Distinct profiles of antibodies to Kaposi sarcoma-associated herpesvirus antigens in patients with Kaposi sarcoma, multicentric Castleman disease, and primary effusion lymphoma.

Antibody responses against lytic and latent Kaposi sarcoma (KS)-associated herpesvirus antigens were investigated in patients with KS, multicentric Castleman disease (MCD), and primary effusion lymphoma. Antibodies against the lytic antigen K8.1 were 5-fold higher in patients with MCD than those with KS, whereas antibodies to the sum of latent antigens v-cyclin and LANA were 27-fold higher in patients with KS, compared with patients with MCD (P < 001). The sum of anti-v-cyclin and anti-LANA antibody titers discriminated patients with KS from those with MCD and KS with 93% sensitivity and 83% specificity. These results suggest that antibody responses to lytic and latent KS-associated herpesvirus antigens differ in these diseases.

Proteome-wide anti-hepatitis C virus (HCV) and anti-HIV antibody profiling for predicting and monitoring the response to HCV therapy in HIV-coinfected patients.

We quantified antibody responses to the hepatitis C virus (HCV) proteome that are associated with sustained virologic response (SVR) in human immunodeficiency virus (HIV)/HCV-coinfected patients treated with pegylated interferon and ribavirin. Analysis of pre- and posttreatment samples revealed significant decreases in the combined anti-core, anti-E1, and anti-NS4 HCV antibody titers in those with SVRs but not in those who experienced relapse or who did not respond. Furthermore, anti-HIV p24 antibody titers inversely correlated with treatment response. These results suggest that profiling anti-HCV antibody is useful for monitoring HCV therapy, especially in discriminating between those who experience relapse and those who have SVRs at 48 weeks.

Rapid serological detection of autoantibodies associated with Sjögren's syndrome.

BACKGROUND: Sjögren's syndrome (SjS) is a relatively common autoimmune disease characterized by oral and ocular dryness. There is an increasing need for simple, sensitive and rapid technologies for the diagnosis of SjS and other autoimmune diseases. Here we investigated whether a quick version of luciferase immunoprecipitation systems (QLIPS) could be used to produce a rapid, specific and quantitative test to detect autoantibodies associated with SjS. METHODS: Using QLIPS, which requires only ten minutes of incubation, a cohort of control and SjS sera were tested for antibodies to three SjS autoantigens (La, Ro60 and Ro52). Sensitivity and specificity of the QLIPS tests were compared with LIPS and existing ELISA data. The QLIPS test for Ro52 was then evaluated with a new validation cohort and its diagnostic performance determined. RESULTS: Using QLIPS, autoantibodies to three SjS antigens, La, Ro60, and Ro52 were detected in 49%, 56% and 70%, respectively, of the SjS patients and none of the controls (100% specificity). With antibody titers in the Ro52-seropositive SjS samples approximately 1,000 times higher than the healthy controls, not only was Ro52 the most informative, but detection of anti-Ro52 antibodies under these non-equilibrium conditions was improved compared to the standard 2 hour LIPS format. Validation of the anti-Ro52 QLIPS test in a new, independent cohort of SjS and control serum samples showed 66% sensitivity and 100% specificity. CONCLUSION: Together these results suggest that the QLIPS format for Ro52 yields both a more rapid and more discriminating test for detecting Ro52 autoantibodies than existing immunoassays and has the potential to be adapted for point-of-care evaluation of patients with SjS and other rheumatologic diseases.

Highly quantitative serological detection of anti-cytomegalovirus (CMV) antibodies.

BACKGROUND: Human cytomegalovirus infection is associated with a variety of pathological conditions including retinitis, pneumonia, hepatitis and encephalitis that may be transmitted congenitally, horizontally and parenterally and occurs both as a primary infection and as reactivation in immunocompromised individuals. Currently, there is a need for improved quantitative serological tests to document seropositivity with high sensitivity and specificity. METHODS: Here we investigated whether luciferase immunoprecipitation systems (LIPS) would provide a more quantitative and sensitive method for detecting anti-CMV antibodies. Four protein fragments of immunodominant regions of CMV antigens pp150 and pp65 were generated as Renilla luciferase (Ruc) fusion proteins and used in LIPS with two cohorts of CMV positive and negative sera samples previously tested by ELISA. RESULTS: Analysis of the antibody responses to two of these antigen fragments, pp150-d1 and pp150-d2, revealed geometric mean antibody titers in the first cohort that were 100-1000 fold higher in the CMV positive sera compared to the CMV negative samples (p < 0.0001) and infection status exactly matched the ELISA results for the 46 samples of the first cohort (100% sensitivity and 100% specificity). Two additional antigen fragments, pp65-d1 and pp65-d2 also showed robust antibody titers in some CMV-infected sera and yielded 50% and 96% sensitivity, respectively. Analysis of a second cohort of 70 samples using a mixture of the 4 antigens, which simplifies data collection and analysis, yielded values which correlated well with the sum of the values from the 4 separate tests (rs = 0.93, p < 0.00001). While comparison of the LIPS results from this second cohort with ELISA showed 100% sensitivity, LIPS detected six additional CMV positive samples that were not detected by ELISA. Heat map analysis revealed that several of the LIPS positive/ELISA negative samples had positive LIPS immunoreactivity with 3-4 of the CMV antigens. CONCLUSION: These results suggest that LIPS provides a highly robust and quantitative method for studying anti-CMV antibodies and has the potential to more accurately document CMV infection than standard ELISA.

Increased neuronal activity in the OVLT of Cyp1a1-Ren2 transgenic rats with inducible Ang II-dependent malignant hypertension.

The contribution of angiotensin II (Ang II) to the pathophysiology of hypertension is established based on facts that high levels of circulating Ang II increase vasoconstriction of peripheral arteries causing a rise in blood pressure (BP). In addition, circulating Ang II has various effects on the central nervous system, including the osmosensitive neurons in the organum vasculosum of the lamina terminalis (OVLT). Osmosensitive neurons in the OVLT transduce hypertonicity via the activation of the nonselective cation channel known as transient receptor potential vanilloid 1 (TRPV1), causing membrane depolarization, followed by increased action potential discharge. This effect is absent in mice lacking expression of the TRPV1 gene. Most observations related to the importance of the OVLT in cardiovascular control are mainly based on models of lesion of the entire preoptic periventricular tissue. However, it remains unclear whether neuronal activity and TRPV1 protein expression levels alter in the OVLT of Cyp1a1-Ren2 transgenic rats with inducible Ang II-dependent malignant hypertension. C-fos was used as a marker of neuronal activity. Immunostaining was used to demonstrate distribution of c-fos positive neurons in the OVLT of Cyp1a1Ren2 transgenic rats. Western blot analysis showed increased c-fos and TRPV1 total protein expression levels in the OVLT of hypertensive rats. The present findings demonstrate increased c-fos and TRPV1 expression levels in the OVLT of Cyp1a1-Ren2 transgenic rats with Ang II-dependent malignant hypertension.

Rapid, simple, quantitative, and highly sensitive antibody detection for lyme disease.

There is currently a need for improved serological tests for the diagnosis and monitoring of Lyme disease, an infection caused by Borrelia burgdorferi. In the present study, we evaluated luciferase immunoprecipitation systems (LIPSs) for use for profiling of the antibody responses to a panel of B. burgdorferi proteins for the diagnosis of Lyme disease. Initially, serum samples from a cohort of patients and controls (n = 46) were used for training and were profiled by the use of 15 different B. burgdorferi antigen constructs. For the patient sera, the antibody responses to several B. burgdorferi antigens, including VlsE, flagellin (FlaB), BmpA, DbpA, and DbpB, indicated that the antigens had high levels of immunoreactivity. However, the best diagnostic performance was achieved with a synthetic protein, designated VOVO, consisting of a repeated antigenic peptide sequence, VlsE-OspC-VlsE-OspC, Analysis of an independent set of serum samples (n = 139) used for validation showed that the VOVO LIPS test had 98% sensitivity (95% confidence interval [CI], 93% to 100%; P < 0.0001) and 100% specificity (95% CI, 94% to 100%; P < 0.0001). Similarly, the C6 peptide enzyme-linked immunosorbent assay (ELISA) also had 98% sensitivity (95% CI, 93% to 100%; P < 0.0001) and 98% specificity (95% CI, 90% to 100%; P < 0.0001). Receiver operating characteristic analysis revealed that the rates of detection of Lyme disease by the LIPS test and the C6 ELISA were not statistically different. However, the VOVO LIPS test displayed a wide dynamic range of antibody detection spanning over 10,000-fold without the need for serum dilution. These results suggest that screening by the LIPS test with VOVO and other B. burgdorferi antigens offers an efficient quantitative approach for evaluation of the antibody responses in patients with Lyme disease.

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

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.

Sensitive and robust luminescent profiling of anti-La and other autoantibodies in Sjogren's syndrome.

Sjogren's syndrome (SjS) patients often have a variety of extraglandular manifestations including neurological and gastrointestinal involvement. In this study we evaluated the diagnostic performance of luciferase immunoprecipitation system (LIPS) that employs mammalian cell-produced recombinant antigens for analyzing SjS autoantibody responses. LIPS testing of mammalian cell-produced La, Ro60 and Ro52 recombinant antigens with defined commercial antibodies demonstrated highly specific immunoprecitation of each antigen without cross-reactivity. Next, sera from 57 SjS and 25 volunteers were evaluated by LIPS against a panel of human autoantigens. LIPS detected robust anti-La antibody levels in 43/57 SjS patients (75% sensitivity) and markedly outperformed an ELISA (46% sensitivity). Profiling of other SjS-associated autoantigens revealed the presence of anti-Ro60, anti-Ro52 in 63% and 61%, of SjS patients, respectively. Interestingly, a C-terminal fragment of Ro52 (Ro52-Delta2), a protein fragment not previously found to be antigenic by ELISA, also showed positive immunoreactivity in 42/57 SjS patients (65% sensitivity). Additional profiling of other autoantigens revealed that certain SjS patients also showed positive immunoreactivity with thyroid peroxidase (14%), AQP-4 (12%) and the H(+)/K(+) gastric ATPase (16%) suggesting potential autoantibody attack of thyroid, neuronal and gastric parietal cells, respectively. These heterogeneous autoantibody responses detected by LIPS in SjS will likely be useful for diagnosis and for evaluating extraglandular manifestations.