Dataset Information

Comparison of radioimmunoprecipitation with luciferase immunoprecipitation for autoantibodies to GAD65 and IA-2beta.

ABSTRACT:

Objective

To compare the sensitivity and specificity of luciferase immunoprecipitation (LIPS) with radioimmunoprecipitation (RIP) for the measurement of autoantibodies to the type 1 diabetes autoantigens glutamic acid decarboxylase 65 (GAD65) and insulinoma-associated protein (IA)-2beta.

Research design and methods

Sera from 49 type 1 diabetic patients and 100 nondiabetic control subjects from Diabetes Antibody Standardization Program 2007 were used to screen for autoantibodies to GAD65. An additional 200 type 1 diabetic patients and 200 nondiabetic control subjects were used to validate the GAD65 results and screen for autoantibodies to IA-2beta.

Results

LIPS showed equal sensitivity and specificity to RIP for detecting autoantibodies to GAD65 and IA-2beta. Receiver-operating characteristic analysis revealed that the detection of autoantibodies to GAD65 and IA-2beta by LIPS and RIP were not statistically different.

Conclusions

The LIPS assay does not require the use of radioisotopes or in vitro transcription/translation and is a practical alternative at the clinical level for the RIP assay.

SUBMITTER: Burbelo PD

PROVIDER: S-EPMC2845022 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Publications

Comparison of radioimmunoprecipitation with luciferase immunoprecipitation for autoantibodies to GAD65 and IA-2beta.

Burbelo Peter D PD Hirai Hiroki H Issa Alexandra T AT Kingman Albert A Lernmark Ake A Ivarsson Sten-A SA Notkins Abner L AL Iadarola Michael J MJ

Diabetes care 20100119 4

<h4>Objective</h4>To compare the sensitivity and specificity of luciferase immunoprecipitation (LIPS) with radioimmunoprecipitation (RIP) for the measurement of autoantibodies to the type 1 diabetes autoantigens glutamic acid decarboxylase 65 (GAD65) and insulinoma-associated protein (IA)-2beta.<h4>Research design and methods</h4>Sera from 49 type 1 diabetic patients and 100 nondiabetic control subjects from Diabetes Antibody Standardization Program 2007 were used to screen for autoantibodies to ...[more]

PMID: 20086252

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Project description:BackgroundEarly detection of porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine is necessary to control this devastating disease. By monitoring host serum antibodies to viral antigens, early virus detection within herds is feasible. In this study, recombinant antigens were generated using recombinant DNA techniques to fuse PRRSV structural protein (N) or nonstructural protein 1α (nsp1α) with the Rellina luciferase gene. Next, fused genes were cloned into plasmids and transfected into HEK-293 T cells for transient expression. Upon co-incubation of lysates with pig sera, antigen-antibody complexes formed that bound to Protein-G coated onto microplates. By further measurement of luminance value, a modified form of Luciferase Immunoprecipitation Systems, namely luciferase-linked antibody capture assay (LACA) was developed for detection of PRRSV-specific antibodies.ResultsKnown anti-PRRSV antibody-positive or -negative serum samples (125 and 122 samples, respectively) were used to validate the LACA and compared it with IDEXX PRRS ×3 ELISA. Based on the result, N-Rluc and nsp1α-Rluc LACA results were 95.3 and 94.4% in agreement with IDEXX ELISA, suggested a similar specificity of LACA to IDEXX ELISA. Moreover, when both LACA and IDEXX ELISA were used to evaluate sequential serum samples obtained from PRRSV experimentally infected pigs, the PRRSV-specific antibody response was detectable as early as 3 days post-inoculation (dpi) using N-Rluc LACA, but undetectable until 7 dpi using IDEXX ELISA, suggesting an improved sensitivity of LACA. Meanwhile, antibodies specific for nsp1α were detected at higher levels overall, but were undetectable until 10 dpi. Furthermore,. Notably, one IDEXX ELISA positive result was not confirmed by LACA or IFA and was thus considered a false-positive result.ConclusionThe LACA exhibited similar specificity but improved sensitivity to that of the commercial IDEXX PRRS ×3 ELISA kit for detection of PRRSV-specific antibodies in pig serum. Importantly, LACA could be adapted for detecting antibodies against other PRRSV targets, such as nsp1α, to achieve earlier detection of PRRSV infection.

Dataset Information

Comparison of radioimmunoprecipitation with luciferase immunoprecipitation for autoantibodies to GAD65 and IA-2beta.

Objective

Research design and methods

Results

Conclusions

Publications

Comparison of radioimmunoprecipitation with luciferase immunoprecipitation for autoantibodies to GAD65 and IA-2beta.

Similar Datasets

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