Improvement of immunoglobulin M capture immunoassay specificity: toxoplasma antibody detection method as a model.

In the Toxoplasma gondii immunoglobulin M (IgM) capture fluorometric enzyme immunoassay used as a model, nonspecific responses due to the binding of human IgM to horseradish peroxidase (HRP) conjugates were observed despite the removal of the Fc portion of the immunoglobulin. This interaction may be mediated through the binding of human IgM to the HRP moiety of the conjugate. Addition of polymerized HRP into the reaction mixture reduced nonspecific signals in the majority of low false-positive serum reactions. Other plausible sites of interaction are conserved epitopes of mouse immunoglobulins presenting antigenic similarities with the allotopes of other species. Fragmentation of mouse antimicrobial IgG to Fab' and selection of proper conjugation procedure improved assay specificity.

Analytical quality control in neonatal screening.

This critical review questions the present understanding in monitoring of analytical quality control in neonatal screening. Current status and historical background of the analytical quality control, particularly of the tests intended for the screening of congenital hypothyroidism and some inborn errors of metabolism, is reviewed. The reasons why attempts to standardize immunoassays through the preparation of a so-called "gold standard" (e.g. for thyrotropin) will not resolve noncomparability of results are discussed. The review presents arguments for the necessity of elaboration of international guidelines for methods assessment and comparison with an emphasis on their clinical relevance.

New neonatal thyrotropin enzyme immunoassay with fluorimetric detection: comparison with time-resolved fluoroimmunoassay.

This short communication compares a novel fluorimetric microplate enzyme immunoassay (FEIA) with a commercial time-resolved fluoroimmunoassay for the determination of thyrotropin in dried blood spots. The evaluation was performed using a retrospective study design with newborn blood samples from three screening centres. Non-parametric Spearman rank correlation analysis revealed highly significant positive correlation between methods: rs = 0.465, p < 0.0001 (Hannover), rs = 0.659, p < 0.0001 (Minsk), rs = 0.755, p < 0.0001 (Helsinki). Wilcoxon signed rank test performed for paired FEIA and time-resolved fluoroimmunoassay showed that the results obtained by both tests represented the same distribution (p < 0.0001). The new method, using fluorimetric detection, can be performed with the instrumentation commonly used for the screening of congenital hypothyroidism and phenylketonuria. Results are obtained within three to four hours after arrival of the sample in the laboratory. Preliminary evaluation indicates the method to be a suitable alternative to time-resolved fluoroimmunoassay for neonatal thyroid function screening.

A rapid fluorometric enzyme immunoassay for the determination of neonatal TSH from blood spots.

We describe a novel method for the detection of thyrotropin from dried blood spots using a horseradish peroxidase-labelled sandwich enzyme immunoassay with fluorometric detection. The detection limit of the present assay is 1.25 mIU/l with within-run and between-run imprecision being in the range 5.2 to 11.4%. The results of the assay correlate well with two commercial methods: an enzyme immunoassay (r = 0.93) and a time-resolved fluorescence assay (r = 0.90). The blood spot values also show a good correlation (r = 0.93) with respective values obtained from plasma using a commercial immunoradiometric method. The assay may also be performed colorimetrically with sensitivity similar to the fluorometric assay. However, the latter provides a wider dynamic range with an upper limit of 400 mIU/l while the colorimetric method reaches a plateau at 25 mIU/l. Due to its simplicity and rapid performance (3 h), the fluorometric assay is suitable for the routine screening of congenital hypothyroidism.

3-p-hydroxyphenylpropionic acid--a sensitive fluorogenic substrate for automated fluorometric enzyme immunoassays.

The application of 3-p-hydroxyphenylpropionic acid (HPPA), a fluorogenic substrate of horseradish peroxidase (HRP) to an automated microplate fluorometric enzyme immunoassay is described. Fluorescence intensity of the end product was highly dependent on the pH of the buffer and on the concentrations of the substrate mixture ingredients. The determination of human thyrotropin (TSH) and recombinant hepatitis B surface antigen (rHBsAg) were performed using a fluorometric enzyme immunoassay (FEIA) with HPPA as the substrate, and a colorimetric one with tetramethylbenzidine (TMB) as the chromogenic substrate. The sensitivity of both types of assays proved comparable. The distinct advantage of a fluorometric assay is the possibility to perform a quantitative detection of analyte over a very wide dynamic range. Clinical evaluation of both assays showed good correlation between the FEIA and conventional methods.

Endogenous interference in immunoassays in clinical chemistry. A review.

The increasing availability and use of immunoassays in clinical chemistry have revealed a number of endogenous interferences. Solid-phase sandwich immunoassays based on monoclonal antibodies are particularly sensitive to any factor able to bridge immunoglobulins together. Heterophilic immunoglobulin antibodies have been demonstrated in up to 40% of patient samples and to cause spuriously elevated results unless certain precautions are taken. Rheumatoid factors belong to the same category, but their affinity is usually too low to cause significant interference. Immunoscintigraphy generates high-titre anti-immunoglobulin responses causing serious interferences in immunoassays. Recently interfering factors of unknown nature causing nonspecific binding of enzyme-labelled antibodies have been observed. Spuriously decreased values can be caused by complement, which may interfere with antigen-binding to solid phase antibody. The aforementioned and other endogenous interferences in immunoassays are reviewed and methods for their elimination discussed.

Rapid determination of C-reactive protein by enzyme immunoassay using two monoclonal antibodies.

Several monoclonal antibodies for human C-reactive protein (CRP) were characterized, and two antibodies binding to separate domains were used to construct a rapid and simple immunoenzymometric assay for CRP. The assay consists of a single 15 min immunological reaction during which CRP forms a complex with a peroxidase-labelled antibody and with another antibody attached to the test-tube wall. The immobilized complex is detected by a 3 min colour reaction using peroxidase substrate. The quantitative measuring range of the assay is 0.04-5 mg/l, and no hook occurs at five-fold higher values. The sensitivity of the method allows reliable determination of low CRP levels, eg. in paediatric samples. The values obtained with the present assay correlated well with turbidimetric results.

Effect of complement binding on a solid-phase immunometric TSH assay.

The binding of serum thyrotropin (TSH) to plastic beads coated with a monoclonal antibody to human TSH was inhibited unless EDTA was present during the incubation. The inhibitory factor in serum was heat labile, and its effect could be abrogated by the addition of human albumin-anti-albumin immune complexes. Subsequently it was shown that the antibody-coated beads were able to bind the first component of complement, C1q, and that this binding was inhibited by addition of albumin-anti-albumin complexes. The results show that a surface coated with a monoclonal murine antibody is able to bind complement, and that binding of complement may interfere in solid-phase immunometric assays.

Immunocatalytic assay of pancreatic alpha-amylase in serum and urine with a specific monoclonal antibody.

In this immunocatalytic assay for alpha-amylase (EC 3.2.1.1) of pancreatic origin, a highly specific monoclonal antibody coupled to plastic beads is used to extract pancreatic amylase from samples, leaving salivary amylase in solution. The catalytic activity of the bound pancreatic amylase is then determined with blocked p-nitrophenyl maltoheptaoside as substrate. The method shows no cross-reactivity with salivary amylase, analytical recovery is 89-109% for pancreatic amylase, and interassay imprecision is 7.1-7.7%. We used the method to determine pancreatic amylase in serum and urine from healthy controls and different patient groups. The reference intervals for 34 supposedly healthy controls were: serum, 10-48 U/L (mean 27 U/L); urine, less than 20-435 U/L (mean 104 U/L). Results by the present assay correlated well with a salivary amylase inhibition assay (Boehringer Mannheim). We conclude that the described immunocatalytic assay is clinically useful for detecting increased activities of pancreatic amylase in serum and urine.

Effects of inhibitors of polyamine biosynthesis on the growth and melanogenesis of murine melanoma cells.

Both 2-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (EC 4.1.1.17), and methylglyoxal bis(guanylhydrazone) (MGBG), a competitive inhibitor of S-adenosylmethionine decarboxylase (EC 4.1.1.50), strikingly stimulated melanotic expression of murine Cloudman S91 melanoma cells. The stimulation of tyrosinase (EC 1.10.3.1) activity and melanin formation by DFMO was closely associated with intracellular depletion of putrescine and spermidine developed in response to the drug. However, little or no evidence was obtained indicating that enhanced melanogenesis in response to MGBG was mediated through an inhibition of polyamine biosynthesis. Indirect inhibitors of ornithine decarboxylase, such as 1,3-diaminopropane and 1,3-diaminopropan-2-ol, but not putrescine, likewise inhibited the growth of the melanoma cells and stimulated their melanin production. The stimulation of melanogenesis by polyamine antimetabolites was not mediated by cyclic adenosine 3':5'-monophosphate, in contrast to the effect elicited by alpha-melanotropin. It is also unlikely that MGBG or the diamines acted as lysosomotropic agents capable of stimulating tyrosinase activity in situ, since the enzyme activity was stimulated by the drugs irrespective of whether assayed in cultured cells or using cell-free homogenates. None of the agents stimulated tyrosinase activity in vitro. The effect of DFMO and MGBG on melanoma cell proliferation was reversible, but the restoration of normal growth and melanin formation, especially in cells exposed to DFMO, was remarkably slow. The present results represent a further experimental model, in which the inhibition of polyamine accumulation is accompanied by signs of terminal differentiation.

Effect of polyamine antimetabolites on cultured human keratinocytes from normal and uninvolved psoriatic skin.

We describe the effect of two polyamine antimetabolites on polyamine and macromolecule synthesis of cultured human keratinocytes obtained by suction blisters from normal skin and the uninvolved skin of psoriatic patients. The concentrations of spermidine and spermine steadily increased during the culture of normal keratinocytes in vitro, whereas the putrescine concentration showed only a transient rise at the beginning of the active growth phase. Treatment with difluoromethylornithine decreased the concentrations of putrescine and spermidine in both normal and uninvolved psoriatic keratinocytes, but had no effect on either DNA or protein synthesis. Methylglyoxal bis(guanylhydrazone) marginally decreased the levels of spermidine and spermine and significantly inhibited the DNA and protein synthetic activities. Pretreatment of uninvolved psoriatic keratinocytes with difluoromethylornithine enhanced the accumulation of methylglyoxal bis(guanylhydrazone), resulting in a profound inhibition of cellular macromolecule synthesis. This synergistic effect was not seen in normal keratinocytes. Thus, although no statistically significant difference was observed between the cells derived from normal and uninvolved psoriatic epidermis, the psoriatic keratinocytes appeared to be more sensitive to the action of polyamine antimetabolites. The inhibition of DNA and protein synthesis by methylglyoxal bis(guanylhydrazone) was prevented by concomitant treatment with spermidine.

Differential effects of 2-difluoromethylornithine and methylglyoxal bis(guanylhydrazone) on the testosterone-induced growth of ventral prostate and seminal vesicles of castrated rats.

2-Difluoromethylornithine totally prevented any increases in putrescine and spermidine concentrations in the ventral prostate of castrated rats during a 6-day testosterone treatment. Prostatic ornithine decarboxylase activity was inhibited by 80%, whereas S-adenosylmethionine decarboxylase was stimulated by more than 9-fold. In seminal vesicle, the inhibition of putrescine and spermidine accumulation, as well as of ornithine decarboxylase activity, was only minimal, and no stimulation of S-adenosylmethionine decarboxylase was observed. Administration of methylglyoxal bis(guanylhydrazone) to castrated androgen-treated rats resulted in a marked increase in concentrations of all prostatic polyamines. Prostatic ornithine decarboxylase activity was nearly 2 times and adenosylmethionine decarboxylase activity 9 times higher than that of the testosterone-treated animals. In contrast with ventral prostate, methylglyoxal bis(guanylhydrazone) treatment inhibited moderately the accumulation of spermidine and spermine in seminal vesicle, although both ornithine decarboxylase and S-adenosylmethionine decarboxylase activities were stimulated. Difluoromethylornithine inhibited significantly the weight gain of ventral prostate, but methylglyoxal bis(guanylhydrazone) produced a substantial increase in prostatic weight. These changes were largely due to the fact that the volume of prostatic secretion was greatly decreased by difluoromethylornithine, whereas methylglyoxal bis(guanylhydrazone) increased the amount of secretion. Treatment with difluoromethylornithine strikingly increased the methylglyoxal bis(guanylhydrazone) content of both ventral prostate and seminal vesicle, but even under these conditions the drug concentration remained low in comparison with other tissues. The results indicate that a combined use of these two polyamine anti-metabolites does not necessarily result in a synergistic growth inhibition of the androgen-induced growth of male accessory sexual glands.

Effect of gossypol on the motility and metabolism of human spermatozoa.

Gossypol, a polycyclic compound isolated from cotton seeds, had a dose-dependent inhibitory effect on human sperm motility. The drug also inhibited powerfully fructolysis and glycolysis by human spermatozoa. Both lactate and CO2 formation from the 14C-labelled sugars was inhibited, and the prevention of CO2 formation from [1-14C]pyruvate and [2-14C]pyruvate by gossypol indicated a direct effect on the tricarboxylic acid cycle. Repeated washing of the sperm cells after gossypol pretreatment failed to abolish the inhibitory effect on CO2 production. The profound disturbances of the sperm energy metabolism induced by gossypol were also reflected by a striking fall of the sperm ATP content. Gossypol had little effect on glucose utilization by minces of human vaginal mucosa, indicating the specificity of gossypol.

Inhibition of DNA and protein synthesis in UV-irradiated mouse skin by 2-difluoromethylornithine, methylglyoxal bis(guanylhydrazone), and their combination.

Exposure of mouse skin to UVB irradiation greatly enhanced the biosynthesis and accumulation of putrescine and spermidine before or concomitantly with stimulation of epidermal macromolecular (DNA and protein) synthesis. Topical treatment of UV-exposed skin with 2 inhibitors of polyamine biosynthesis, 2-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) (MGBG) prevented the enhanced epidermal accumulation of polyamines, especially spermidine, and also inhibited the incorporation of radioactive precursors into DNA and protein. When applied in combination, these 2 antimetabolites of polyamines produced an inhibition of macromolecular synthesis that was at least additive: [3H]thymidine incorporation decreased by 80% and [14C]leucine incorporation by 44% as compared with the UVB-irradiated control mice. A slight decrease in the ratio of [3H]histidine/[14C]leucine incorporation indicated that protein synthesis of the differentiating cell layers was also affected by the inhibitors. The effects of the combined DFMO and MGBG treatment were partially reversed by concomitant topical application of spermidine.

Stimulation of melanotic expression in murine melanoma cells exposed to polyamine antimetabolites.

An exposure of cultured Cloudman S91 melanoma cells to inhibitors of polyamine biosynthesis, 2-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) (MGBG), distinctly promoted the expression of differentiated biochemical functions of the tumor cells. Slight to moderate growth inhibition produced by the compounds was associated with a stimulation of melanogenesis, as reflected by a striking enhancement of tyrosinase (EC 1.10.3.1) activity and an increase in cellular melanin content. Both antimetabolites acted synergistically with alpha-melanotropin (MSH), as regards the stimulation of melanogenesis. Exposure of the melanoma cells to MSH resulted in most experiments in a marked decrease of the intracellular polyamine pools, usually involving all three polyamines (putrescine, spermidine and spermine). The DFMO-induced stimulation of melanogenesis was totally suppressed by the administration of putrescine, whereas the MSH-stimulated tyrosinase activity was not influenced by the diamine. Although many recent reports indicate that terminal differentiation is accompanied by a distinct stimulation of polyamine biosynthesis, our results suggest that in certain cells polyamine deprivation may lead to an enhanced expression of differentiated phenotype.

Arginase activity and polyamine biosynthesis in psoriasis.

Polyamine biosynthesis and arginase activity in psoriasis were studied using keratotome strips and suction blister roofs as specimens. In the uninvolved psoriatic skin a slight (1.3-fold; p less than 0.05) increase in the spermidine level was observed compared with control skin. There was also a 1.2-fold increase (p less than 0.05) in the spermidine/spermine molar ratio, which is considered to be an indicator of proliferation activity. No changes were noted in other polyamines or polyamine-synthesizing enzymes ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (AMDC) in the uninvolved psoriatic skin vs. control skin. Neither was there any significant difference in the arginase activity. In psoriatic lesions the levels of all polyamines, as well as the activities of biosynthetic enzymes, were significantly (p less than 0.001) elevated. The putrescine level was elevated to 2.5-fold, spermidine to 2.7-fold and spermine to 1.4-fold. The enzyme activities expressed severalfold increases. The enhancement of arginase activity was less prominent than that of polyamine-synthesizing enzymes, but the increase from 271 +/- 88 to 354 +/- 126 mumol/g/h (1.3-fold) was statistically significant (p less than 0.001; paired t-test). The increase in arginase activity in suction blister roofs, which represents pure epidermis, was more pronounced than that in keratotome strips, i.e. about double. The results show thus that polyamine biosynthesis is significantly enhanced in psoriatic lesions, but there is only a slight difference between the uninvolved psoriatic skin and control skin.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of polyamines and their antimetabolites in clinical medicine.

Polyamine research, which began with a clinical observation more than 300 years ago, has progressed for several decades as pure basic research, sometimes considered as an academic triviality. The role of polyamines in clinical medicine is coming of age. The fruits of polyamine research are just now entering into the realm of practical application and in a very multidisciplinary manner. Basic research on polyamine metabolism and the elucidation of their physiologic functions has involved many academically interesting, even revolutionary, aspects, but the imagination of biochemists and cell biologists may no longer be sufficient to discover the best ways to translate the results of this basic research into clinical practice. It is almost certain that polyamine antimetabolites will soon find their place among the drug regimens used for the treatment of human malignancies and, possibly, also of hyperproliferative skin diseases. The elucidation of the role of polyamines in cell differentiation may offer fundamental applications regarding the regulation of cell cycle events. The discovery of the antiparasitic properties of polyamine antimetabolites may have a major impact on the well-being of millions of people in the developing world. The potential application of polyamine research in microbial and viral diseases is an area in which investigational insight is just beginning. Finally, the clinical chemistry of extracellular polyamines, although initially disappointing, has not yet been explored in depth and may offer applications useful for the diagnosis or follow-up of a variety of common diseases.