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Background: Isatuximab, an IgG-kappa (IgGκ) anti-cluster of differentiation 38 (CD38) monoclonal antibody approved for use in patients with relapsed or refractory multiple myeloma (MM), can potentially interfere with the visualization of endogenous monoclonal protein (M-protein) on standard immunofixation electrophoresis (IFE) and lead to inaccurate classification of a patient's response to therapy. The Hydrashift 2/4 isatuximab IFE assay (Hydrashift isatuximab assay) removes isatuximab interference from IFE. Using samples from patients enrolled in clinical trials of isatuximab-based therapy for MM, we demonstrate how the Hydrashift isatuximab assay improves the ability to detect residual M-protein and offer recommendations for when the assay is most useful., Methods: Samples from 141 patients with a variety of known M-protein isotypes were selected and analyzed by standard IFE and the Hydrashift isatuximab assay. A positive control containing isatuximab was run on every standard IFE and Hydrashift gel., Results: The Hydrashift isatuximab assay reliably shifted the migration of isatuximab in patient samples. Standard IFE was adequate for determining 104 patients' M-protein status, and the Hydrashift isatuximab assay confirmed these results. In samples from 37 patients with a history of IgGκ MM and a single IgGκ band visible on standard IFE near the isatuximab migration site, the Hydrashift isatuximab assay was able to separate isatuximab from endogenous M-protein, identifying residual M-protein in 17 samples and preventing false-positive interpretations of standard IFE in 20 samples., Conclusions: The Hydrashift isatuximab assay is most useful in patients with known IgGκ MM when a single IgGκ band appears near the isatuximab migration site on standard IFE during isatuximab-based therapy., Clinicaltrials.gov Registration Numbers: NCT03275285 and NCT03319667., (© Association for Diagnostics & Laboratory Medicine 2024.)

Background: Plasma cell disorders (PCDs) are typically characterized by excessive production of a single immunoglobulin, defined as a monoclonal protein (M-protein). Some patients have more than one identifiable M-protein, termed biclonal. Traditional immunofixation electrophoresis (IFE) cannot distinguish if two bands of the same isotype represent biclonal proteins or M-proteins with some other feature. A novel assay using immunoenrichment coupled to matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (Mass-Fix) was applied to determine whether two bands of the same isotype represented (1) monomers and dimers of a single M-protein, (2) an M-protein plus a therapeutic monoclonal antibody (t-mAb), (3) an M-protein with light chain glycosylation, or (4) two distinct biclonal M-proteins., Methods: Patient samples with two bands of the same isotype identified by IFE were enriched using nanobodies against IgG, IgA, IgM, or κ and λ light chains then analyzed by Mass-Fix. Light chain masses were used to differentiate IgGκ M-proteins from t-mAbs. Mass differences between peaks were calculated to identify N-glycosylation or matrix adducts. High-resolution mass spectrometry was used as a comparator method in a subset of samples., Results: Eighty-one residual samples were collected. For IgA, 93% (n = 25) were identified as monoclonal. For IgG, 67% (n = 24) were monoclonal, and 33% (n = 12) were truly biclonal. Among the monoclonal IgGs, the second band represented a glycosylated form for 21% (n = 5), while 33% (n = 8) had masses consistent with a t-mAb. 44% (n = 8) of IgM samples were biclonal, and 56% (n = 10) were monoclonal, of which one was glycosylated., Conclusions: We demonstrate the utility of mass spectrometry in the characterization of multiple IFE bands of the same isotype. Improved reporting accuracy of M-proteins is useful for monitoring of patients with PCDs., (Copyright © 2021 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Accurate determination of complement component C1q is hampered by the fact that C1q is an immune complex binding protein. Consequently, immunochemical techniques which rely on immune complex formation in fluid phase such as nephelometry and turbidimetry tend to give results which differ from those obtained by, for example, ELISA and other solid phase-based assays. In this chapter, we discuss the pros and cons of different techniques for the quantification of C1q and present a comprehensive protocol for a newly developed magnetic bead-based sandwich immunoassay which has replaced nephelometry in our complement diagnostic laboratory at the University Hospital in Uppsala.

Objective: Measurement of monoclonal immunoglobulins is a reliable estimate of the plasma cell tumor mass. About 15% of plasma cell myelomas secrete light chains only. The concentration of serum free light chains is insufficient evidence of the monoclonal light chain burden. A sensitive quantitative estimate of serum free monoclonal light chains could be useful for monitoring patients with light chain myeloma. We describe such an assay that does not require mass-spectrometry equipment or expertise., Methods: Serum specimens from patients with known light chain myelomas and controls were subjected to ultrafiltration through a membrane with pore size of 50 kDa. The filtrate was concentrated and tested by immunofixation electrophoresis. The relative area under the monoclonal peak, compared to that of the total involved light chain composition, was estimated by densitometric scanning of immunofixation gels. The proportion of the area occupied by the monoclonal peak in representative densitometric scans was used to arrive at the total serum concentration of the monoclonal serum free light chains., Results: Using an ultracentrifugation and concentration process, monoclonal serum free light chains were detectable, along with polyclonal light chains, in all 10 patients with active light chain myelomas. Monoclonal light chains were identified in serum specimens that did not reveal monoclonal light chains by conventional immunofixation electrophoresis. The limit of detection by this method was 1.0 mg/L of monoclonal serum free light chains., Conclusion: The method described here is simple enough to be implemented in academic medical center clinical laboratories and does not require special reagents, equipment, or expertise. Even though urine examination is the preferred method for the diagnosis of light chain plasma cell myelomas, measurement of the concentration of serum free light chains provides a convenient, albeit inadequate, way to monitor the course of disease. The method described here allows effective electrophoretic differentiation of monoclonal serum free light chain from polyclonal serum free light chains and provides a quantitation of the monoclonal serum free light chains in monitoring light chain monoclonal gammopathies., (© American Society for Clinical Pathology 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Serum protein electrophoresis (SPE) is a widely used laboratory technique to diagnose patients with multiple myeloma (MM) and other disorders related to serum protein. In patients with MM, abnormal monoclonal protein can be detected by SPE and further characterized using immunofixation electrophoresis (IFE). There are several semi-automated agarose gel-based systems available commercially for SPE and IFE. In this study, we sought to evaluate the analytical performance of fully automated EasyFix G26 (EFG26) and semi-automated HYDRASYS 2 SCAN (H2SCAN) for both SPE and IFE., Methods: Both instruments were operated according to manufacturer's instructions. Samples used include a commercially available normal control serum (NCS) and patients' specimens. The following were evaluated: precision and comparison studies for SPE, and reproducibility and comparison studies for IFE. Statistical analyses were performed using Microsoft Excel., Results: For SPE repeatability study, our results showed that EFG26 has higher coefficient of variation (%CV) compared with H2SCAN for both samples except for monoclonal component with %CV of 0.97% and 1.18%, respectively. Similar results were obtained for SPE reproducibility study except for alpha-1 (4.16%) and beta (3.13%) fractions for NCS, and beta fractions (5.36%) for monoclonal sample. Subsequently, reproducibility for IFE was 100% for both instruments. Values for correlation coefficients between both instruments ranged from 0.91 to 0.98 for the five classic bands., Conclusion: Both instruments demonstrated good analytical performance characterized by high precision, reproducibility and correlation., (© 2020 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals, Inc.)

Background: Daratumumab, a monoclonal antibody used to treat relapsed or refractory multiple myeloma, can interfere with protein electrophoresis and immunofixation assays. False-positive immunofixation results due to daratumumab can cause uncertainty regarding the status of a patient's disease and lead to potential misclassification of their response to therapy. The Hydrashift 2/4 Daratumumab assay (Sebia) was recently cleared by the Food and Drug Administration for resolving daratumumab interference on immunofixation. Here, we evaluate the performance of the Hydrashift assay in multiple myeloma patients receiving treatment with daratumumab-based regimens., Methods: Waste serum samples from multiple myeloma patients (n = 40) receiving daratumumab were analyzed by standard immunofixation and the Hydrashift assay. Results from these tests were compared and were evaluated along with pretreatment serum protein electrophoresis and immunofixation results, if available., Results: The Hydrashift assay shifted the migration of daratumumab in patient samples. In 27 cases, the patient's M protein was distinguishable from daratumumab by standard immunofixation. In these cases, the Hydrashift assay confirmed that the IgGκ band was daratumumab and helped identify the presence of treatment-related oligoclonal bands. There were 11 instances in which the patient's IgGκ M protein comigrated with daratumumab. In all 11 cases, the Hydrashift assay confirmed the presence of residual M protein. Finally, in 2 patients whose pretreatment immunofixation results were not available, the Hydrashift assay confirmed that the IgGκ band visible on immunofixation was due to daratumumab alone., Conclusions: The Hydrashift 2/4 Daratumumab assay is a useful tool to clarify the source of an IgGκ band on immunofixation and allow a patient's M protein to be viewed without interference., (© 2018 American Association for Clinical Chemistry.)

Background: Monoclonal free light chains (FLC) commonly exist in monomeric or dimeric forms but rarely as larger molecules. Little is known about whether polymeric molecules can affect urine protein electrophoresis (UPE) results., Methods: Urine samples were collected from 72 multiple myeloma (MM) patients with Bence Jones protein (BJP). Urine protein and immunofixation electrophoresis were analyzed on Sebia SDS "agarose" gel electrophoresis system (SDS-AGE), and immunoglobulin free light chains were measured on the BNII nephelometric assay., Results: A type of disulfide-bound FLC dimer shows a pattern shift to the position of the "albumin" band in urine protein electrophoresis in multiple myeloma (MM) patients according to the Sebia agarose gel-based detection system, which was validated by immunofixation, SDS-PAGE, and mass spectrometric methods. Similar cases were found in 21 (29.17%) of 72 MM patients with BJP, and 19 (90.5%) of 21 patients were the lambda type., Conclusions: These results indicate that BJP with lambda type has a strong tendency to abnormally migrate, which may increase the risk of misinterpretation of protein electrophoresis in clinics. Thus, when the urine protein electrophoresis is inconsistent with the result by nephelometric method, urine protein electrophoresis needs to be repeated on the deduced condition to confirm the essence of the originally identified "albumin.", (© 2018 Wiley Periodicals, Inc.)

Polyclonal antibodies including purified antibody fractions and animal or human antisera may react with unknown antigens or antigens other than their main specificity in reactions that are best visualized by gel electroimmunoprecipitation methods (e.g., when analyzing complex antigen mixtures). The great advantage of gel immunoprecipitation approaches is that each immunoprecipitate contains antigen in a pure form and that the precipitate is separated by position, shape, and size from other precipitates in the complex patterns of crossed immunoelectrophoresis. The identification of the antigen content of such immunoprecipitates is important but challenging because of the very stable, high-affinity complex formation leading to precipitation in the gels. Here, we present detailed step-by-step recipes for identifying the antigen content of electroimmunoprecipitates.

Immunoelectrophoresis (IEP) was the first practical method that combined electrophoresis and immunoprecipitation for identifying and characterizing proteins within complex mixtures. Over the years, IEP has been extended to include a variety of techniques and, as a general name, has been applied to virtually any technique that involves electrophoresis and antigen-antibody precipitin reaction for proteins. Because of the diversity in technical details of different IEP versions, the method described here deals only with classic IEP. Although it requires some manual expertise, IEP is versatile, relatively easy to customize, and economical with no need for expensive instrumentation. Further, it can discern identity, partial identity, and nonidentity of the proteins. Any low-viscosity body fluid specimen or, possibly, culture fluid and tissue extract could be tested with IEP if proper antibodies are available. With these attributes, classic IEP remains a valuable tool for clinical diagnostic testing, purity checking of biochemical and pharmaceutical products, and research.

Immunofixation is currently very used in medical laboratories. The interpretation of the results is usually easy, but some cases raise interpretative problems. We here report two cases difficult to interpret. In the first case, we report a case of nonspecific precipitation of the protein on each track, in the second case we report a case of double monoclonal band on immunofixation electrophoresis. Reducing agent such as β2-mercaptoethanol used in these two cases allowed to solve the problem and to make a diagnosis. A comparison between clinical radiological and laboratory test data is necessary before making a diagnosis of monoclonal immunoglobulin.

Crossed immunoelectrophoresis (C-IE) is used to detect and quantify specific proteins. An application allowed the evaluation of complement system activation by nanomaterials. The work aimed to improve the C-IE toward a higher throughput and less tedious method. A new concept was implemented to prepare and run agarose gels. The first and the second dimension of electrophoresis were performed on a single gel plate, prepared before the beginning of the analysis. Several samples were migrated simultaneously on the same migration line. Up to 35 analyses were run at once, providing stamp-sized electrophoregrams (2.8 × 3 cm(2) ) maintaining the performance of the original method performed on 5 × 7 cm(2) gel slabs. Robustness and precision of the method were demonstrated through a validation approach using ANOVA. Handling, experimental duration, amount of reagents, and overall cost of one analysis were considerably reduced compared to the original method. With the same equipment, seven times more analyses can be performed in one run. C-IE can be used to analyze many types of proteins. The new experimental modalities were suitable for the application developed in the present work that was to evaluate activation of protein C3 of the complement system triggered by nanomaterials., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Monoclonal gammopathies are characterised by the production of a monoclonal immunoglobulin or free light chains by an abnormal plasma cell or B-cell clone and may indicate malignancy or a precursor (MGUS). There is currently no consensus on the initial test or combination of tests to be performed in suspected monoclonal gammopathies but serum protein electrophoresis and urine protein electrophoresis are commonly requested as initial investigations. If abnormal, immunofixation electrophoresis is then performed to confirm the presence of paraprotein and to determine its heavy and light chain type. Recently, some groups have developed simplified "screening" IFE methods for use in parallel to SPEP for the detection monoclonal gammopathies. We argue here that screening IFE may be of benefit in clinical laboratories using SPEP with poor resolution in the β-region, assisting in the detection of mainly IgA paraprotein, but may be of less benefit in laboratories utilising higher resolution gels. Further it may increase the detection of trace bands of questionable clinical significance, representing transient phenomena in infectious and auto-immune conditions or very low risk MGUS. The increased detection of these bands using screening IFE would require further patient follow up, possibly causing unnecessary patient anxiety and additional follow up healthcare costs.

The reliable detection of paraprotein in serum and urine is the primary purpose of electrophoretic procedures in clinical laboratories. Screening immunofixation electrophoresis (sIFE) employs a single application of antisera directed against heavy and light chains that facilitates the detection of paraproteins that migrate in the non-γ region or that are below the detection limit of protein electrophoresis. These paraproteins that are missed by routine electrophoresis occur in up to 27.3% of newly investigated and 13.6% of monitored patients. Small paraproteins missed by conventional electrophoretic techniques are clinically important in the diagnosis and monitoring of malignant plasma and B-cell disorders. The superior diagnostic performance of sIFE makes it suitable as the initial laboratory procedure to investigate paraproteins in complex serum and urine matrices.

The cellular prion protein (PrPC) is a glycoprotein, anchored to the plasma membrane and abundantly expressed in the central nervous system. The expression of PrPC in the peripheral tissues is low and only little information is available on its functions in the nonneuronal tissues. The antioxidant function of PrPC during the activation of hepatic stellate cells has already been reported. Therefore, the aim of the study was to expand our knowledge on the functions of PrPC by detailed characterization of its expressional profile in the liver. In a combined strategy by using capillary immunoelectrophoresis and standard techniques, we have shown a sexually dimorphic expression of PrPC in mice and human liver tissues. Further, we showed a significant age-dependent upregulation of PrPC expression in the liver of 14- and 9-month-old mice as compared to 3 months of age. Therefore, this study may provide new insights into the gender-specific role of PrPC in the liver, which may further be linked to its protective role against oxidative stress during aging. In addition, the current study also shows an application of immunoelectrophoresis with a low coefficient of variation to analyze the miniscule amount of PrPC in the mouse liver tissue., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Low-density lipoprotein (LDL) particle (P, or molar) concentration has been shown to be a more sensitive marker of cardiovascular disease (CVD) risk than LDL cholesterol. Although elevated circulating lipoprotein(a) [Lp(a)] cholesterol and mass have been associated with CV risk, no practicable method exists to measure Lp(a)-P. We have developed a method of determining Lp(a)-P suitable for routine clinical use., Methods: Lipoprotein immunofixation electrophoresis (Lipo-IFE) involves rigidly controlled electrophoretic separation of serum lipoproteins, probing with polyclonal apolipoprotein B antibodies, then visualization after staining with a nonspecific protein stain (Acid Violet). Lipo-IFE was compared to the Lp(a) mass assay for 1086 randomly selected patient samples, and for 254 samples stratified by apo(a) isoform size., Results: The Lipo-IFE method was shown to be precise (CV <10% above the 50 nmol/l limit of quantitation) and linear across a 16-fold range. Lipo-IFE compared well with the mass-based Lp(a) assay (r=0.95), but was not affected by variations in apo(a) isoform size. With a throughput of 100 samples in 90 min, the assay is suitable for use in the clinical laboratory., Conclusions: The Lipo-IFE method will allow Lp(a)-P to be readily tested as a CVD risk factor in large-scale clinical trials., (Copyright © 2014. Published by Elsevier B.V.)

Polyclonal antibodies including purified antibody fractions and animal or human antisera may react with unknown antigens or antigens other than their main specificity in reactions that are best visualized by gel electroimmunoprecipitation methods, e.g., when analyzing complex antigen mixtures. The great advantage of gel immunoprecipitation approaches is that each immunoprecipitate contains antigen in a pure form and that the precipitate is separated by position, shape, and size from other precipitates in the complex patterns of crossed immunoelectrophoresis. The identification of the antigen content of such immunoprecipitates is important but challenging because of the very stable, high affinity complex formation leading to precipitation in the gels. Here, we present detailed step-by-step recipes for identifying the antigen content of electroimmunoprecipitates.

Cerebrospinal fluid (CSF) rhinorrhea is a serious condition that may result in severe complications. Various laboratory tests, relying on the detection of CSF-specific proteins in nasal secretions, have been developed but diagnosis remains challenging. The aim of this study was to evaluate two new methods targeting either ß2-transferrin or beta-trace-protein. Rhinorrhea samples from patients suspected of CSF leakage (n=36) were analyzed using two-dimensional gel electrophoresis (2-DE) for CSF rhinorrhea diagnosis. Twelve patients with rhinorrhea strongly suggestive of a CSF leak also underwent a fluorescein test. The same cohort was retrospectively analyzed with a beta-trace protein immunoblot developed in-house (n=36) and a new commercial ß2-transferrin immunofixation assay (Sebia, Evry, France) (n=33). 2-DE was positive in 9 patients suffering from rhinorrhea following skull base fracture (n=3), post-surgery (n=4), or spontaneously (n=2). The 27 remaining cases were negative. These results were confirmed by the beta-trace protein immunoblot and ß2-transferrin immunofixation tests, except for one sample found negative with 2-DE but positive with the two other assays. Results from the three analytical methods were concordant with fluorescein tests. Beta-trace protein immunoblot and ß2-transferrin immunofixation assays are fast and reliable methods that allow detecting CSF leakage in nasal fluid with high sensitivity and specificity., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Articles have debated whether or not urine analysis remains valuable for identifying monoclonal gammopathies. A general impression is that the newer serum free light chain (FLC) assay is more analytically sensitive, more quantitative and simpler to perform. Many laboratory directors may have seized on the idea of eliminating urine analysis because it is a tedious procedure and requires expert interpretation while most laboratories can perform automated serum FLC assay. Others have concluded that urine immunofixation electrophoresis (IFE) optimizes the diagnostic sensitivity and should be included when there is a clinical indication. Here, I show that papers faulting urine analysis often used inappropriate urine methodology and this helps explain why there was misinterpretation. Moreover, the literature, shows urine IFE is often more sensitive for identifying low-level monoclonal FLC than the serum assay because urine IFE is as sensitive when performed appropriately and generally more specific. Besides, the reference range for serum FLC assay is unclear which is a great problem in assessing response to treatment and in identifying diseases when there is low concentration monoclonal FLC. I conclude that urine IFE remains important and is complementary to serum FLC assay, although the best algorithms for use remains to be elucidated.

An important step in vaccine production and quality control is the analysis of identity of different lots. For that purpose chicken was immunized with acellular Pertussis components (Pertussis toxoid, Filamenteous haemagglutinin, Pertactin, Fimbriae 2/3 antigen). The resulting antibodies (IgY) were non-invasive extracted from egg yolk and used for rocket immunoelectrophoresis (RIE). We demonstrated that the Ab reacted with characteristic peaks ("rockets") with the corresponding antigen. The shape of the peaks varied depending on the manufacturer and the nature of antigen (adsorbed or non-adsorbed). The coefficients of variation was about 20% during a year period. In summary, our data illustrate that an IgY-based RIE is not only a cost-effective method but also proficient for monitoring Pertussis vaccines., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A robust and highly reproducible capillary isoelectric focusing (cIEF) method for the evaluation of charge heterogeneity of monoclonal antibody (mAb) pharmaceutical which contains covalently bound antitumor compounds was developed using a combination of commercially available dimethylpolysiloxane-coated capillary and carrier ampholyte. In order to optimize major analytical parameters for robust mobilization, experimental responses from three pI markers were selected. The optimized method gave excellent repeatability and intermediate precision in estimated pI values of charge variants with relative standard deviations (RSDs) of not more than 0.06% and 0.95%, respectively, when using IgG(4) as a model. Furthermore, RSDs of charge variant compositions were less than 5.0%. These results suggest that the proposed method can be a powerful tool for reproducible evaluation of charge variants of both naked mAbs and their conjugates with high resolution, and it is applicable to quality testing and detailed characterization in the pharmaceutical industry. In addition, it should be noticed that the method provided non-linear pH gradient within the tested ranges, from pI 9.50 to 3.78, and the pH gradient caused the inconsistency of estimated pI ranges between cIEF and gel IEF. This result indicates that selecting appropriate pI markers based on the target pI ranges of charge variants for each mAb related pharmaceutical is highly recommended for the precise determination of pI values., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In this study, novel polysulfone-based cation-exchange membranes with strong mechanical strength have been developed and applied in ion-exchange-membrane-partitioned free-flow IEF (IEM-FFIEF) to replace the conventional immobiline membranes. A fundamental understanding of protein mass transfer in the IEM-FFIEF process has been revealed experimentally with the aid of membrane-based boundary effect model contributed by Ennis et al. we have proven experimentally the existence of a pH gradient across the membrane cross-section when an IEM-FFIEF system is in operation. The boundary effects on particle velocities are calculated based on the IEF assumption and various characterizations, and are compared with the experimental results. In the IEM-FFIEF experiments, a protein mixture (BSA and myoglobin (Mb)) and sulfonated polysulfone membranes with different ion-exchange capacities are applied. Experimental results show that the real velocity and real mobility (of Mb in this study) are comparable with the mathematic model developed by Ennis et al. This suggests that the equation proposed by Ennis et al., is sufficient to capture the mass transfer through membrane in the IEM-FFIEF system after considering the effects of pore size distribution and effects of disturbed electric field. The charge properties of the membrane surface play a dominant role on the separation performance of the membranes. The newly developed porous solid-phase ion-exchange membranes may potentially and effectively replace immobilines to perform the selective function for protein separation.

Objective: To compare the clinical value of microcentrifugalcolumn method with crossed affinity immunoelectrophoresis autoradiography method for the measurement of alpha-fetoprotein variant (AFP-L3) in differentiation of benign and malignant liver., Methods: Serum AFP-L3 variants in 102 primary hepatocellular carcinoma patients and 41 chronic and cirrhosis patients were separated by microcentrifugalcolumn method and crossed affinity immunoelectrophoresis autoradiography method and the clinical value of both method were compared., Results: In 102 primary hepatocellular carcinoma patients, the sensitivity of AFP-L3 was 79.4% and 91.2% respectively, in 41 chronic and cirrhosis patients. The specificity of AFP-L3 was 70.7% and 29.3% respectively. The diagnostic accuracy was 76.9% and 73.4% respectively. The area in the ROC curve was 0.791 and 0.758 respectively. In the 4 primary hepatocellular carcinoma patients with lower AFP-L3, the AFP-L3 was positive by useing microcentrifugalcolumn method, but none of AFP-L3 were found by useing crossed affinity immunoelectrophoresis autoradiography method., Conclusion: The micro centrifugalcolumn method is more simple and rapid, it may be more useful in differentiation of benign and malignant liver than traditional crossed affinity immunoelectrophoresis autoradiography method.

Our objective was to evaluate a gel system that uses unconcentrated urine specimens for protein electrophoresis (PEL) and immunofixation electrophoresis (IFE) in patients with monoclonal gammopathies. For the study, 222 urine specimens were analyzed by our current PEL method (Helena Laboratories, Beaumont, TX) and by a system that recommends use of unconcentrated urine (Sebia, Norcross, GA). M protein concentrations were compared in the 43 cases with a measurable M spike. IFE was performed on 111 of the samples using both methods. There was a 97% concordance for detection of PEL abnormalities. The concordance for IFE was 98%. M protein concentrations by the 2 methods correlated well (r2=0.99; slope, 1.04). Cases with insufficient urine volumes for concentration (PEL, 7; IFE, 20) were analyzed in the Sebia gel system, and in 11 cases (PEL, 2; IFE, 9) an M protein was identified.High-resolution gel electrophoresis of urine using the Sebia system offers similar performance for detection, characterization, and quantification of M proteins when compared with our current gel system. Testing unconcentrated urine specimens will mean fewer sample rejections owing to insufficient sample volume.

A method for the identification of protein antigens captured in electroimmunoprecipitates was developed. Different antigen-antibody precipitates were generated by agarose gel immunoelectrophoresis. The immunoprecipitates were excised and various methods for extracting and dissociating the precipitates were systematically studied by analyzing for protein components of the extracts using peptide mass fingerprinting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal recovery of antigen was obtained by 24-h extraction at 37 degrees C using a minimal volume of 0.06 M Tris-HCl, 10% SDS (pH 7). This simple and robust method is useful for the characterization of antibody specificity. It can also be used to identify antigens generating unknown precipitates in crossed immunoelectrophoresis with polyspecific antisera, including human IgG-antigen complexes electroimmunoprecipitated by secondary antibodies. Thus, the method may prove useful as an additional technique in biomarker discovery.

Immunoelectrophoresis can be used for analysis of individual proteins in complex mixtures. The conditions involved in immunoelectrophoresis are mild, avoiding the risk of denaturation, and it is possible to perform relative quantification of individual components. The principle disadvantage is the dependence on rabbit antisera as reagents. The usefulness of immunoelectrophoresis in allergy research is greatly enhanced by the possibility of identification of allergens to which the individual in question has IgE. The common principle is characterized by two independent electrophoreses having direction of current perpendicular to each other, i.e., crossed immunoelectrophoresis (CIE). This ultimately results in the formation of characteristic bell-shaped precipitates, each precipitate representing one antigen. There is a linear relationship between the amount of antigen and size of precipitate for a given antibody concentration for each precipitate and so relative quantification can be performed. The sensitivity and resolution power of CIE is very high and there are multiple variations of the technique, some of which will be illustrated in this chapter.

Objective: Gc globulin (vitamin D-binding protein) is a component of the extracellular actin scavenger system. The level of Gc globulin is reduced in patients with fulminant hepatic failure, septic shock and trauma. Furthermore, low levels of Gc globulin in patients with fulminant hepatic failure and multiple trauma have been found to correlate with the morbidity and mortality of patients. Owing to a large increase in the turnover of Gc globulin upon complex formation with actin, it may be important to determine both the total Gc globulin concentration and the degree of complexing with actin for estimating the clinical prognosis of a patient. For this reason, we have compared a crossed immuno-electrophoresis method (CIE), suitable for visualizing the degree of complexing with actin, with a rocket immuno-electrophoresis method (RIE), previously used for determination of the complex degree., Material and Methods: Sera from healthy donors and from patients with acetaminophen-induced liver disease or trauma were investigated using CIE, RIE and enzyme-linked immunosorbent assay (ELISA)., Results: Using the CIE, no Gc globulin-actin complexes were detected among healthy donors. Complexes were present in 21 of 39 patients with liver disease and 3 of 37 trauma patients. High complex ratios (> 20 %) were found in 6 of 7 patients with hepatic encephalopathy. Using the RIE, complexes were detected in most samples., Conclusion: The results show that the CIE method may be used for determining the degree of actin complexing in conjunction with ELISA or RIE in determining the levels of total Gc globulin.

Recently, turbidimetric immunoassays for detecting and quantifying kappa and lambda free light chains (FLC) have become available and are promoted as being more sensitive than immunofixation electrophoresis (IFE) in detecting FLC monoclonal proteins. In this study, we assessed the ability of these turbidimetric assays to detect serum monoclonal proteins involving both free and heavy-chain-bound kappa and lambda light chains compared to standard immunofixation electrophoresis. Sera demonstrating a restricted band of protein migration (other than a definite M spike) by serum protein electrophoresis (SPE), which may represent early monoclonal proteins, were also examined. When compared to IFE, percent agreement, sensitivity, and specificity for the kappa-FLC and lambda-FLC were 94.6, 72.9, and 99.5% and 98.5, 91.4, and 99.7%, respectively, in detecting monoclonal proteins involving free and heavy-chain-bound light chains. The majority of sera (73.7%) demonstrating a restricted band of protein migration on SPE demonstrated abnormal IFE patterns suggestive of multiple myeloma or monoclonal gammopathy of unknown significance, but gave normal kappa/lambda FLC ratios using the turbidimetric immunoassays. In conclusion, the kappa and lambda FLC assays are significantly less sensitive (72.9 to 91.4%) than IFE, but specific in detecting serum monoclonal proteins. Moreover, the kappa/lambda ratio has little value in routine screening since the majority of sera with abnormal IFE patterns had normal kappa/lambda FLC ratios.

Purpose: Retrospective studies have shown that immunoassays measuring free light chains (FLC) in serum are useful for diagnosis and monitoring of multiple myeloma. This study prospectively evaluates the use of FLC assays and, for the first time, investigates the relationship between serum FLC concentrations and the presence and detectability of Bence Jones (BJ) proteins in the urine., Patients and Methods: Three hundred seventy-eight paired samples of serum and urine were tested from 82 patients during the course of their disease. The sensitivities of serum FLC analysis and urine immunofixation electrophoresis (IFE) in detecting monoclonal FLC were compared. Serum FLC concentrations required for producing BJ proteins detected by IFE were determined., Results: Abnormal FLC were present in 54% of serum samples compared with 25% by urine tests. In abnormal serum samples for kappa or lambda, the sensitivity of IFE to detect the respective BJ proteins in urine were 51% and 35% and the median serum FLC concentrations required to produce detectable BJ proteins were 113 and 278 mg/L. Renal excretions of monoclonal FLC increased with serum concentrations, but excretions significantly decreased at high serum concentrations combined with renal dysfunction., Conclusion: Serum FLC assays are significantly more sensitive for detecting monoclonal FLC than urine IFE analysis. They also have the advantage of FLC quantification and are more reliable for monitoring disease course and response to treatment.

A new kind of affinity electrophoresis called antibody affinity electrophoresis is a technique used to capture protein antigens based on their interactions with specific monoclonal or polyclonal antibodies incorporated in the polyacrylamide gel. Polyclonal anti-glutathione-S-transferase (anti-GST), monoclonal anti-bovine serum albumin (anti-BSA), and polyclonal anti-human alpha-lactalbumin are embedded in distinct areas of a 7.5% native polyacrylamide gel. Some of the embedded antibodies get covalently and/or noncovalently incorporated into the gel matrix network. Under electrophoresis conditions, these antibodies do not show significant electrophoretic mobility, as compared to their specific protein antigen analytes. We observed that electrophoretic migration of GST, BSA, and protein G ceases when they encounter anti-GST, anti-BSA, and immunoglobulin G, respectively.

The structural flexibility of antithrombin is essential for its molecular trapping mechanism but also makes it vulnerable to even minor changes affecting its conformational stability, which influences hemostasis significantly. The conformational transformation of this serpin has been poorly investigated in biologic samples because available immunologic methods hardly differentiate between different conformations of this protein. Crossed immunoelectrophoresis (CIE) in presence of heparin has been classically used to identify mutant antithrombins with low heparin affinity. We demonstrate that this method also separates native and relaxed antithrombin, permitting the analysis of conformational variations of this potent anticoagulant with just a few microliters of plasma. However, CIE does not distinguish between antithrombin conformations with reduced heparin affinity: latent, cleaved, thrombin-antithrombin complexes, or heparin-binding mutants. Therefore, clinical interpretation of CIE results should be examined with caution. Using this and other methods, and evaluating the functional activity of antithrombin, we analyzed the conformational transformation of antithrombin in biologic samples. We confirmed its transformation to the latent configuration by incubating it at 50 degrees C. This conformational change also occurs at 37 degrees C, supporting the idea that this process is involved in the senescence of antithrombin. However, fresh plasma contains only traces of latent antithrombin, suggesting that this conformation is rapidly cleared in vivo. Finally, small increases in temperature (to 40 degrees C) resulted in a faster conformational transformation of antithrombin. Fever has been suggested to have key structural, functional, and clinical consequences in patients with conformational mutations in antithrombin. Our results support a role for small changes in temperature in nonmutated antithrombin, suggesting that fever is a general risk factor for thrombosis.

The interest of serum protein immunofixation in myeloma and Waldenstr m's macroglobulinemia is widely known. It is not so well defined in other malignant hemopathies. The purpose of this study was to determine immunofixation abnormalities in malignant hemopathies other than multiple myeloma and Waldenstr m's macroglobulinemia. We selected serum immunofixations of 61 patients affected by malignant hemopathies and 53 patients affected by other pathologies susceptible to give immunofixation's alterations. We showed that the frequency of immunofixation abnormalities was more important in patients affected by malignant hemopathies than in patients affected by other pathologies (70.5% vs 35.8%). A high frequency of monoclonal immunoglobulins was found in patients with lymphoma (53.3%) and oligoclonal immunoglobulins in other hemopathies (48.2%). No significant difference of the frequency of the monoclonal immunoglobulin isotypes was found. In summary, this retrospective study demonstrates a high frequency of immunofixation abnormalities in malignant hemopathies other than multiple myeloma and Waldenstr m's macroglobulinemia and different immunofixation characteristics between lymphomas and other hemopathies.

Detection of benzo[a]pyrene diol epoxide (BPDE)-damaged DNA in a human lung carcinoma cell line (A549) has been performed using free zone affinity capillary electrophoresis with laser-induced fluorescence (LIF). Using BPDE as a model carcinogenic compound, the speed, sensitivity and specificity of this technique was demonstrated. Under free zone conditions, an antibody bound adduct was baseline-resolved from an unbound adduct in less than 2 min. The efficiencies of separation were in excess of 6 x 10(5) and 1 x 10(6) plates per meter for the antibody-bound and unbound adducts, respectively. Separation using a low ionic strength buffer permitted the use of a high electric field (830 V/cm) without the loss of resolving power. Using LIF detection, a concentration detection limit of roughly 3 x 10(-10) M was achieved for a 90-mer oligonuleotide containing a single BDPE. The use of formamide in the incubation buffer to enhance denaturing of DNA did not affect the stability of the complex between the antibody and the adducts. Using a fluorescently labeled BPDE-modified DNA adduct probe, a competitive assay was established to determine the levels of BPDE-DNA adducts in A549 cells.

beta-Trace protein is an immunological marker for the detection of cerebrospinal fluid traces. The aim of the study was to evaluate the predictive value of the beta-trace protein test. We investigated 145 specimens for cerebrospinal fluid using beta-trace protein. All case notes were retrospectively reviewed and tabulated for indication and procedure with focus on outcome. The specimens were analyzed by immunoelectrophoresis. beta-Trace protein was detected in 62 specimens and was absent in 83. In correlation with the clinical course, the clinical findings, the results of high-resolution computed tomography of the paranasal sinuses or petrous bone, computed tomographic cisternography, magnetic resonance imaging, indium 111-pentetic acid radionuclide cisternography, intraoperative findings, and visualization using sodium-fluorescein, there were no false-positive results. In 6 cases, a false-negative result occurred. The overall accuracy was 95.68%. The beta-trace test has a specificity of near 100% and a sensitivity of 91.17%. Analysis of beta-trace protein is a valuable test and has some advantages in comparison with the beta2-transferrin assay.

There has long been the requirement for a marker of well being and chronic stress that is, itself, unaffected by the transient stressors associated with sampling. Work done in the human, the rat and the dog suggests that salivary IgA may fulfil these criteria. This is the first report on the establishment of a saliva sampling technique and immunoelectrophoretic assay for quantification of secretory Immunoglobulin A (s-IgA) in the squirrel monkey (Saimiri sciureus). A safe and effective sampling method is described, as well as a detailed description of an accurate rocket immunoelectrophoretic assay developed for this study. Preliminary results demonstrate a large intra-individual variation, as well as inter-individual variation in s-IgA levels between successive days. This indicates that if stress is associated with decreased s-IgA levels in the squirrel monkey, the usefulness of s-IgA as a stress marker may be limited because analyses of large numbers of samples may be necessary in order to obtain significance.

Capillary zone electrophoresis (CZE) and immuno-subtraction electrophoresis (ISE) were evaluated for ability to detect and immunotype monoclonal proteins, compared with agarose gel electrophoresis (AGE) and immunofixation electrophoresis (IFE), respectively. Six hundred seventeen serum samples were analyzed with CZE and AGE to determine sensitivity and specificity in detecting IFE-confirmed monoclonal gammopathies. Both techniques detected all monoclonal spikes due to IgM (n = 8), IgG (n = 38), and free light chains (n = 3). Agarose gel electrophoresis, however, detected only 11 of 14 (79%) IgA monoclonal spikes detected with CZE. In a second study, 78 serum samples, 48 of which had a monoclonal gammopathy confirmed with IFE, were evaluated with ISE. Only 60% to 75% of the monoclonal gammopathies were correctly immunotyped with ISE by 4 readers blinded to the IFE immunotype. Thus CZE was more sensitive than AGE in detecting low concentrations of monoclonal proteins, but ISE is less accurate than IFE in determining the immunotype of the monoclonal gammopathy.

alpha 2-Macroglobulin (alpha 2M) is a major protease inhibitor in human plasma. In this study, serum alpha 2M was determined by means of immunoelectrophoresis (IEP), rocket immunoelectrophoresis (R-IEP), laser nephelometry (LN) and single radial immunodiffusion (SRID) in 133 subjects. The values obtained by these methods coincided well in most specimens, but some specimens showed disagreement. The sera of alpha 2M deficiency found by IEP were analyzed by the other methods. We found that R-IEP gave the results of alpha 2M deficiency most frequently while the other methods gave the results of normal alpha 2M level for some specimens. We studied on the presumption that the phenomenon is due to heterogeneity in molecular weight of alpha 2M. After alpha 2M was purified by affinity chromatography and gel filtration chromatography, we further employed gel-filtration on a high-performance liquid chromatography (HPLC) to isolate alpha 2M with different molecular size. Two peaks of immunoreactive alpha 2M were found by the gel-filtration HPLC. When we performed IEP for both portions, the first portion with higher molecular size showed larger rate of migration than the second portion. Serum alpha 2M quantitation with SRID is not appropriate because molecular weight influences on the determination by SRID. We conclude that R-IEP is best suitable for the detection of decrease of serum alpha 2M, although this method requires expertness of manipulation.

The first test for recognition of monoclonal gammopathies should be serum protein electrophoresis with high-resolution agarose gel. Serum protein electrophoresis should be performed whenever multiple myeloma, Waldenström's macroglobulinemia, primary amyloidosis, or a related disorder is suspected. Immunofixation is critical for the differentiation of a monoclonal from a polyclonal increase in immunoglobulins. Quantitation of immunoglobulins should be performed with a rate nephelometer. The viscosity of serum should be measured if the patient has signs or symptoms of hyperviscosity syndrome. A 24-hour urine specimen should be obtained for determination of the total amount of protein excreted each day. Immunofixation of the urine should be performed on every patient who has an M-protein level greater than 1.5 g/dL (15 g/L) in the serum or in whom multiple myeloma, Waldenström's macroglobulinemia, primary amyloidosis, or a related disorder is suspected.

Multimodal application of lectin affinity electrophoresis of alpha-fetoprotein (AFP) glycoforms is reviewed. Crossed affinity immunoelectrophoresis developed by Bøg-Hansen and others was extended to tandem-lectin affinity electrophoresis by tandem lining of two different lectin gels and to mixed-lectin affinity electrophoresis. By introducing an antibody-affinity blotting technique for detection of separated glycoforms of AFP, several two-dimensional combinations of lectin affinity electrophoresis became possible: two different lectins for the first and second dimension electrophoresis, lectin-gradient affinity electrophoresis, and electrophoretic separation of lectin isoforms in the first-dimension electrophoresis, followed by affinity electrophoresis against the separated lectin isoforms. Usefulness of the different modalities of lectin affinity electrophoresis for several analytical purposes has been described.