Synthesis and evaluation of luminescent tracers and hapten-protein conjugates for use in luminescence immunoassays with immobilised antibodies and antigens. A critical study of macro solid phases for use in immunoassay systems, Part II

This article describes the synthesis of labels and hapten-protein conjugates for use in bio- and chemiluminescent immunoassay systems, together with the problems encountered. The effects of maleimide upon acetate-, adenylate- and pyruvate kinase activity have been studied, as well as upon the luciferin-luciferase monitoring system. Maleimide inhibited both acetate and adenylate kinase but showed no inhibition of pyruvate kinase and the monitoring reagent. Four heterobifunctional reagents were tested for their capability in forming pyruvate kinase-donkey-anti-rabbit IgG conjugates which retained enzyme and antibody activity. The best results were obtained with succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and succinimidyl-6-(p-maleimidophenyl)-hexanoate. The relationship between the amounts of succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and IgG was studied with respect to enzymic activity of the conjugate. The Michaelis-Menten constants for both conjugated and non-conjugated pyruvate kinase were calculated and compared. It was found that the maximal velocity (Vmax) of the conjugated enzyme was lower than that of the non-conjugated enzyme although the "apparent" Km value was the same for both conjugated and non-conjugated pyruvate kinase. The pyruvate kinase-anti rabbit IgG conjugate was tested for its ability to bind to rabbit-IgG coated polystyrene balls. In addition to bioluminescent labels, the synthesis of chemiluminescent markers was undertaken and optimised. The three substances used for labelling were diazoluminol, diazoisoluminol and N-(4-aminobutyl)-N-ethylisoluminol hemisuccinamide the latter being used as an N-hydroxysuccinamide "active" ester. The ratio of label to IgG was studied for diazoluminol and N-(4-aminobutyl)-N-ethylisoluminol hemisuccinamide active ester after it had been discovered that diazoisoluminol was not suitable for coupling to antibodies. The optimal molar ratios label: IgG were for diazoluminol 40:1 and for N-(4-aminobutyl)-N-ethylisoluminol hemisuccinamide active ester 60:1. Increasing the substitution rate led to a lessening of the dynamic range, shown by an increase in the ratio between unspecific binding (noise) to maximal binding (signal) in an assay. The synthesis of hapten-protein conjugates for covalent coupling to polystyrene balls was undertaken as this formed part of the preparation for the assays described in Part III. The optimal production of gentamicin-bovine serum albumin and thyroxine-transferrin conjugates has been described in detail.     

Magnetocardiography in the evaluation of fetuses at risk for sudden cardiac death before birth

Background

We hypothesized that fetuses at risk for sudden death may have abnormal conduction or depolarization, ischemia, or abnormal heart rate variability (HRV) detectable by magnetocardiography.

Methods

Using a 37-channel biomagnetometer, we evaluated 3 groups of fetuses at risk for sudden death: group 1, critical aortic stenosis (AS); group 2, arrhythmias; and group 3, heart failure and in utero demise. Five to 10 recordings of 10-minute duration were recorded, and signal was averaged to determine rhythm, conduction intervals, HRV, and T-wave morphology.

Results

In group 1, 2 of 3 had atrial and ventricular strain patterns. In (n = 53) group 2, 15% had prolonged QTc and 17% had T-wave alternans (TWA). Of 23 group 2 fetuses with atrioventricular block, 74% had ventricular ectopy, 21% had junctional ectopic tachycardia, and 29% had ventricular tachycardia. Group 3 (n = 2) had abnormal HRV and TWA.

Conclusion

Repolarization abnormalities, unexpected arrhythmias, and abnormal HRV suggest an arrhythmogenic mechanism for “sudden cardiac death before birth.”     

Development of ACRODAT<Superscript>®</Superscript>, a new software medical device to assess disease activity in patients with acromegaly

Purpose

Despite availability of multimodal treatment options for acromegaly, achievement of long-term disease control is suboptimal in a significant number of patients. Furthermore, disease control as defined by biochemical normalization may not always show concordance with disease-related symptoms or patient’s perceived quality of life. We developed and validated a tool to measure disease activity in acromegaly to support decision-making in clinical practice.

Methods

An international expert panel (n?=?10) convened to define the most critical indicators of disease activity. Patient scenarios were constructed based on these chosen parameters. Subsequently, a panel of 21 renowned endocrinologists at pituitary centers (Europe and Canada) categorized each scenario as stable, mild, or significant disease activity in an online validation study.

Results

From expert opinion, five parameters emerged as the best overall indicators to evaluate disease activity: insulin-like growth factor I (IGF-I) level, tumor status, presence of comorbidities (cardiovascular disease, diabetes, sleep apnea), symptoms, and health-related quality of life. In the validation study, IGF-I and tumor status became the predominant parameters selected for classification of patients with moderate or severe disease activity. If IGF-I level was ≤1.2x upper limit of normal and tumor size not significantly increased, the remaining three parameters contributed to the decision in a compensatory manner.

Conclusion

The validation study underlined IGF-I and tumor status for routine clinical decision-making, whereas patient-oriented outcome measures received less medical attention. An Acromegaly Disease Activity Tool (ACRODAT) is in development that might assist clinicians towards a more holistic approach to patient management in acromegaly.

     

Children, adolescents, and television

As we have indicated, children's television has either a documented or probable effect on a variety of health-related behaviors in children and adolescents in the United States. Studies of cognitive development indicate that television provides a stimulus for learning and that children learn from television. The adverse effects of television appear related to both the time spent watching television and the content of the programs that are viewed. The reviewed observations suggest that a variety of initiatives are warranted to alter the time children spend watching television, the content of programs, and the types of programs for children and adolescents that are produced and broadcast. These initiatives require the development of effective techniques and materials for counseling parents, as well as continued political and legislative activities at the local and national level. We must promote the conviction that time spent in activities other than television viewing will provide our children with the values necessary to understand and interact with an increasingly complex world. Effective governmental action on behalf of children to change television will require a reaffirmation and enforcement of the Public Interest Standard. For half a century, the broadcast media have been licensed to use the airwaves in the public interest. The diversity and magnitude of the adverse effects of television on the health of children strongly suggest that the use of television has not been in the public interest. Although cable television offers multiple alternatives, less than 60% of American households receive cable. Broadcast television still represents the only alternative for 40% of American children. Substantial regulatory change by the current administration is unlikely. Therefore, legislative activity to mandate broadcast practices responsive to the needs of children appears the most appropriate national approach.     

Adolescent contraception. Review and recommendations

In the light of the increase in teenage sexual activity over the past decade, the authors review current social, psychological, and educational attitudes. Together with a summary of the legal ramifications, they provide recommendations for treatment of this patient group.     

Contrast-dependent Dissociation of Visual Recognition and Detection Fields

Seeing an object'as something'is different from simply seeing it (see Watanabe, S., 1985, Pattern Recognition: Human and Mechanical , John Wiley). This distinction between recognition and detection often goes unnoticed in physiology and clinical practice, where visual performance is characterized in terms of acuity, visual field and contrast sensitivity. The corresponding functions of stimulus detection are consistent with the neural projection properties from the retina to the striate cortex, i.e. the'cortical magnification theory'. Yet recognition performance for characters (Strasburger, H. et al. , 1994, Eur. J. Neurosci. , 6, 1583-1588) and grey-level patterns (Jüttner, M. and Reutschler, I., 1996, Vision Res. , 36, 1007–1022) does not fit into this scheme. Here we show that this discrepancy results in the dissociation of visual recognition and detection fields, which is dramatic at low pattern contrast. Form proper can be appreciated exclusively within the much narrower field of recognition, the window of visual intelligence. Its function is, at low contrast, probably mediated by the magnocellular pathway and at all contrasts is determined by the processing characteristics of higher stages of the ventral visual pathway.