Discovery of Two Novel Immunoepitopes and Development of a Peptide-based Sarcoidosis Immunoassay

Abstract

Rationale: Sarcoidosis is a systemic granulomatous disorder associated with hypergammaglobulinemia and the presence of autoantibodies. The specific antigens initiating granulomatous inflammation in sarcoidosis are unknown, and there is no specific test available to diagnose sarcoidosis. To discover novel sarcoidosis antigens, we developed a high-throughput T7 phage display library derived from the sarcoidosis cDNA and identified numerous clones differentiating sarcoidosis from other respiratory diseases. After clone sequencing and a homology search, we identified two epitopes (cofilin μ and chain A) that specifically bind to serum IgGs of patients with sarcoidosis. Objectives: To develop and validate an epitope-specific IgG-based immunoassay specific for sarcoidosis. Methods: We chemically synthesized both immunoepitopes (cofilin μ and chain A) and generated rabbit polyclonal antibodies against both neoantigens. After extensive standardization, we developed a direct peptide ELISA and measured epitope-specific IgG in the sera of 386 subjects, including healthy control subjects (n = 100), three sarcoidosis cohorts (n = 186), pulmonary tuberculosis (n = 70), and lung cancer (n = 30). Measurements and Main Results: To develop a model to classify sarcoidosis distinctly from other groups, data were analyzed using fivefold cross-validation when adjusting for confounders. The cofilin μ IgG model yielded a mean sensitivity, specificity, and positive and negative predictive value of 0.97, 0.9, 0.9, and 0.96, respectively. Those same measures for chain A IgG antibody were 0.9, 0.83, 0.84, and 0.9, respectively. Combining both biomarkers improved the area under the curve, sensitivity, specificity, and positive and negative predictive value. Conclusions: These results provide a novel immunoassay for sarcoidosis. The discovery of two neoantigens facilitates the development of biospecific drug discovery and the sarcoidosis-specific model.

Keywords: T7 phage display; antigen; chain A; cofilin; sarcoidosis.

Figure 1.

Patients with sarcoidosis exhibit increased levels of anti-cofilin μ–specific IgGs. (A) Cofilin μ–specific IgGs were measured via standardized cofilin μ peptide ELISA in the sera of subjects with sarcoidosis (n = 186) and healthy control subjects (n = 100). All measurements were duplicates and standardized in our laboratory. The optical density (OD) value at 450 nm was a measure of cofilin μ–specific IgGs. The OD values (mean ± SD) of cofilin μ–specific IgG in sera of patients with sarcoidosis was 1.5 ± 0.7, whereas the healthy control subjects had a mean OD of 0.4 ± 0.4. There were significant differences in the ODs between sarcoidosis cohorts and healthy control subjects (P = 4.7 × 10⁻³⁴). The horizontal lines represent the mean OD values of healthy subjects (black line) and subjects with sarcoidosis (red line). (B) Polyclonal anti-cofilin μ IgG antibodies were generated by immunizing rabbits with cofilin μ peptide. After three immunizations with cofilin μ peptide, the immunization achieved a high concentration of polyclonal IgG antibody that was purified. The polyclonal rabbit anti-cofilin μ IgG was used to obtain a standard curve for cofilin μ peptide ELISA. Anti-cofilin μ IgG standard curve showing OD at 450 nm versus anti-cofilin μ IgG concentration (ng/ml). (C) Sarcoidosis cohorts displayed significantly higher concentrations of serum anti-cofilin μ IgGs (μg/ml). The mean value of cofilin μ–specific IgGs in healthy control subjects was 27 ± 31 μg/ml, and the mean values in different sarcoid cohorts were 152 ± 61 μg/ml (Detroit), 81 ± 38 μg/ml (San Francisco [SF]), and 122 ± 46 μg/ml (ACCESS). The horizontal lines represent the mean anti-cofilin μ IgG values of healthy subjects (black line), the Detroit cohort (purple line), the SF cohort (green line), and the ACCESS cohort (blue line). There was a significant difference between the healthy control subjects and the Detroit (P = 1.8 × 10⁻²⁷), SF (P = 4.0 × 10⁻¹²), and ACCESS (P = 8.9 × 10⁻¹⁶) cohorts. Within the sarcoidosis cohorts, the differences were as follows: Detroit versus SF (P = 1.87 × 10⁻⁶) and ACCESS (P = 7.3 × 10⁻³) and ACCESS versus SF (P = 2.6 × 10⁻⁵). (D) Patients with sarcoidosis displayed significantly higher concentrations of serum anti-cofilin μ IgGs than control subjects. The values (mean ± SD) of anti-cofilin μ IgGs in sarcoidosis were 127 ± 60 μg/ml, 23 ± 12 μg/ml in healthy control subjects, 18 ± 17 μg/ml in tuberculosis (TB), and 5 ± 2 μg/ml in lung cancer (LC). The horizontal lines represent the mean anti-cofilin μ IgG values of healthy control subjects (black line), subjects with sarcoidosis (red line), and subjects with TB or LC (gray line). There was a significant difference between the subjects with sarcoidosis and healthy control subjects (P = 1.5 × 10⁻³⁶), between subjects with sarcoidosis and those with TB (P = 3.2 × 10⁻³²), and between subjects with sarcoidosis and those with LC (P = 1.6 × 10⁻¹⁸).

Figure 2.

Classification and receiver operating characteristic (ROC) curve of cofilin μ. ROC curves for anti-cofilin μ IgG were generated by using fivefold cross-validation (CV). CV models were obtained from the entire dataset by stepwise inclusion of confounders (sex, race, and smoking) and selecting confounders to minimize the Akaike information criterion to obtain the optimal model. Cofilin μ ROC curve yielded areas under the curve (AUC) of 0.98 (black), 0.96 (red), 0.98 (green), 0.93 (blue), and 0.96 (light blue).

Figure 3.

Patients with sarcoidosis exhibit increased levels of anti-chain A–specific IgGs. (A) Chain A–specific IgGs were measured via standardized chain A peptide ELISA in the sera obtained from subjects with sarcoidosis (n = 186) and healthy control subjects (n = 100). The optical density (OD) value at 450 nm was a measure of chain A–specific IgGs. The OD values (mean ± SD) of chain A–specific IgGs in patients with sarcoidosis was 1.3 ± 0.5, whereas in healthy control subjects, it was 0.4 ± 0.2. There were significant differences in the ODs between sarcoidosis cohorts and healthy control subjects (P = 7.5 × 10⁻³⁴). The horizontal lines represent the mean OD values of healthy subjects (black line) and subjects with sarcoidosis (red line). (B) Polyclonal anti-chain A IgGs were generated by immunizing the rabbits with chain A peptide. After three immunizations of rabbits with chain A peptide, the immunization achieved a high concentration of polyclonal IgG antibody that was purified to develop a standard curve for chain A peptide ELISA. A standard curve showing OD at 450 nm versus anti-chain A IgG concentration (ng/ml) is presented. (C) Sarcoidosis cohorts displayed significantly higher concentrations of serum anti-chain A IgGs. The mean value of chain A–specific IgGs in healthy control subjects was 6 ± 4 μg/ml, and the mean values in different sarcoid cohorts were 22 ± 11 μg/ml (Detroit), 26 ± 6 μg/ml (San Francisco [SF]), and 17 ± 6 μg/ml (ACCESS). The horizontal lines represent the mean anti-chain A IgG values of healthy subjects (black line), the Detroit cohort (purple line), the SF cohort (green line), and the ACCESS cohort (blue line). There was a significant difference between the healthy control group and the Detroit cohort (P = 1.34 × 10⁻²²), the SF cohort (P = 1.26 × 10⁻²²), and the ACCESS cohort (P = 5.82 × 10⁻¹⁶). Among the sarcoidosis cohort, differences were as follows: Detroit versus SF (P = 0.06) and ACCESS (P = 9.29 × 10⁻³), ACCESS versus SF (P = 1.62 × 10⁻⁵). (D) Sarcoidosis cohorts displayed significantly higher concentrations of serum anti-chain A IgGs than control subjects. The values (mean ± SD) of anti-chain A IgGs were 22 ± 9 μg/ml (sarcoidosis), 6 ± 4 μg/ml (healthy control subjects), 11 ± 4 μg/ml (tuberculosis [TB]), and 5 ± 3 μg/ml (lung cancer [LC]). The horizontal lines represent the mean anti-cofilin μ IgG values of healthy control subjects (black line), subjects with sarcoidosis (red line), and subjects with TB or LC (gray line). There was a significant difference between the subjects with sarcoidosis and healthy control subjects (P = 1.8 × 10⁻³³), between subjects with sarcoidosis and subjects with TB (P = 7.2 × 10⁻¹⁶), and between subjects with sarcoidosis and subjects with LC (P = 1.1 × 10⁻¹⁵).

Figure 4.

Classifications and receiver operating characteristic (ROC) curve of chain A. ROC curves for anti-chain A IgGs were generated by using fivefold cross-validation (CV). CV models were obtained from the entire dataset by integrating confounders (sex, race, and smoking) in the best classification model. Chain A ROC curve yielded areas under the curve (AUC) of 0.97 (black), 0.94 (red), 0.95 (green), 0.91 (blue), and 0.94 (light blue).

Figure 5.

Classification and receiver operating characteristic (ROC) for combined epitopes (cofilin μ and chain A). Cross-validation (CV) models were obtained from the entire dataset by stepwise inclusion of confounders (sex, race, and smoking) to achieve the optimal model by minimizing the Akaike information criterion. The combined ROC curve for cofilin μ and chain A yielded areas under the curve (AUC) of 0.99 (black), 0.98 (red), 0.99 (green), 0.93 (blue), and 0.97 (light blue).

Figure 6.

Schematic representation of the discovery and development of the highly specific and sensitive peptide ELISA for detecting cofilin μ– or chain A–specific IgG antibodies in serum. Flowchart depicting the steps involved in the discovery of sarcoid-specific clones, using the T7 phage display library with subtractive panning and immunoscreening, the identification of cofilin μ and chain A epitopes, and the development of cofilin μ and chain A peptide ELISA. MΦ = macrophages; PBMC = peripheral blood mononuclear cell; ROC = receiver operating characteristic; TB = tuberculosis.