Comparison of oligoclonal immunoglobulin G bands in multiple sclerosis cerebrospinal fluid by immunoblotting and immunofixation

We previously analyzed unconcentrated cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) and other neurologic diseases by isoelectric focusing in agarose gel. We have now developed an immunoblot method for detection of oligoclonal IgG bands in unconcentrated MS CSF. The oligoclonal IgG band patterns seen after immunoblotting were compared with those of conventional immunofixation. Although immunoblotting was found to be rapid the resolution and intensity of oligoclonal IgG bands were somewhat better after immunofixation. Since immunofixation is simpler than immunoblotting, we recommend that clinical laboratories use immunofixation after isoelectric focusing to detect oligoclonal IgG bands in CSF.     

Immobilized pH gradient isoelectric focusing and immunoblotting for investigations of anti-Borrelia burgdorferi IgG antibodies.

Anti-Borrelia burgdorferi immunoglobulin G (IgG) responses in cerebrospinal fluid, serum, and joint fluid from Lyme disease patients were investigated by immobilized pH gradient (IPG) isoelectric focusing (IEF) in pH 4-10 and pH 4-7 gels. After focusing, the anti-B.-burgdorferi antibodies were blotted by affinity-driven transfer to antigen-coated polyvinylidene difluoride membranes (immunoblot) and the IgG antibodies were immunoenzymatically stained. IPG-IEF gels gave an excellent resolution of IgG and the immunoblot proved advantageous for the detection of anti-B. burgdorferi IgG antibodies. These antibodies, as judged from the electromigration characteristics, were found to contain oligoclonal as well as polyclonal subpopulations. This latter group included IgG antibodies that were inadequately resolved when separated by conventional carrier ampholyte IEF.     

Capillary isoelectric focusing immunoassay as a new nanoscale approach for the detection of oligoclonal bands

The detection of oligoclonal bands (OCBs) in cerebrospinal fluid is an indicator of intrathecal synthesis of immunoglobulins which is a neurochemical sign of chronic inflammatory brain diseases. Intrathecally synthesized IgGs are typically observed in patients with multiple sclerosis. The current standard protocol for the detection of OCBs is IEF on agarose or polyacrylamide gels followed by immunoblotting or silver staining. These methods are time consuming, show substantial interlaboratory variation and cannot be used in a high throughput‐approach. We have developed a new nanoscale method for the detection of OCBs based on automated capillary IEF followed by immunological detection. Evidence for intrathecal IgG synthesis was found in all tested patients (n = 27) with multiple sclerosis, even in two subjects who did not have oligoclonal bands according to standard methods. The test specificity was at 97.5% (n = 19). Our findings indicate that the novel OCB‐CIEF‐immunoassay is suitable for the rapid and highly sensitive detection of OCBs in clinical samples. Furthermore, the method allows for a higher sample throughput than the current standard methods.     

Detection of oligoclonal IgG bands in cerebrospinal fluid by immunofixation after isoelectric focusing on polyacrylamide gels with the PhastSystem.

This paper describes the detection of oligoclonal bands of IgG immunoglobulins in cerebrospinal fluids (CSF) using isoelectric focusing (IEF) and immunofixation with the PhastSystem equipment. The proteins were separated by IEF on polyacrylamide gels, pH 3-9. The IgG immunoglobulins were then fixed by overlaying the gel with antihuman IgG antiserum. Fixed immunoglobulins were detected with silver staining in the PhastSystem Development Unit. One microliter of serum or CSF containing 15 mg/L of IgG was found to be optimal. The procedure had good reproducibility both with regard to IEF and silver staining of the fixed immunoglobulins. The manpower need was reduced, and the procedure has been found to be useful in the clinical laboratory.     

Analysis of cerebrospinal fluid proteins by electrophoresis

The cerebrospinal fluid (CSF) is a specific ultrafiltrate of plasma, which surrounds the brain and spinal cord. The study of its proteins and their alteration may yield useful information on several neurological diseases. By using various electrophoretic separation techniques, several CSF proteins have been identified derived from plasma or from brain. Different one-dimensional methods, such as agarose gel electrophoresis and isoelectric focusing, are of similar value in identifying the non-specific oligoclonal bands, which are mainly helpful in the diagnosis of multiple sclerosis and other inflammatory diseases. Isoelectric focusing has a greater resolution than other one-dimensional methods, and it yields additional data about disease-associated proteins occurring in Alzheimer's disease, Huntington's chorea and amyotrophic lateral sclerosis. Silver-stained two-dimensional gels provide more information about the complex protein composition of CSF, particularly about proteins produced in the brain, such as apolipoprotein E and neuron-specific enolase. For the detection of oligoclonal antibodies, the investigation of protein changes revealed by Parkinson's disease, schizophrenia and Creutzfeldt-Jakob disease, and the analysis of CSF immune complexes, two-dimensional electrophoresis has a greater sensitivity.     

Subtyping of group specific component (GC) in micro-bloodstains and semen stains by isoelectric focusing in ultrathin immobilized pH gradient gels followed by enzyme immunodetection

A powerful method for group specific component (GC) subtyping with good resolution of GC bands by isoelectric focusing on ultrathin immobilized pH gradients is described. After separation, GC detection is achieved using a highly sensitive alkaline phosphatase conjugated enzyme-immuno system. The efficiency of the method in forensic case work for subtyping GC from diluted bloodstain extracts, blood micro-stains and semen stains is demonstrated. Furthermore, GC subtyping and selective detection of human GC provides the evidence for human origin of the stain. This is important when analyzing microstains with limited stain consumption.     

Analysis of human apolipoproteins C by isoelectric focusing in immobilized pH gradients

Apolipoproteins C are involved in many ways in the metabolism of plasma lipoproteins. Apolipoproteins C from the delipidated VLDL of 35 controls and 165 normo- and hyperlipoproteinemic patients were analyzed by isoelectric focusing on an immobilized pH gradient, pH 4.0-5.0, with 7 M urea, which raised the apparent pH range to 4.8-5.7. This method is an improvement over conventional isoelectric focusing with carrier ampholytes with regard to both resolution and reproducibility. Due to the high resolution (0.1 pH units per cm) additional apolipoprotein C-III bands: C-III0 A1, C-III0 A2, C-III1 C and C-III2 C (the designations A, anodic, and C, cathodic, refer to direction of migration on IEF in relation to the main band) are described for the first time. The possible artifactual nature of these protein bands could be excluded. Cleavage with neuraminidase and peptidases, immunological detection and/or two-dimensional electrophoresis were used to obtain more information. The additional bands seem, in part, to be hydrolysis products of carboxypeptidase A (C-III1 C, C-III2 C). The appearance of C-III1 C and C-III2C was dependent upon the serum triglyceride concentration. The percent distribution of C apolipoproteins in very low density lipoproteins (VLDL) from control serum agreed with previously published data. Apolipoproteins C can also be focused in immobilized pH gradients from VLDL and serum without delipidation.     

Screening of Cerebrospinal Fluid and Blood Sera of Multiple Sclerosis Patients for Oligoclonal Immunoglobulins by Capillary Electrophoresis

Summary Multiple sclerosis (MS) is an autoimmune disease characterized by the production of specific types of immunoglobulins into the central nervous system. These immunoglobulins appear as oligoclonal bands (OCBs) in agarose isoelectric focusing (IEF) of cerebrospinal fluid (CSF). Among the cases with clinically definite MS, up to 95% have oligoclonal IgG bands in their CSF. In this report, we describe a micellar electrokinetic capillary chromatography (MEKC) method for the separation of CSF and serum proteins. MEKC was performed using 25 mM borate buffer, pH 10, containing 25 mM SDS at 20 kV and normal polarity. High values of repeatability in migration times and of reproducibility in peak areas were obtained (R.S.D. values were less than 2%). Calibration graphs were linear up to 2000 mg L^–1. LOQ was 6.5 mg L^–1 and LOD determined as a signal to noise ration of 3:1 was 4.5 mg L^–1. Analysis of CSF and serum samples from patients with clinical definite MS and healthy individuals demonstrated the presence of two peaks migrating as -globulins in the CSF samples of patients. These peaks were absent from controls and the serum of the same patients. Correlation of the data obtained from IEF and MEKC analysis for 25 patients showed that the diagnostic sensitivity and specificity of MEKC were ca 89% and 92% respectively. The obtained results indicate that this MEKC method may be helpful for the diagnosis of multiple sclerosis. Capillary electrophoresis compared to flat bed IEF provides reproducible results, requires shorter analysis time, and allows direct quantitative determination.Presented at: International Symposium on Separation and Characterization of Natural and Synthetic Macromolecules, Amsterdam, TheNetherlands, February 5–7, 2003     

Molecular heterogeneity,detected by two electrophoretic micro procedures,of IgG in human cerebrospinal fluid (CSF)

Semi-automated electrophoretic procedures in the PhastSystem (Amersham Pharmacia Biotech) with micro polyacrylamide gels (PAGs) and SDS-PAG gradients were modified to analyze IgG in human cerebrospinal fluid (CSF) and matched serum samples with respect to the molecular IgG structure L-H-H-L. Isoelectric focusing (IEF) with specific immunofixation detected discrete IgG bands in CSF standing out against a polyclonal and monoclonal background pattern in CSF and serum; they were denoted oligoclonal bands (IgG OBs) (OB assay positive) indicating IgG synthesis in the central nervous system (CNS) of patients with subacute and chronic processes of inflammatory CNS disorders; assay was negative with identical (mirror) bands in CSF and serum for other CNS processes. IgG OBs were specified as lambda (kappa) IgG subfractions, precipitated with the anti-light (L) chains lambda (kappa) and anti-heavy (H) chain fragments (Fd, Fc, C(H)2) as well as with anti-F(ab')(2), and as duplex IgGs with kappa and lambda OBs at the same pI. With SDS-PAG gradient electrophoresis and specific immunofixation more than six IgG fractions were detected and classed according to apparent molecular weights of a S-sulfonated human IgG standard; they were characterized with the monospecific antibodies against the L and H chain fragments as 25, 50, 75, 100, 125 and 150 kD fractions containing combinations of L and H chains as well as mixtures of both L and H chain fragments of varying dimensions. Generally, this molecular IgG heterogeneity could not be connected with the IgG OB heterogeneity revealed by IEF; but single OBs in the strongly alkaline pH region of PAG may correspond to H fragments with basic pI. Nevertheless, evidence for the existence of both free L chains and the free H chain were revealed as specific OBs with IEF and with the anti-L and anti-H antibodies in the 25 kD and 50 kD fractions, respectively, of CSF samples of six patients with diverse CNS diseases. Further experiments are needed to elicit the origin of the molecular IgG heterogeneity during the immune response of subacute and chronic inflammatory processes in human CNS.     

Immunoblotting techniques with picogram sensitivity in cerebrospinal fluid protein detection

Agarose isoelectric focusing followed by blotting with nitrocellulose, nylon or polyvinylidene difluoride membranes, and immunochemical detection of cerebrospinal fluid IgG with various combinations of antisera, was evaluated. Polyvinylidene difluoride proved to be an easy-to-handle and reliable membrane for protein blotting. Among immunochemical visualization reactions, the most sensitive employed biotinylated goat anti-human IgG followed by streptavidin colloidal gold conjugate and silver enhancement in 20% w/v urea, allowing a sensitivity of less then 1 picogram IgG/band.     

Dynamic labeling of diagnostically significant microbial cells in cerebrospinal fluid by red chromophoric non-ionogenic surfactant for capillary electrophoresis separations

During bacterial infections of the central nervous system the number of microorganisms in the cerebrospinal fluid is often ranging from few up to hundreds of cells per milliliter. The electrophoretic techniques with the UV-detection reach a detection limit for whole cells of approximately 10⁷ cells per milliliter. The coupling of the filtration cartridge with capillary isoelectric focusing can improve the detection limit by four orders of magnitude. In order to improve the detection limit the red non-ionogenic surfactant 1-[[4-(phenylazo)phenyl]azo]-2-hydroxy-3-naphthoic acid polyethylene glycol ester, PAPAN 1000, has been prepared and used for the dynamic labeling of analytes before filtration of the sample with a concentration modulation in the analysis of proteins or microorganisms. Values of isoelectric points of labeled analytes have been calculated using pI markers detectable at 515 nm and have been found comparable with pI of the native compounds. Minimum detectable amounts of proteins and microorganisms were lower than nanograms and a hundred labeled cells, respectively. The introduced method, coupling of the filtration cerebrospinal fluid spiked with microorganisms and labeled by PAPAN, facilitates their rapid CIEF separation in the pH gradient pH range of 2–5 at their clinically important level 10¹ to 10² cells per milliliter.     

Dodecyl maltoside detergent improves resolution of hepatic membrane proteins in two-dimensional gels.

This report describes the incorporation of an alkyl maltoside detergent in two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) sample lysis buffer in order to improve resolution of protein patterns separated by nonequilibrium pH gradient electrophoresis. Membrane-associated proteins with alkaline isoelectric points form horizontal streaks on two-dimensional electrophoretograms when solubilized with conventional nonionic detergent. Dodecyl maltoside enhances protein delipidation during solubilization and improves pattern resolution and protein mobility.     

Reliable phenotyping of alpha-1-antitrypsin by hybrid isoelectric focusing in an ultranarrow immobilized pH gradient.

Genetically determined phenotypes of the highly polymorphic human alpha 1-antitrypsin were examined by hybrid isoelectric focusing in a narrow immobilized pH gradient. The chosen pH range from 4.45 to 4.75 was useful for identification and classification of the common PI M subtypes and a number of PI variants in the microheterogeneous regions of m6, m7, and m8. A high degree of resolution and an improved sharpness of PI bands was achieved with this excellent technique. It allowed the distinction of a new PI M variant, which has been designated M8, or Mingolstadt, according to the PI nomenclature. The pI difference of this mutant to the slightly cathodically located subtype M3 is approximately 0.001 pH unit. In addition, some common as well as rare phenotypes are presented.     

Horizontal two-dimensional electrophoresis of cerebrospinal fluid proteins with immobilized pH gradients in the first dimension.

A horizontal two-dimensional electrophoresis method with immobilized pH gradient isoelectric focusing supplemented with carrier ampholytes in the first dimension was applied to cerebrospinal fluid (CSF) proteins. About 300 protein spots could be detected on the silver-stained two-dimensional maps of CSF samples. This high-resolution method is a tool worthy of consideration for the research of CSF proteins and disease-specific changes in different neurological disorders.     

Advanced online mass spectrometry detection of proteins separated by capillary isoelectric focusing after sequential injection

Capillary isoelectric focusing hyphenated with mass spectrometry detection, following the sequential injection of the carrier ampholytes and the sample zone, is highly efficient for the characterization of proteins. The main advantage of the sequential injection protocol is that ampholytes, with pH ranges, which are not supposed to cover the isoelectric points of the sample components, can be used for separation. The method then allows online mass spectrometry detection of separated analytes either in the absence (substances that have left the pH gradient) or in the presence of low‐level ampholytes (substances that are migrating within the pH gradient). The appearance of the substances within, or outside the pH gradient depends on, e.g., the composition of the ampholytes (broad or narrow pH range) or on the composition of electrolyte solutions. The experiments performed in coated capillaries (with polyvinyl alcohol or with polyacrylamide) show that the amount and the injection length of the ampholytes influence the length of the pH gradient formed in the capillary.     

Determination of cefepime in plasma and cerebrospinal fluid by micellar electrokinetic chromatography with direct sample injection

A simple micellar capillary electrokinetic chromatography (MEKC) with UV detection is described for analysis of cefepime in plasma and cerebrospinal fluid by direct injection without any sample pretreatment. The separation of cefepime from biological matrix was performed at 25 degrees C using a background electrolyte consisting of tris(hydroxymethyl)aminomethane (Tris) buffer with sodium dodecyl sulfate (SDS) as the electrolyte solution. Under optimal MEKC condition, good separation with high efficiency and short analyses time is achieved. Several parameters affecting the separation of the drug were studied, including the pH and concentrations of the Tris buffer and SDS. Using cefazolin as an internal standard, the linear ranges of the method for the determination of cefepime in plasma and cerebrospinal fluid were 1-50 and 1-20 microg/mL, respectively; the detection limits of plasma (signal-to-noise ratio = 3; injection, 5 kV, 5 s) and cerebrospinal fluid (signal-to-noise ratio = 3; injection, 0.5 psi, 3 s) were 0.2 microg/mL and 0.3 microg/mL, respectively. Application of the proposed method for determination of cefepime in plasma and cerebrospinal fluid collected after intravenous administration of 2 g cefepime in patients with meningitis was demonstrated.     

The simplified technique of high resolution two-dimensional polyacrylamide gel electrophoresis: biomedical applications in health and disease

The application of our simplified technique of high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to human body fluids is reviewed. Serum/plasma protein changes associated with alcohol abuse, familial dyslipoproteinemia ("fish-eye" disease), and myocardial infarction are demonstrated. High resolution 2-D PAGE of amniotic fluid, cerebrospinal fluid, urine, and saliva is shown with reference to the work of others, and the detection of pink-violet staining "lumicarmines" in sweat and tear fluid is reported for the first time. General aspects relating to the methodology are discussed. These include sample preparation, the choice of electrophoresis conditions (denaturing or nondenaturing) and detection method (Coomassie Brilliant Blue or silver), and the effects of native protein pretreatment with sodium dodecyl sulfate prior to silver staining or isoelectric focusing gel shrinkage in glycerol prior to second-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Isoelectric focusing of human parotid salivary proteins in hybrid carrier ampholyte-immobilized pH gradient polyacrylamide gels.

Isoelectric focusing of human salivary proteins with carrier ampholyte-isoelectric focusing systems requires prior desalting and concentration of samples, a procedure which is time-consuming and requires relatively large volumes of samples. By contrast, immobilized pH gradient gels are more tolerant to salt loads. Thus pretreatment of samples consists only of centrifugation prior to isoelectric focusing. If larger loads (greater than 50 micrograms) are required, the samples may be concentrated by lyophilization and reconstitution in a smaller volume of water or by dialysis against 30% w/v polyethylene glycol. Immobilized pH gradient polyacrylamide gels (incorporating a hybrid carrier ampholyte system) of two pH ranges (pH 4-9 and pH 3.5-5.0) have been used to separate the proteins in human parotid saliva. The effects of urea on focused patterns were studied; in pH 4-9 gels it gave improved resolution of protein bands, whereas in pH 3.5-5.0 gels it prevented protein precipitation. The salivary proteins were then visualized by staining with Coomassie Brilliant Blue G-250 or a silver procedure. Using the latter, 25-30 well-resolved bands were formed on a pH 4-9 gel loaded with 20 micrograms of proteins. The method offers considerable advantages compared with carrier ampholyte-isoelectric focusing.     

A turning point in proteome analysis: sample prefractionation via multicompartment electrolyzers with isoelectric membranes

Sample prefractionation, as obtained via multicompartment electrolyzers with isoelectric membranes, greatly enhanced the load ability, resolution and detection sensitivity of two-dimensional (2-D) maps in proteome analysis. This was demonstrated with different samples. In an Escherichia coli total cell extract, analysis by a 2-D map run in a pH 4-5 gradient showed many more spots when prefractionated, as compared with standard maps available in databases such as SWISS-2DPAGE. Analysis of human plasma in the pH 3-6 range showed an increase in the number of highly acidic proteins in the fractionated sample compared to whole plasma. With both samples no protein precipitation or smears occurred and much larger sample amounts could be loaded upon prefractionation, so that a large number of spots could be visualized by Coomassie staining, which is fully compatible with subsequent matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis.     

Behaviour of substituted aminomethylphenol dyes in capillary isoelectric focusing with electroosmotic zone displacement

The behaviour of six substituted aminomethylphenol dyes, having pI values between 5.3 and 10.4, in capillary isoelectric focusing with electroosmotic zone displacement is described. Using untreated fused-silica capillaries and different neutral capillary conditioners in the catholyte, the low-molecular-mass dyes are shown to focus and elute reproducibly in the order of decreasing pI values. In the absence of proteins, the detection times of the dyes are independent of the sampled amount. Hence these substances permit the characterization of the pH gradient produced in this capillary isoelectric focusing method. With concurrent focusing of dyes and test proteins, a macromolecular impact on detection times (reduction of electroosmosis)‘ is revealed. The effect is shown to be dependent on the type and amount of proteins applied and has been observed with three different capillary conditioners. Nevertheless, mapping of the pH gradient with these dyes and determining the pI values of known proteins is shown to provide values in agreement with those in the literature. Hence the substituted aminomethylphenol dyes can be employed as pI markers in capillary isoelectric focusing with electroosmotic zone displacement. Further, focusing and separation of two of the six dyes by preparative recycling free fluid isoelectric focusing is described, illustrating that the substituted aminomethylphenol dyes are also applicable to other free fluid focusing methods.