After overnight fast, 10 mL blood samples were collectedfrom both groups in test tubes containing sodium heparin asanticoagulant. Tubes were centrifuged at 3,500 rpm at roomtemperature for 15 minutes. Plasma and red blood cells wereobtained and deep frozen (at
80°C) until analysis time.
Determination of serotonin
The ELISA diagnostic kit used for serotonin was a productof IBL International, Germany. The assay procedure fol-lows the basic principle of competitive ELISA whereby there is competition between a biotinylated and a non-biotinylated antigen for a fixed number of antibody bindingsites. The amount of biotinylated antigen bound to theantibody is inversely proportional to the analyte concentra-tion in the sample. When the system is in equilibrium, thefree biotinylated antigen is removed by a washing step andthe antibody-bound biotinylated antigen is determined by use of anti-biotin alkaline phosphatase as marker and p-nitro phenyl phosphate as a substrate. Quantification of unknowns is achieved by comparing the enzymatic activity of unknowns with a response curve prepared by usingknown standards.
Determination of dopamine
The quantitative determination of dopamine in humanplasma was measured using ELISA diagnostic kit, a prod-uct of American Laboratory Products Company (ALPCO),United States.Dopamine was extracted by using a cis-diol-specificaffinity gel, acylated and then derivatized enzymatically.The competitive ELISA kit used the microtiter plateformat. The antigen is bound to the solid phase of themicrotiter plate. The derivatized standards, controls, andsamples and the solid phase-bound analytes competedfor a fixed number of antiserum binding sites. After thesystem was in equilibrium, free antigen and free antigen-antiserum complexes were removed by washing. Theantibody bound to the solid phase was detected by ananti-rabbit IgG-peroxidase conjugate using tetramethyl-benzidine as a substrate. The reaction was monitored at450 nm. Quantification of unknown samples was achievedby comparing their absorbance with a reference curve pre-pared with known standard concentrations.
Determination of gamma aminobutyric acid (GABA)
The quantitative determination of GABA in human plasmawas measured using ELISA diagnostic kit, a product of Immunodiagnostic AG, Germany. This assay was based onthe method of competitive enzyme linked immunoassays.The sample preparation included the addition of a derivati-zation reagent for GABA. Afterwards, the treated sampleswere incubated in wells of a microtiter plate coated with apolyclonal antibody against GABA-derivative, together withassay reagent containing GABA-derivative (tracer). Duringthe incubation period, the target GABA in the sample com-peted with the tracer for the binding of the polyclonal anti-bodies on the wall of the microtiter wells. GABA in thesample displaced the tracer out of the binding to the anti-bodies. Therefore, the concentration of antibody-boundtracer was inversely proportional to the GABA concentra-tion in the sample.During the second incubation step, a peroxidase conju-gate was added to each microtiter well to detect the tracer.After being washed away, the unbound components tetra-methylbenzidine were added as a peroxidase substrate. Fi-nally, the enzymatic reaction was terminated by an acidicstop solution. The color changed from blue to yellow, andthe absorbance was measured at 450 nm using a spec-trophotometer. The intensity of the yellow color was in- versely proportional to the GABA concentration in thesample; therefore, a high GABA concentration in the sam-ple reduced the concentration of antibody-bound tracerand lowered the photometric signal. A dose responsecurve of absorbance unit (optical density at 450 nm) vs.concentration was generated using the values obtainedfrom the standards. GABA present in the patient samples(EDTA plasma) was determined directly from this curve.
Determination of oxytocin
The ELISA assay kit was a product of BioSource, USA. Thisimmunoassay kit allows the
quantitative determin-ation of human oxytocin concentrations in plasma. The mi-crotiter plate provided in this kit had been pre-coated withan antibody specific to oxytocin. Standards or samples werethen added to the appropriate microtiter plate wells with ahorseradish peroxidase (HRP)-conjugated antibody and in-cubated. Substrate solutions were then added to each well.The enzyme-substrate reaction was terminated by the ad-dition of a sulfuric acid solution and the color change wasmeasured spectrophotometrically at 450 nm. The concen-tration of oxytocin in the samples was then determinedby comparing the optical density of the samples to thestandard curve.
Determination of interferon-
-inducible protein 16 (IFI16)
The human IFI16 ELISA kit was provided with by Bio-Source. This immunoassay kit allows for the
quantitative determination of human IFI16 concentra-tions in plasma. The IFI16 ELISA kit applied the quanti-tative sandwich enzyme immunoassay technique. Themicrotiter plate had been pre-coated with a monoclonalantibody specific for IFI16. Standards or samples werethen added to the microtiter plate wells and IFI16, if present, bound to the antibody pre-coated wells. Inorder to quantitatively determine the amount of IFI16present in the sample, a standardized preparation of
et al. Journal of Neuroinflammation
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