Fluoroimmunologic analysis of proteins

Review contains new data on protein fluoroimmunoassay (FIA), which combines advantages of RIA and EIA. Basic approaches to antibody production, principles of homogeneous and heterogeneous FIA as well as of time-resolved FIA properties of 28 fluorescent labels presenting various classes of organic compounds (of which most prospective are aminoquinolone, cyanocoumarines, chlorophylls, phycobiliproteins, metallorganic compounds) are described. Some examples of FIA application for quantitative evaluation of proteins in biological samples, properties of some FIA kits, and specifications of 6 fluorimeters developed for FIA are given. The nearest perspectives of FIA development and application in medical and biological research are considered.     

Serotonergic modulation of footshock induced aggression in paired rats.

Footshock induced aggression (FIA) was induced in paired rats and three paradigms of aggressive behaviour were recorded, namely, latency to fight (LF), total period of physical contact (TPP) and cumulative aggression scores (CAS). The effects of increasing or decreasing central serotonergic activity, by using a number of pharmacological agents with well defined effects on rat brain serotonin, were investigated on FIA and on FIA augmented by apomorphine, a dopamine receptor agonist. The results show that centrally administered serotonin, the serotonin precursor, 5-hydroxytryptophan administered with clorgyline, a selective MAO A inhibitor, quipazine, a serotonin receptor agonist, and fluoxetine, a selective inhibitor of neuronal re-uptake of serotonin, attenuated all paradigms of FIA and apomorphine induced potentiation of FIA. On the contrary, the other re-uptake inhibitor used, citalopram, appeared to have a dual effect and decreased LF and CAS, while increasing TPP. The serotonin synthesis inhibitor, p-chlorophenylalanine and the selective serotonin receptor (5-HT2) antagonist, ketanserin, augmented all paradigms of FIA per se and apomorphine induced augmentation of FIA. However, the other serotonin receptor antagonist used, metergoline, which blocks both 5-HT1 and 5-HT2 receptor subtypes, attenuated FIA per se but decreased only CAS in apomorphine induced increase in FIA. The data confirm the inhibitory effect of the central serotonergic system on aggressive behaviour and the inverse relationship existing between it and the central dopaminergic system in the modulation of FIA, as has also been confirmed in earlier biochemical investigations from this laboratory. The data has been discussed in the light of existing knowledge on serotonin receptor subtypes and the presence of modulatory serotonergic heteroreceptors on central dopaminergic neurones.     

Isolation and characterization of an acetyl group-recognizing agglutinin from the serum of the Indian white shrimp Fenneropenaeus indicus

A natural agglutinin from the serum of the Indian white shrimp Fenneropenaeus (Penaeus) indicus was purified to electrophoretic homogeneity by a single-step affinity chromatography on N-acetylglucosamine-Sepharose 6B. The expression of hemagglutinating (HA) activity of F. indicus agglutinin (FIA) was independent of the presence of divalent cations and insensitive to their chelators. FIA gave a single symmetrical peak in its native form with a molecular mass estimate of 200 kDa on gel filtration in HPLC, and SDS-PAGE under reducing conditions revealed that it is a homo-oligomer of a 27-kDa subunit protein. The pattern of reactivity of FIA against anti-FIA rabbit serum in immunodiffusion and immunoelectrophoretic analysis provided additional evidence for its purity and homogeneity. HA-inhibition studies documented exclusive specificity of FIA for acetyl groups in carbohydrates independently of the presence of these groups at the C-2 or C-5 position and its stereochemical arrangement in the axial or equatorial orientation. The unique ability of FIA to recognize acetyl groups was also explicitly demonstrated with sialo- and asialo-glycoproteins. Strikingly, FIA also interacted equally with amino acids and chemicals containing acetyl groups, thereby unambiguously demonstrating the exquisite specificity of FIA for an acetyl group, irrespective of the presence of this group in carbohydrate or noncarbohydrate ligands. The susceptibility of HA activity of FIA to inhibition by lipopolysaccharides from diverse gram-negative bacteria as well as its ability to selectively agglutinate several bacterial species isolated from infected shrimps implicate a potential role of this humoral agglutinin of F. indicus in the host immunodefense reactions against microbial invaders.     

Dopaminergic modulation of footshock induced aggression in paired rats.

Footshock induced aggression (FIA) was induced in weight matched paired rats and three paradigms of aggressive behaviour was recorded, namely, the latency to fight (LF), total period of physical contact (TPP) and cumulative aggression scores (CAS). Dopamine (DA), administered centrally, and peripherally administered L-dopa (with benserazide, a peripheral decarboxylase inhibitor), a DA precursor, and the postsynaptic D2 receptor agonists, apomorphine, N-n-propyl-norapomorphine (PNA), bromocriptine, lisuride and pergolide, induced a dose-related facilitation of FIA characterized by decrease in LF and increase in TPP and CAS. However, the DA presynaptic receptor agonist, BHT-920, induced a biphasic effect with inhibition of FIA being induced by a lower dose and facilitation of the aggressive behaviour produced by a higher dose. The postsynaptic D2 receptor antagonists, haloperidol, spiperone and pimozide, induced a dose-related attenuation of FIA, an effect not seen with domperidone, a peripheral DA receptor antagonist. The results indicate that central dopaminergic postsynaptic D2 receptors have a modulatory facilitative effect on FIA, while the presynaptic DA autoreceptors mitigate aggressive behaviour. However, the presynaptic DA receptor agonist, BHT-920, appears to lose its receptor specificity on dose increment. Long term administration of haloperidol, followed by withdrawal, or desipramine, induced per se augmentation of FIA and potentiated the aggression-facilitative effects of L-dopa, apomorphine and PNA. Since both these treatments are known to induce supersensitivity of central postsynaptic dopamine D2 receptors, the effects are likely to be related to augmented function of dopamine neurones. The findings, in conjunction with a recent report from this laboratory indicating an increase in rat brain DA levels in FIA, support the contention that the central DA system has a facilitative effect on FIA.     

On-line determination of intracellular beta-galactosidase activity in recombinant Escherichia coli using flow injection analysis (FIA).

A flow injection analysis (FIA) system was developed for the determination of cytoplasmic beta-galactosidase activity in recombinant Escherichia coli. The FIA system and its application for on-line monitoring of beta-galactosidase production during cultivation of recombinant E. coli in a 60-l airlift tower loop reactor is described. The results demonstrate that an FIA assay in conjunction with a cell disintegration step can be applied successfully for on-line monitoring of intracellular protein formation.     

Sensitive flow-injection method with peroxyoxalate chemiluminescence detection combined with preparative high-performance liquid chromatography for determination of choline-containing phospholipids in human serum

A sensitive and rapid flow-injection analysis (FIA) of total choline-containing phospholipids (PLs) and a selective FIA method for the class assay of choline-containing PLs combined with preparative HPLC were described. The FIA method is based on peroxyoxaxalate chemiluminescence (PO-CL) detection of hydrogen peroxide enzymatically formed from choline-containing PL. The linear standard curves were obtained up to 1 nmol/20-μl injection (r>0.999) with the detection limits of 1.3–1.6 pmol at a signal-to-noise ratio of 2. The total amounts of choline-containing PLs in human serum were ranged from 1.63 to 3.19 mg/ml. The HPLC separation of choline-containing PLs was achieved with an aminopropyl-modified silica gel column using a mixture of acetonitrile-methanol-10 mM ammonium phosphate buffer pH 5.8 as eluent. The eluate corresponding to each choline-containing PL was collected, evaporated, dissolved in 0.1% Triton X-100 aqueous solution, and then injected into FIA system. The FIA method combined with preparative HPLC was applied to the assay of human serum.     

Differences in salt tolerance of three sugar beet genotypes

The effect of increasing NaCl concentrations (up to 150 m M ) on growth and mineral composition of three genotypes of sugar beet ( Beta vulgaris L., MONOHILL, ADA and FIA) has been studied. Growth was stimulated or little affected in water culture by 50 m M NaCl in all 3 genotypes. Further increase in NaCl concentration depressed growth in ADA more than in MONOHILL, whereas in FIA growth did not significantly differ from the untreated control. In all 3 genotypes, particularly in FIA, increasing NaCl concentrations decreased potassium content in the shoots more than in the fibrous and storage roots. Simultaneously, the accumulation of sodium and chloride in the shoots was considerably higher in FIA than in ADA, where in contrast larger proportions of these ions were retained in the roots. The results demonstrate considerable genotypic differences in salt tolerance of sugar beet and indicate a positive correlation between salt tolerance and accumulation of sodium and chloride in the shoots. FIA but not ADA may be suited for a breeding programme of sugar beet for improved salt tolerance.     

An improved method for quantitative trait loci detection and identification of within-line segregation in F2 intercross designs

We present a new flexible, simple, and powerful genome-scan method (flexible intercross analysis, FIA) for detecting quantitative trait loci (QTL) in experimental line crosses. The method is based on a pure random-effects model that simultaneously models between- and within-line QTL variation for single as well as epistatic QTL. It utilizes the score statistic and thereby facilitates computationally efficient significance testing based on empirical significance thresholds obtained by means of permutations. The properties of the method are explored using simulations and analyses of experimental data. The simulations showed that the power of FIA was as good as, or better than, Haley-Knott regression and that FIA was rather insensitive to the level of allelic fixation in the founders, especially for pedigrees with few founders. A chromosome scan was conducted for a meat quality trait in an F(2) intercross in pigs where a mutation in the halothane (Ryanodine receptor, RYR1) gene with a large effect on meat quality was known to segregate in one founder line. FIA obtained significant support for the halothane-associated QTL and identified the base generation allele with the mutated allele. A genome scan was also performed in a previously analyzed chicken F(2) intercross. In the chicken intercross analysis, four previously detected QTL were confirmed at a 5% genomewide significance level, and FIA gave strong evidence (P < 0.01) for two of these QTL to be segregating within the founder lines. FIA was also extended to account for epistasis and using simulations we show that the method provides good estimates of epistatic QTL variance even for segregating QTL. Extensions of FIA and its applications on other intercross populations including backcrosses, advanced intercross lines, and heterogeneous stocks are also discussed.     

Reaction of macrophage disappearance as a method for the evaluation of cellular immunity in experimental plague.

The reaction of macrophage disappearance (RMD) was found to develop one day after the immunization of experimental animals with the antigen FIA of Y. pestis and to persist for 21 days, correlating with a pronounced protective effect of the above antigen over this period. There were differences between white mice and guinea pigs in the intensity of the RMD, particularly when immunization was carried out using an FIA preparation without prolonged action. These differences are thought to reflect varying degrees of immunity elicited by FIA immunization. It is recommended that the RMD be used to quantify cellular immunity to Y. pestis.     

Determination of l-glutamate in various commercial soy sauce products using flow injection analysis with a modified electrode

A flow injection analysis (FIA) system with a modified electrode has been developed and optimized for determination of l-glutamate using l-glutamate oxidase (GLOD) (EC 1.4.3.11). GLOD was immobilized on controlled-pore glass using glutaraldehyde. The optimal potential applied on the working electrode was +700mV against a platinum (Pt) reference electrode. The optimal pH and flow rate of the carrier buffer were 7.4 and 1.5ml/min, respectively. A modified electrode was integrated into the FIA system in order to eliminate electroactive interference and it was used to determine l-glutamate in 39 samples of Thai commercial soy sauce products. The results obtained were compared with those obtained from enzymatic assay using glutamate dehydrogenase and those from a chromatographic assay using an amino acid analyser. Good correlations were observed amongst these methods. The results indicated that use of an FIA system with a modified electrode was able to eliminate electroactive interference and was applicable to the determination of l-glutamate in food samples. The modified FIA was faster and simpler than the more common methods of enzymatic and chromatographic analysis.     

Growth and nutrition of three Salix clones in low conductivity solutions

The effect of replacing 50% or 95% of the potassium by sodium on growth and on potassium and sodium levels in three genotypes of sugar beet (MONOHILL; ADA; FIA) has been studied in water culture over a period from 2 to 9 weeks.
In all three genotypes there was a preferential uptake of potassium compared to sodium. Nevertheless, at high sodium supply most of the potassium was replaced by sodium, particularly in the leaves. At the same supply the accumulation of sodium in the leaves increased in the following order: ADA < MONOHILL < FIA. Even with high dominance of sodium in the medium, the youngest leaves of FIA held about 0.5 mmol potassium per g dry matter, and potassium was evidently translocated from old leaves to the new growth.
Effects of sodium on growth became more important with time. After 9 weeks, 50% replacement of potassium by sodium increased growth of all plant organs of the three genotypes. Replacing 95% of potassium by sodium depressed growth of the storage root in MONOHILL and particularly in ADA, with simultaneous enhancement of leaf growth in the latter. In FIA, however, this treatment further stimulated both leaf and, particularly, storage root growth. Sodium in comparison with potassium increased the sucrose concentration in leaves and storage roots. The highest sucrose concentration in the storage roots of ADA and FIA was obtained in the treatment with 95% sodium.
The results demonstrate pronounced genotypic differences in sugar beet with respect to the response to sodium. FIA has the most natrophilic behaviour and might be a promising genotype for breeding programmes for adaptation of sugar beet plants to soils high in sodium.     

Highly effective protein detection for avidin-biotin system based on colloidal photonic crystals enhanced fluoroimmunoassay

There has been immense interest in both instruments and methods to enhance fluorescence signal and achieve highly sensitive fluoroimmunoassay (FIA). In this paper, we present a facile, low-cost and general method of biotinylated colloidal photonic crystal (PC) to improve the FIA of avidin (avidin FIA). The fluorescence signal intensity of the avidin FIA on the colloidal PC can be enhanced over two orders of magnitude relative to the control sample, attributed to the large surface area, resonance field and coherent scattering effect of the colloidal PC. The detection limit is shrunk to 1/69 of that of the control sample. Furthermore, the signal to interference ratio (S/I ratio) is increased because the band-edge induced fluorescence enhancement is wavelength-selective. The interference fluorescence does not go up proportionally while the signal is significantly enhanced by the colloidal PCs. It is believed that the colloidal PC modified with biotin can act as an effective material for a general and sensitive fluoroimmunoassay.     

Solubilized substrates for the on-line measurement of lipases by flow injection analysis during chromatographic enzyme purification.

A flow injection analysis (FIA) system for the on-line measurement of lipases in chromatographic processes has been developed. The photometrically detectable substrates para-nitrophenylpalmitate, S,O,O'-tripropyryl-1-thioglycerol, and 1,2-O-dilauryl-rac-glycero-3-glutaric-resorufinester were investigated. Different detergents and qualities of assay emulsions were tested for optimal results in FIA applications. Emphasis was placed on increasing the stability of the assay emulsion. Lipases of different origin and specificity were detected. The linear detection range was adapted to the requirements of the chromatographic purification procedures. The connection of the FIA with a fast protein liquid chromatography system permitted the automatization of lipase purification by monitoring protein content, salinity, and enzyme activity of the effluent from column chromatography.     

Bienzymatic amperometric sensor for protein assay in milk

A new bienzymatic amperometric sensor is proposed for the assay of the protein content of milk. The sensor is based on two enzymes: carboxypeptidase A and L-amino acid oxidase. The response characteristics obtained for this sensor (detection limit of 1.5 micromol/L, linear concentration range between 1.8 and 2.8 micromol/L), as well as high selectivity over possible interferences from milk, made it applicable as a detector in flow injection analysis (FIA). The response characteristics obtained in the non-equilibrium conditions (FIA system) are: detection limit of 1.5 micromol/L and linear concentration range between 2 and 3.5 micromol/L. Without FIA, the average recovery of proteins from milk and milk products is 99.06 +/- 0.07% and, by utilization of FIA, it increased to 99.73 +/- 0.03. The sensor proved a good reliability for the assay of proteins in milk and milk products.     

Development of time-resolved immunofluorometric assays for rat follicle-stimulating hormone and luteinizing hormone and application on sera of cycling rats

The aim of this study was to develop time-resolved immunofluorometric assays (TR-IFMA) for measuring rat (r)FSH and rLH. The advantages of these IFMAs are higher sensitivity due to lower background values, higher specificity as only intact molecules of FSH and LH can be measured, and a very long shelf life of the nonradioactive biotin antigens compared with radiolabeled iodine antigens. For rFSH, IFMAs are lacking, while for rLH, if present, the resources for antibodies are scarce or the mouse monoclonal antibodies (mMAbs) against LHalpha are inactive with FSH. Thus specific antibodies need to be obtained. With the final TR-IFMAs, rFSH and rLH levels were assessed during the estrous cycle and compared with those obtained with the more classical RIAs and fluoroimmunoassays (FIAs). Two IFMAs for rFSH were developed with mMAbs against the recombinant human (rec h)FSHbeta subunit (FSH56A) attached to the wall and two different rabbit polyclonal antibodies (PAbs) against the alpha subunit of rec hFSH (R93-2705) or recombinant rat (rec r)LH (R95-2715) conjugated with biotin as signal antibody. With both IFMAs, rFSH holo-molecules can be measured. Rat FSH standards could be assessed between 0.02 and 10 ng/ml with a detection limit of 0.05 and 0.24 ng/ml in buffer and serum, respectively. These detection limits in four IFMAs were 8- to 16-fold lower than those in RIAs and FIAs. This detection level allowed the measurement of FSH levels in serum of hypophysectomized (HYPEX) rats at 0.18 ng/ml. In serum of cycling rats, the FSH levels of the IFMA were 2-fold lower than those of the FIA, while in ovariectomized (OVX) rats the IFMA levels were comparable. A peak level of FSH was found during proestrus of Day 2 and gestation with both RIA and FIA, but with IFMAs at gestation only. An IFMA for rLH was set up with mMAb (hCG77A) reacting with rLHbeta as capture and rabbit PAb to rec rLHalpha (R95-2712) as signal antibody. Rat LH standard could be assessed between 0.001 and 10 ng/ml with a detection limit of 0.012 and 0.1 ng/ml in buffer and serum, respectively, which was 8-fold lower than that in RIA/FIA. In serum of HYPEX rats, LH was undetectable (< 0.04 ng/ml), whereas a high background level of 2.5 ng/ml was measured in the FIA. In serum of cycling rats, only a very low LH level of 0.14 ng/ml was measured, which strongly deviated from the level of 3.46 ng/ml with an FIA. The load of LH in serum of OVX rats was 2.91 ng/ml, which was 12-fold lower than that for the FIA. The peak level of LH was detected on proestrus Day 2 with RIA, FIA, and IFMA. In conclusion, two IFMAs for rFSH and one for rLH have been developed with high sensitivity and specificity for intact gonadotropins. The LH pattern during the estrous cycle was comparable between IFMA, RIA, and FIA, although the overall level in the IFMA was much lower, as were HYPEX levels. The FSH pattern differed only on proestrus Day 2 in the IFMA from that of RIA/FIA, showing a peak level with RIA/FIA and a basal level with the IFMA. This implies that in RIA/FIA measurements, proteins other than intact FSH and LH interfere with the analysis at proestrus Day 2 for FSH and in HYPEX, cycling, and OVX rats for LH.     

Specialized functional domains in hemoglobin: dimensions in solution of the apohemoglobin dimer labeled with fluorescein iodoacetamide

The fluorescence characteristics of 8-anilino-naphthalene-1-sulfonic acid (ANS) coupled to apohemoglobin and to apohemoglobin labeled with fluorescein iodoacetamide (FIA) at beta-93 have been compared. The quenching of emission of ANS produced by FIA was measured both with steady-state and with time-resolved techniques. In this system the emission of ANS in the beta-heme pockets was totally quenched by FIA at beta-93. Steady-state measurements indicated a 57% efficiency of energy transfer between ANS in the alpha-heme pockets and FIA at beta-93. Time resolution showed that the initial (unquenched) lifetime of ANS was 18.2 ns. In the presence of FIA two new components were generated with lifetimes of 2.0 and 6.6 ns. Assuming a random orientation of the probes, the distances inferred from these measurements were near 4.6 and 3.6 nm for the time-resolved and near 48 A for the steady-state measurements. In the tridimensional model of hemoglobin the distance between the iron atom of the alpha 1 chains and the SH group of the beta 1 chains at position 93 is 3.6 nm in oxyhemoglobin and 4.1 nm in deoxyhemoglobin. To these distances 0.5-1.0 nm may be added to allow for the dimensions of the probes. Thus it appears that removal of the heme fails to produce any important enlargement of the molecule. On the contrary, the data suggest a slight shrinking of apohemoglobin, which may be consistent with a collapse of the heme pocket when heme is removed. The rest of the molecule does not seem to be greatly affected.(ABSTRACT TRUNCATED AT 250 WORDS)     

On-line monitoring of enzymes in downstream processing by flow injection analysis (FIA)

Flow injection analysis (FIA) has been employed to automate enzyme assays for formate dehydrogenase (FDH) and l-leucine dehydrogenase (l-LeuDH). Coupled to a special sampling device the FIA assays were used to monitor on-line downstream processes, e.g. disintegration of microbial cells and cross-flow filtration of cell homogenates.     

Development of a sensitive quantitative focal assay for human immunodeficiency virus infectivity.

Accurate and sensitive quantitation of infectious human immunodeficiency virus (HIV) has been difficult to achieve. In this report, a quantitative focal immunoassay (FIA) for HIV was developed using human HeLa cells rendered susceptible to HIV infection by introduction of the CD4 gene via a retrovirus vector. Infected cells were identified by using human anti-HIV antibodies or mouse monoclonal antibodies specific for HIV together with secondary fluorescein- or peroxidase-conjugated antibody specific for mouse or human immunoglobulins. The assay identified cells infected with either wild-type or culture-adapted HIV isolates and was capable of detecting 1 positive cell in 10(6) cells. The FIA was also effective at detecting cell-free HIV, and in contrast to assays using A3.01, CEM, and other human leukemia cells, the FIA detected most wild-type HIV isolates. HIV neutralization could be determined by using the FIA, and two monoclonal antibodies reactive with HIV gp120 were found to neutralize only the LAV-IIIB strain of HIV. These monoclonal antibodies, as well as antibodies in serum samples from patients with acquired immune deficiency syndrome, were able to inhibit the spread of HIV infection in human lymphocyte suspension cultures but not in CD4-positive HeLa cells growing attached to plastic dishes.     

Comparison of the action of pristan and Freund's incomplete adjuvant on the development of ascites in mice--the recipients of hybridoma cells

Production of immunodiagnostic preparations based on monoclonal antibodies requires large amounts of such antibodies. Most frequently preparative quantities of monoclonal antibodies are provided by ascitic fluid from hybridoma-carrying mice. Prior to intraperitoneal administration of hybridoma cells mice are subjected to stimulation with pristan, a mineral oil component. The authors showed that the Freund's incomplete adjuvant (FIA) may be as well used for this purpose. The effect of pristan and the FIA on ascites development in mice with hybridomas producing monoclonal antibodies to the Ia-like antigens of man was studied. The stimulants were administered in amounts of 0.5 ml per a mouse. It was shown that irrespective of the stimulant injection time and the number of the administered hybridoma cells the amount of the ascitic fluid formed in female mice BALB/c stimulated with the FIA was 1.5-3 times higher as that in the animals stimulated with Pristane, the antibody titer in the ascitic fluid being unchanged.     

Atmospheric pressure chemical ionization-mass spectrometry method to improve the determination of dansylated polyamines

Determination of polyamine pools is still a step impossible to circumvent in studies aimed at determining the pathophysiological role of natural polyamines. In addition, polyamine measurement in biological fluids and tissues may have clinical relevance, especially in cancer patients. Among the wide panel of analytical methods developed for the quantification of polyamines, high-performance liquid chromatographic (HPLC) separation of polyamines after derivatization with dansyl chloride remains the most commonly used method. In this work, we show that atmospheric pressure chemical ionization-mass spectrometry (MS) can be used to detect and quantify biologically relevant polyamines after dansylation, without chromatographic separation. Positive-ion mass spectra for each dansylated polyamine were generated after optimization by flow injection analysis (FIA). FIA coupled with MS detection by selected ion monitoring greatly increased the sensitivity of the polyamine detection. The method is linear over a wide range of polyamine concentrations and allows detection of quantities as low as 5 fmol. The FIA/MS method is about 50-fold more sensitive than the conventional HPLC/fluorimetry procedure. A good correlation (r>0.98) between these two methods was observed. The FIA/MS method notably reduces the time of analysis per sample to 1.5 min and turns out to be rapid, efficient, cost saving, reproducible, and sufficiently simple to allow its routine application.