Quantitative Serum Free Light Chain Assay – Analytical Issues

Serum free light chain (FLC) assay is an important advance in the diagnosis and monitoring of monoclonal light chain diseases and a complementary test to serum protein electrophoresis and immunofixation. Immunoturbidimetric and immunonephelometric assays for serum FLC are available on routine chemistry analysers and can detect FLC down to ~1 mg/L. These assays use polyclonal anti-human FLC antisera and require acceptable imprecision, specificity, accuracy, and reproducibility between reagent batches to prevent under- or over-estimation of FLC concentration.Assay imprecision determined between reagent lots has a variation of 8–45% for FLC concentrations and 17–32% for the calculated κ/λ FLC ratio. Dilution studies indicate some over-recovery of FLC, which may depend upon the dilution matrix. However, greater discrepancies are underestimation from nonlinear reactions and overestimation possibly from interferences or multi-reactivity to polymeric FLC. Nonlinear monoclonal FLC give concentrations which are 2- to 6-fold increased at higher sample dilution and FLC measured on different platforms may not give the same results.Laboratory staff and clinicians should be aware of the analytical limitations of the FLC assay. Assay imprecision, especially with different lots of FLC reagent, may have a significant effect on changes in the FLC concentration and κ/λ FLC ratio. Sample dilution anomalies have the potential to confound result interpretation for patients with monoclonal light chain disease. These issues, if not adequately appreciated, have the potential to mislead clinical diagnosis and assessment of response to therapy.Serum free light chain (FLC) assay came into routine clinical laboratories following the publication in 2001 describing the presence of monoclonal FLC in 19/28 non-secretory myeloma (NSMM) patients at diagnosis.¹ Use of the assay has grown globally since that time and other retrospective clinical studies have shown the clinical utility of FLC in serum as a complementary test to serum protein electrophoresis (SPEP) for the diagnosis and monitoring of monoclonal light chain diseases.Monoclonal immunoglobulin free light chains are important tumour markers often present in serum and urine of patients with monoclonal gammopathies. Serum kappa (κ) and lambda (λ) FLC assays measure total polyclonal and monoclonal FLC in serum and the calculated κ/λ FLC ratio is a surrogate measure of clonality. Retrospective studies have shown serum FLC is clinically indicated for diagnosis and prognosis in plasma cell proliferative disorders, including primary amyloidosis (AL), NSMM, light chain multiple myeloma (LCMM), light chain deposition disease (LCDD) and solitary plasmacytoma; in documenting stringent complete response in multiple myeloma (MM); and for routine serial measurement to assess response in the oligosecretory diseases, NSMM, AL and LCDD.² Serial measurements may also be indicated in MM when serum M-protein is <10 g/L or urinary Bence Jones protein (BJP) excretion is <200 mg/24h.³ However, there is currently no data to support its use in the monitoring of MM where disease is measurable by other methods such as SPEP. (Refer also to the review on FLC by J Katzmann in this issue).     

Homogeneous Enzymatic Assay for L-Cysteine with βC-S Lyase

We have developed a new enzymatic assay for determining L-cysteine concentration. The method involves the use of βC-S lyase from Streptococcus anginosus, which catalyzes the α,β-elimination of L-cysteine to hydrogen sulfide, pyruvate, and ammonia. The production of pyruvate is measured by D-lactate dehydrogenase and NADH. The decrease in NADH was proportional to the L-cysteine concentration up to 1.0 mM. When serum samples were used, within-day and day-to-day coefficient variations were below 4%. This method is simple, and can easily and reliably be used for accurate determination of L-cysteine concentration in serum or other samples.     

A β-glucuronidase (GUS) Based Cell Death Assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, β-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.     

Immuno-PCR Assay for Quantitation of Antibodies to Epstein–Barr Virus

Successful disease prevention and therapy critically depend on timely diagnosis of infections. Quantitative immuno-PCR (qiPCR) technology improves the sensitivity in the detection of antibodies to pathogens. A qiPCR-based assay was developed to determine IgG antibodies to Epstein–Barr virus (EBV) in the human blood serum. EBV nuclear protein 1 fragment (pEBV) was expressed in Escherichia coli. A synthetic single-stranded deoxyribonucleotide was conjugated to streptavidin, and the conjugate was used to detect рEBV–IgG1–biotin complexes by qiPCR. The IgG1 titers determined by qiPCR were compared to the results of enzyme-linked immunosorbent assay (ELISA). The sensitivity of qiPCR was one order of magnitude higher than that of ELISA. Thus, a highly sensitive qiPCR-based assay was developed to quantitate antibodies specific to the recombinant EBV antigen.     

Antioxidant Activity of Supramolecular Water-Soluble Fullerenes Evaluated by β-Carotene Bleaching Assay

We investigated the antioxidant activity of supramolecular water-soluble fullerenes, polyvinylpyrrolidone (PVP)-entrapped C₆₀, and γ-cyclodextrin (CD)-bicapped C₆₀, based on comparable β-carotene bleaching assay. Antioxidant activity against reactive oxygen species (ROS) generated by three different methods, (i) autoxidation of linoleic acid, (ii) hydrogen peroxide promoter, and (iii) photoirradiation, was evaluated as percent of inhibition relative to a control experiment in view of the bleaching rate constant (k _obs) as well as the persistent absorbancy of β-carotene. Water-soluble fullerenes exhibit significant inhibitory effects on the oxidative discoloration of β-carotene in any system.     

Assay of β-galactosidase activity by Flow Injection Analysis (FIA)

Summary A novel method to analyze -galactosidase by Flow Injection Analysis is presented with a linear working range extended to at least 2150 U/mL, being the detection limit 25 U/mL with 55 samples per hour frecuency and a BSD of 0.954% versus 2.4% obtained by manual assay. The method was tested with optimal results with samples from Escherichia coli cultures producing -galactosidase.     

Genetic Assay for Small Fragments of Bacteriophage φX174 Deoxyribonucleic Acid

The double-stranded replicative form deoxyribonucleic acid (RF-DNA) of bacteriophage phiX174 was fragmented by pancreatic deoxyribonuclease, and the complementary strand fragments were then annealed to intact viral single strands. When such complexes infected Escherichia coli spheroplasts, some of the progeny virus bore genetic markers derived from the RF-DNA fragments. In this way, genetic markers have been salvaged from DNA fragments less than 50 nucleotides in length. This method is potentially useful as a specific assay to aid in the purification of genetically defined DNA fragments and also as a mechanism for the incorporation of small chemically synthesized DNA sequences into viral genomes.     

How do Detergents Work? A Qualitative Assay to Measure Amylase Activity

We present a practical activity focusing on two main goals: to give learners the opportunity to experience how the scientific method works and to increase their knowledge about enzymes in everyday situations. The exercise consists of determining the amylase activity of commercial detergents. The methodology is based on a qualitative assay using a colorimetric process. Quantitative results are also obtained by measuring the halo formed. This activity is suitable for and adaptable to learners at different levels of education: primary school, secondary education or even for pre-service teachers, which is the group the version described in this paper was intended for. This laboratory activity was designed to include the scientific method as a learning outcome. This was especially important in pre-service teachers, as increasing scientific literacy is one of the primary goals of science education. Through the activity, students also learn about micro-organisms and their applications in their daily lives, which is one of the tenets of Science–Technology–Society–Environment programs. A case study was conducted with a group of learners made up of 75 pre-service teachers from Universitat Rovira i Virgili in Tarragona, in order to verify whether this lab activity is well designed and can be satisfactorily implemented.     

Radioreceptor Assay for α;-Msh Using Mouse B16 Melanoma Cells

Abstract

A radioreceptor assay for α;-MSH is described which is based on cultured mouse B16 melanoma cells and bioactive monoiodinated [Nle⁴]-α;-MSH tracer. The assay was used (1) to study the binding characteristics of α;-MSH to B16 cells, (2) to determine the relative binding activity of MSH peptides, and (3) to measure MSH in tissue extracts. The association of α;-MSH to B16 cells reached a stable plateau after 3 h at 15°C. At 25° or 37°C, the binding was transient and at 0-1°C, the association was very slow. The hormone-receptor complex was relatively stable between 0° and 15°C whereas a 50% dissociation was reached after 90 min at 25°C and after 35 min at 37°C. The mean K_D for α;-MSH of four saturation experiments was 1.3 nM and the number of receptors 9570 per cell. 1,10-Phenanthroline had a stabilizing effect in the binding assay when used at a 0.3 mM concentration. From the MSH peptides tested in the binding assay, some showed similar potencies in three bioassays (tyrosinase, melanin and Anolis skin), whereas others displayed considerably     

Analysis of Alpha-Synuclein in Malignant Melanoma – Development of a SRM Quantification Assay

Globally, malignant melanoma shows a steady increase in the incidence among cancer diseases. Malignant melanoma represents a cancer type where currently no biomarker or diagnostics is available to identify disease stage, progression of disease or personalized medicine treatment. The aim of this study was to assess the tissue expression of alpha-synuclein, a protein implicated in several disease processes, in metastatic tissues from malignant melanoma patients. A targeted Selected Reaction Monitoring (SRM) assay was developed and utilized together with stable isotope labeling for the relative quantification of two target peptides of alpha-synuclein. Analysis of alpha-synuclein protein was then performed in ten metastatic tissue samples from the Lund Melanoma Biobank. The calibration curve using peak area ratio (heavy/light) versus concentration ratios showed linear regression over three orders of magnitude, for both of the selected target peptide sequences. In support of the measurements of specific protein expression levels, we also observed significant correlation between the protein and mRNA levels of alpha-synuclein in these tissues. Investigating levels of tissue alpha-synuclein may add novel aspect to biomarker development in melanoma, help to understand disease mechanisms and ultimately contribute to discriminate melanoma patients with different prognosis.     

Development of a High-Throughput Assay for Identifying Inhibitors of TBK1 and IKKε

IKKε and TBK1 are noncanonical IKK family members which regulate inflammatory signaling pathways and also play important roles in oncogenesis. However, few inhibitors of these kinases have been identified. While the substrate specificity of IKKε has recently been described, the substrate specificity of TBK1 is unknown, hindering the development of high-throughput screening technologies for inhibitor identification. Here, we describe the optimal substrate phosphorylation motif for TBK1, and show that it is identical to the phosphorylation motif previously described for IKKε. This information enabled the design of an optimal TBK1/IKKε substrate peptide amenable to high-throughput screening and we assayed a 6,006 compound library that included 4,727 kinase-focused compounds to discover in vitro inhibitors of TBK1 and IKKε. 227 compounds in this library inhibited TBK1 at a concentration of 10 μM, while 57 compounds inhibited IKKε. Together, these data describe a new high-throughput screening assay which will facilitate the discovery of small molecule TBK1/IKKε inhibitors possessing therapeutic potential for both inflammatory diseases and cancer.     

A Luminex Assay Detects Amyloid β Oligomers in Alzheimer’s Disease Cerebrospinal Fluid

Amyloid beta (aβ) protein assembles into larger protein aggregates during the pathogenesis of Alzheimer’s disease (AD) and there is increasing evidence that soluble aβ oligomers are a critical pathologic species. Diagnostic evaluations rely on the measurement of increased tau and decreased aβ42 in the cerebrospinal fluid (CSF) from AD patients and evidence for oligomeric aβ in patient CSF is conflicting. In this study, we have adapted a monoclonal single antibody sandwich ELISA assay to a Luminex platform and found that this assay can detect oligomerized aβ42 and sAPPα fragments. We evaluated oligomeric aβ reactivity in 20 patients with AD relative to 19 age matched controls and compared these values with a commercially available Alzbio3 kit that detects tau, phosphorylated tau and aβ42 on the same diagnostic platform. We found that CSF samples of patients with AD had elevated aβ oligomers compared to control subjects (p < 0.05) and the ratio of aβ oligomers to aβ42 was also significantly elevated (p < 0.0001). Further research to develop high sensitivity analytical platforms and rigorous methods of developing stable assay standards will be needed before the analysis of oligomeric aβ becomes a routine diagnostic assay for the evaluation of late onset AD patients.