation for maximal overall accuracy, maximal test sensitivity, and maximal test specificity.
ELISA All incubation steps were carried out at room temperature. Polysorb 96-well microassay plates (Nunc Thermo Scientific, Schwerte, Germany) were coated with 100 mL of 0.5-mg or 1-mg mL21 purified biologically active BNT-A1 or BNT-A1 complex (miprolab, G€ ottingen, Germany) in coating buffer (10 mM phosphate buffer, pH 7.2, 145 mM NaCl). Plates were incubated for 1 hour, and excess coating solution was removed by three washing cycles with washing buffer (10 mM phosphate buffer, pH 7.2; 145 mM NaCl, 0.05% Tween 20) using the automated ScanWasher (Molecular Devices, Ismaning, Germany). A volume of 300 mL commercial blocking buffer (miPROBLOCK C, miprolab) was added to each well and incubated for 1 hour. Plates were washed as described. All serum samples were diluted 1:40 or 1:100 in blocking buffer and 100 mL added to the wells. Blocking buffer alone was used as blank control. The plates were incubated for 1 hour followed by five washing cycles. Antihuman immunoglobulin G, peroxidase-labeled (A8792, Sigma, Munich, Germany), was used as secondary detection antibody. The labeled antibody was diluted 1:40,000 in blocking buffer, and an aliquot of 100 mL was added to each well. After incubating the plates for 1 hour, eight washing cycles were applied to carefully remove unbound secondary antibodies. 3,30 ,5,50 -Tetramethylbenzidine Substrate (Sigma) was diluted according to the manufacturer’s instructions, and 100 mL was used in each well. The enzymatic reaction was stopped with 50 mL 1 M H2SO4 after 30 minutes. Absorbance was measured at 405 nm with a SpectraMax photometer (Molecular Devices) against the blank control. The parameter settings of the various ELISA versions are shown in Table 1. The HDA was performed as described previously.4
Mathematical Modeling The sera were classified in antibody-negative and antibody-positive sera according to the HDA results. The ELISA sensitivity was calculated as the probability that a true positive result was obtained (HDA positive samples/HDA positive samples 1 ELISA false-negative samples). Specificity of the ELISA was the probability of a true negative result (HDA negative samples/HDA negative samples 1 ELISA false-positive samples). In receiver operating characteristic (ROC) analyses, data of specificity and sensitivity are plotted to identify suitable assay systems or cutoff values compared with a reference method, in this case the HDA. In this binary classification system of false-positive and falsenegative samples the random guess is shown as the diagonal in the graph. The area under each curve combines sensitivity and specificity data and is thus a measure of the accuracy of the assay. Absorbance data of
FIG. 1. Receiver operating characteristic (ROC) curves of the various ELISA versions. Sensitivity and specificity of the various enzymelinked immunosorbent assay versions are shown. The area under the ROC curve (A) is the measure of the accuracy of each test.
Movement Disorders, Vol. 29, No. 10, 2014
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FIG. 2. Comparison of hemidiaphragm assay (HDA) results and the results of the various enzyme-linked immunosorbent assay (ELISA) versions. Samples are grouped in HDA-negative (black marks) and HDA-positive (white marks) results. The horizontal line indicates the calculated cutoff based on a pretest probability of 10% and an optimized specificity. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
the ELISA testing were submitted to ROC analyses to identify the best ELISA version using SigmaPlot (Systat Software, San Jose, CA, USA).
scale testing of BNT-AB becomes necessary. We therefore developed an ELISA for BT-AB testing, compared it with the HDA, and optimized it with respect to overall accuracy, sensitivity, and specificity. In the past, several ELISA tests have been used to detect BNT-AB.7-11 Most of them have not been quality assessed properly. Using the HDA as a reference, our ELISA could be evaluated with respect to various test parameters. This allows customization for special purposes. Operating the ELISA at its best overall accuracy with BNT-A1 as coating antigen in a concentration of 1mg mL21, a serum dilution of 1:100 and a cut-off at 0.01 produces 100% sensitivity and 55% specificity. With this parameter setting, our ELISA reaches the maximum sensitivity of all BNT-AB tests available.12 Setting the cutoff at 0.02 produces 100% specificity and 90% sensitivity. For detection of BNTAB in large series of BT-treated patients, our ELISA can be adjusted to maximum sensitivity to function as a screening test, and HDA testing could be used for subsequent confirmation. With this strategy, testing for BNT-AB in large numbers of patients receiving BT drugs could become more economical, faster, and more animal friendly.
Results The results obtained by ROC analyses are shown in Figure 1. Table 1 summarizes overall accuracy, sensitivity, and specificity of the various ELISA versions. The best overall accuracy, that is, the largest area under the curve (A 5 0.96), is seen with ELISA 2, using BNT-A1 as coating antigen in a concentration of 1 mg mL21, a serum dilution of 1:100, and a cutoff of 0.02. When our ELISA is optimized for sensitivity, a sensitivity of 100% and a specificity of 55% can be reached. When our ELISA is optimized for specificity, a specificity of 100% and a sensitivity of 90% can be obtained. All other ELISA versions tested have less favorable values for their primary or secondary quality parameters. Figure 2 shows the dot histogram of the ELISA absorbance values of the samples grouped according to HDA-positive and HDA-negative for all tested ELISA versions. With a single false-negative sample, ELISA 2 shows again the best performance. Minimized background and low cutoff values were achieved in this test.
Discussion The current gold standard for detection of neutralizing BNT-AB is the MPA. Previously, we demonstrated that the HDA can replace the MPA for this purpose.6 Although the HDA has an advantage over the MPA with respect to costs and animal protection, it still consumes animals and still is technically challenging. With the rapid development of new BT drugs, large-
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