MOG encephalomyelitis: international recommendations on diagnosis and antibody testing.

Over the past few years, new-generation cell-based assays have demonstrated a robust association of autoantibodies to full-length human myelin oligodendrocyte glycoprotein (MOG-IgG) with (mostly recurrent) optic neuritis, myelitis and brainstem encephalitis, as well as with acute disseminated encephalomyelitis (ADEM)-like presentations. Most experts now consider MOG-IgG-associated encephalomyelitis (MOG-EM) a disease entity in its own right, immunopathogenetically distinct from both classic multiple sclerosis (MS) and aquaporin-4 (AQP4)-IgG-positive neuromyelitis optica spectrum disorders (NMOSD). Owing to a substantial overlap in clinicoradiological presentation, MOG-EM was often unwittingly misdiagnosed as MS in the past. Accordingly, increasing numbers of patients with suspected or established MS are currently being tested for MOG-IgG. However, screening of large unselected cohorts for rare biomarkers can significantly reduce the positive predictive value of a test. To lessen the hazard of overdiagnosing MOG-EM, which may lead to inappropriate treatment, more selective criteria for MOG-IgG testing are urgently needed. In this paper, we propose indications for MOG-IgG testing based on expert consensus. In addition, we give a list of conditions atypical for MOG-EM ("red flags") that should prompt physicians to challenge a positive MOG-IgG test result. Finally, we provide recommendations regarding assay methodology, specimen sampling and data interpretation.

Non-invasive, ultra-sensitive, high-throughput assays to quantify rare biomarkers in the blood.

Many diseases are easier to treat and control when detected at an early stage of disease progression. Often, disease-related antigens or biomarkers are shed from the primary site and present in the blood. Unfortunately, there are very few tests capable of detecting these rare biomarkers in the blood. A blood test would be very useful to diagnose the disease earlier, monitor effectiveness of treatments, predict recurrence, and monitor recurrence. There is certainly a need to develop assays that are ultra-sensitive, non-invasive, and high-throughput. Here we describe several highly sensitive immunological assays we have developed to detect rare serum antigens. Initially we created an assay named immuno-detection amplified by T7 RNA polymerase (IDAT). To enhance the effectiveness and streamline the procedure, this assay was amended to the facile amplification system termed fluorescent amplification catalyzed by T7 polymerase technique (FACTT). These assays have been used to analyze the tumor antigen HER2 and the prion protein PrPSc. They can also be applied to other tumor markers or antigens from a variety of diseases such as cardiovascular disease, rheumatoid arthritis, Alzheimer's disease, Parkinson's disease, and hepatitis. These tests are not limited to testing only serum, but may also be applicable to detecting biomarkers in tissue, saliva, urine, cerebrospinal fluid, etc. Clearly, the FACTT-based technology represents an important step in the detection of rare molecules in fluids or tissues for a variety of diseases.

A study of high-, middle- and low-molecular weight adiponectin in urine as a surrogate marker for early diabetic nephropathy using ultrasensitive immune complex transfer enzyme immunoassay.

Background For the early identification of patients at risk of developing diabetic nephropathy, we have developed an ultrasensitive immune complex transfer enzyme immunoassay to measure adiponectin in urine. Methods We developed immune complex transfer enzyme immunoassay for adiponectin and measured urinary adiponectin from 70 healthy subjects, 35 obese non-diabetic subjects and 20 patients with diabetes. Results The urinary adiponectin concentrations in patients with diabetes (3.3 ± 10.7 ng/mg creatinine) were significantly higher than those in obese subjects (0.54 ± 0.44; P < 0.01) and healthy subjects (0.46 ± 0.42; P < 0.001). The gel filtration elution profile of urine from healthy subjects showed traces of four immunoreactive peaks (high-, medium-, low-molecular weight and monomer molecules), despite the majority of blood adiponectin being high-molecular weight. However, urinary adiponectin molecules were more frequent in low-molecular weight as the estimate glomerular filtration rate decreased. Furthermore, as blood glucose concentrations rose, middle-molecular weight and high-molecular weight increased in urine. Further, urinary adiponectin concentrations correlated with estimate glomerular filtration rate ( r = -0.61, P < 0.001), but not urinary albumin. In addition, our analysis showed a significantly ( P < 0.001) higher value for urinary adiponectin in the G2 stage of chronic kidney disease classification where urinary albumin is not elevated. Conclusion Adiponectin increases in urine as renal function decreases, and urinary adiponectin may be useful as a surrogate marker for diabetic nephropathy risk.

Measurement of CGRP in dried blood spots using a modified sandwich enzyme immunoassay.

Calcitonin gene-related peptide (CGRP) has roles as a neurotransmitter, neuromodulator and trophic factor. CGRP has been reported to be elevated in neonatal blood of children with autism or mental retardation as compared with normal subjects by recycling immuno-affinity chromatography (RIC). While CGRP detection in neonatal blood is thus important, it is not easy to detect CGRP in dried blood spots because of the limitations of sample volume and the specificity and the sensitivity of available assay systems. In the present study, we modified a "Sandwich Enzyme Immunoassay" for the purpose of detecting CGRP in blood spot eluate. We have prepared blood spots from blood collected from normal human subjects and measured CGRP level in eluates from these blood spots. Instead of a purification step, we have introduced a pre-incubation step and used washed erythrocytes as a dilution solution. The modified assay has good recovery and specificity and appropriate dilution curves. We have compared the eluate levels with levels in serum and plasma from the same individuals and find that CGRP levels in blood spot eluate were similar to those of serum and plasma. Thus, the newly modified EIA may be a useful method for the detection of CGRP in blood spot eluate.

Newborn screening for cystic fibrosis.

Since the late 1970s when the potential of the immunoreactive trypsinogen assay for early identification of infants with cystic fibrosis was first recognised, the performance of newborn blood spot screening (NBS) has been continually assessed and its use has gradually expanded. NBS for cystic fibrosis is a cost-effective strategy and, if standards of care are fully implemented and robust management pathways are in place, has a positive effect on clinical outcomes. In the past decade, NBS has undergone rapid expansion and an unprecedented number of infants with cystic fibrosis have access to early diagnosis and care. Cystic fibrosis NBS has now moved on from the development phase and is entering an era of consolidation. In the future, research should focus on the rationalisation and optimisation of existing programmes, with particular attention to bioethical implications such as unwanted detection of carriers and inconclusive diagnoses.

Lung Carcinoma Predictive Biomarker Testing by Immunoperoxidase Stains in Cytology and Small Biopsy Specimens: Advantages and Limitations.

CONTEXT: - In the burgeoning era of molecular genomics, immunoperoxidase (IPOX) testing grows increasingly relevant as an efficient and effective molecular screening tool. Patients with lung carcinoma may especially benefit from the use of IPOX because most lung carcinomas are inoperable at diagnosis and only diagnosed by small tissue biopsy or fine-needle sampling. When such small specimens are at times inadequate for molecular testing, positive IPOX results still provide actionable information. OBJECTIVE: - To describe the benefits and pitfalls of IPOX in the detection of biomarkers in lung carcinoma cytology specimens and small biopsies by summarizing the currently available commercial antibodies, preanalytic variables, and analytic considerations. DATA SOURCES: - PubMed. CONCLUSIONS: - Commercial antibodies exist for IPOX detection of aberrant protein expression due to EGFR L858R mutation, EGFR E746_A750 deletion, ALK rearrangement, ROS1 rearrangement, and BRAF V600E mutation, as well as PD-L1 expression in tumor cells. Automated IPOX protocols for ALK and PD-L1 detection were recently approved by the Food and Drug Administration as companion diagnostics for targeted therapies, but consistent interpretive criteria remain to be elucidated, and such protocols do not yet exist for other biomarkers. The inclusion of cytology specimens in clinical trials would expand patients' access to testing and treatment, yet there is a scarcity of clinical trial data regarding the application of IPOX to cytology, which can be attributed to trial designers' lack of familiarity with the advantages and limitations of cytology. The content of this review may be used to inform clinical trial design and advance IPOX validation studies.

CEDIA in vitro diagnostics with a novel homogeneous immunoassay technique. Current status and future prospects.

CEDIA assays represent a state of the art technique utilizing two genetically engineered, enzymatically inactive fragments of beta-galactosidase as the basis for a homogeneous enzyme immunoassay. The smaller, amino-terminal polypeptide, designated the enzyme donor (ED), can recombine spontaneously with the large residual fragment, called the enzyme acceptor (EA), to form active beta-galactosidase, in a process called complementation. ED have been designed in such a way that a ligand, such as a hormone or drug, can be chemically attached to a specific amino acid residue without affecting the enzyme complementation. However, the binding of a ligand-specific antibody to the ED-ligand conjugate will inhibit complementation. If a sample containing ligand is added to the reaction mixture, the ligand will compete with the ED-ligand conjugate for the limited number of antibody binding sites. Thus, the ligand concentration in the sample will modulate enzymatic activity by influencing the amount of free ED-ligand conjugate available for complementation. The basic technology of CEDIA assays has a number of inherent advantages, the most important of these being a linear calibration curve with high precision over the whole assay range, lack of endogeneous enzyme activity and minimal serum interference, chemically defined conjugates and flexibility in assay design. These provide significant advantages in comparison to other homogeneous immunoassay techniques. As a result, CEDIA assays have been successfully developed for high concentration drugs such as theophylline, phenobarbital and phenytoin as well as for very low concentration analytes such as digoxin, B12 and folate. In a modified assay format, even the determination of binding proteins has been accomplished, an example being thyroxine binding proteins in the CEDIA T-uptake assay. More recently, the methodology has been extended to the measurement of high molecular weight analytes like ferritin.

Laboratory findings in HIV infection in infancy.

The incidence of heterosexual HIV transmission continues to increase, especially among women of childbearing age. This has obvious implications for the care of pregnant women and their children, which has become as much a social issue as a medical one. Progress in the development of assays for both HIV diagnosis and clinical evaluation has given us the tools with which to begin dealing with this epidemic. Problems intrinsic to neonatal diagnosis are currently being addressed. In the next few years, assays that achieve rapid diagnosis in infancy should be readily available at reasonable cost. The appropriate utilization of this technology is a matter of active debate.

Therapeutic drug monitoring for the 1990s: meeting the laboratory needs of a changing environment.

The need for monitoring serum and plasma concentrations for therapeutic drugs given to hospitalized and outpatients will continue to growth during the next few years, both in terms of the number of tests ordered each year and the number of different drugs that are monitored. The clinical laboratory will play an important role in implementing and maintaining accurate, cost-effective drug monitoring and treatment programs. To meet changing needs, further development and evaluation of automated analytical assays will be necessary.

[Isozyme analysis by enzyme immunoassay (EIA)--clinical significance and future prospect].

Serum isozymes are widely used for estimating the origin or the degree of injury. The significance and future prospect of enzyme immunoassay (EIA) for the determination of isozymes are summarized in this paper. EIAs were developed for isozymes, e.g., unstable or labile, and indistinguishable by electrophoresis or ion-exchange column chromatography EIA can measure isozyme fragments with no catalytic activity. The catalytic activity of isoenzymes released from tissues disappears immediately but immunologically active isozymes or its fragment may remain in the blood for a longer period. The determination of isozymes by EIA provides an accurate mass, released from the injury. The combination assay of catalytic activity with isozyme mass by EIA may open a new approach for the disease.

[Past, present and future of enzyme immunoassay].

Enzyme immunoassay (EIA) is one of the most popular non-isotopic immunoassay methods, with various clinical applications. After radioimmunoassay was developed by Yalow and Berson, its competitive binding principle was combined with enzyme labeling techniques and its application has been expanded by developing various procedures. Theoretically, EIA is more sensitive than RIA, being able to measure levels as minute as some attomole levels. Automated enzyme immunoassay systems have further improved its intra-laboratory variation, but relatively large variations among different reagent kits must be improved. Highly sensitive EIA systems should allow wider clinical applications.

Effects of hematocrit value on microparticle enzyme immunoassay of tacrolimus concentration in therapeutic drug monitoring.

The effects of hematocrit (Ht) value on microparticle enzyme immunoassay (MEIA) of tacrolimus concentration were examined in 1063 whole-blood samples from 42 transplant recipients (13 liver, 20 kidney, and 9 bone marrow transplantations). MEIA guarantees the test's assay quality for blood tacrolimus in samples with Ht values of 25% to 45%. However, 129 samples (29.3%) obtained from liver transplant recipients and 107 samples (61.5%) from bone marrow transplant recipients had lower Ht (<25%). Further, 81 blood samples (18.1%) with Ht > 45% were observed in kidney transplant patients. Twenty-five whole-blood samples with low Ht were tested by 3 assay methods for tacrolimus: MEIA, modified, corrected MEIA (cMEIA), and enzyme-linked immunosorbent assay (ELISA). MEIA gave higher blood concentrations of tacrolimus than ELISA (16.1 versus 11.0 ng/mL, P < 0.001). This difference was generated by overestimation in MEIA and was not observed in samples with normal Ht. This overestimation was eliminated by using cMEIA on samples with low Ht values: there was no difference in blood tacrolimus concentration between cMEIA and ELISA (12.3 versus 11.0 ng/mL). ELISA or cMEIA should be used for tacrolimus assay in samples obtained from bone marrow transplant recipients with anemia and from liver and kidney transplant recipients with unstable Ht values.

Enzyme immunoassays in clinical chemistry: present status and trends.

The most important enzyme immunoassay techniques are described. The enzymes currently used as labels, the methods of coupling them to antigens or haptens and the possible applications of these assays are reviewed. Furthermore, an overview is given of the reliability and practicability of commercially available enzyme immunoassay kits used in clinical chemical laboratories. Special consideration is given to possible interferences, the detection limits and the mechanization of these tests. Various methods for curve-fitting are listed. It is concluded that most of the currently commercially available enzyme immunoassays are suitable for routine application in appropriate centers like clinical chemical laboratories of larger hospitals. The future role of enzyme immunoassays in clinical chemistry is briefly discussed.

A critical evaluation of enzyme immunoassay kits for detection of antinuclear autoantibodies of defined specificities. II. Potential for quantitation of antibody content.

OBJECTIVE: To analyze the performance of different commercial enzyme immunoassay (EIA) kits for measuring antibody levels of antinuclear antibodies (ANA) specific for double stranded (ds) DNA, SSB/La, Sm, and Scl-70. METHODS: Twenty companies that were known major purveyors of EIA kits for detection of ANA were approached to determine their interest and willingness to participate in this study. The manufacturers were advised that they would be sent coded sera containing mixtures of the Arthritis Foundation/Centers for Disease Control reference reagents, and that they were to use their own test kits to analyze the antibody specificities of these sera and to report the data, in optical density (OD) units, or their equivalent. The analysts were blinded to the concentration of the antibodies and the specificities. RESULTS: Initially, 11 manufacturers out of 20 agreed to participate, but 2 subsequently withdrew. The commercial EIA kits have the potential of being able to quantitate specific autoantibody content to ds-DNA, SSB/La, Sm, and Scl-70. However, certain deficiencies in these kits were also detected, the most obvious being lack of uniformly good performance, with kits of certain manufacturers showing exceptional accuracy in 3 out of 4 of their antibody-specific kits and poor accuracy for a 4th kit. CONCLUSION: It is important for clinicians to appreciate that there is marked inter-manufacturer variation in the performance of EIA kits used as an aid in the diagnosis of systemic rheumatic diseases. Manufacturers need to exercise constant surveillance of kit performance and to provide assurance that such is being done. Improved EIA kits would lend themselves to reliable quantitation of antibody levels in human sera and help to determine whether serial measurement of antibody levels might be useful in monitoring disease activity.