Horse IgG (T) ELISA Set

Changes in levels of cytokines or soluble receptors in biological fluids may provide information on immunological pathomechanisms underlying the respective diseases. Here, we studied cytokine patterns of patients with autoimmune encephalitis (AE) before and after immunosuppressive treatment in order to identify possible biomarker candidates and to look for putatively involved pathomechanisms. We performed measurements in Cerebrospinal fluid (CSF) and serum of 7 patients suffering from AE with antibodies (ab) against Leucine-rich glioma-inactivated-protein 1 (LGI1) and 9 AE patients with Contactin-associated protein-like 2 (Caspr2) ab recruited from two tertiary AE centers in Magdeburg and Berlin, Germany. In the Magdeburg samples before and after treatment were available for the measurements and in the Berlin cohort samples were collected after treatment was initiated. First, we used a human cytokine array comprising 36 cytokines or soluble receptors to screen for biomarkers in CSF samples of 8 AE (before and after treatment), 4 herpes-simplex virus meningoencephalitis patients and 4 controls without neuroinflammation. Next, CCL2, CXCl10, CXCl13, Il -6 and sICAM1 were chosen as candidates and measured in CSF and serum with specific ELISA systems in all 16 AE patients, 14 controls without neuroinflammation and 7 herpes-simplex virus meningitis patients. Clinical outcome was assessed via modified Rankin scale. LGI1 and Caspr2 abs from the Magdeburg cohort were purified by chromatography. IgG subclasses of these LGI1 or Caspr2 abs were identified by immunoblot analysis. The levels of most candidate parameters were higher in the CSF of Caspr2 than of LGI1 AE patients and controls, but there were no significant changes of cytokine concentrations before and after initiating treatment. Thus, these parameters seem unsuited as surrogate biomarkers of disease. Significantly higher levels were observed in the CSFs of Caspr2 AE patients (CXCL13 and sICAM1) as well as in the serum of Caspr2 (CXCL10) and LGI1 AE patients (CXCL13) in comparison to control samples. These results suggest that neuro-immunological pathomechanisms may differ between Caspr2 and LGI1 AE patients. Caspr2 AE seems to elicit a higher immune response than LGI1 AE, which has no correlation to the respective IgG subclass combination of AE specific abs involved in each type of disease.

The screening for IgG subclass donor-specific antibodies (DSAs) in allograft recipients uses IgG1-4 subclass-specific monoclonal antibodies (mAbs) that should be mono-specific. The cross-reactivity discrepancies reported for IgG subclass-specific mAbs warranted a critical cross-reactivity pattern analysis of the IgG subclass-specific mAbs most commonly used to detect DSAs. We tested the reactivity of 2 anti-IgG1-, 3 anti-IgG2-, 1 anti-IgG3-, and 2 anti-IgG4-specific PE-conjugated mAbs against microbeads coated with IgG1-4 proteins separately. Each IgG subclass protein was coated at three densities on the beads (0.5, 1, and 2 pg of protein per 10(6) beads), and the PE-conjugated mAbs were titrated from 0.04 mu g/mL to 5 mu g/mL. The IgG subclass reactivity of the sample was acquired on the Luminex multiplex platform. Among the IgG subclass-specific mAbs, only the anti-IgG3 (clone: HP6050) mAb was mono-specific. All other mAbs tested were binding to IgG subclass proteins other than their respective immunogen, thereby being cross-reactive. IgG subclass cross-reactivity patterns were dependent on the concentration of both IgG subclass-specific mAbs and IgG1-4 protein targets coated onto the beads. With the current IgG subclass mAbs available, 3 of the 15 possible combinations of IgG1-4 subclass protein could be identified. While the remaining 12 unique combinations cannot be distinguished clearly, 6 groups that corresponded to two different unique combinations of IgG1-4 subclass protein could be identified. The dilution of serum samples and IgG subclass-specific mAbs, other than the anti-IgG3 (clone: HP6050), must be further optimized before their implementation in IgG subclass DSA screening in allograft recipients.