Pooled Human Serum Research Articles

Production of Vibrio vulnificus hemolysin in vivo and its pathogenic significance

Hemolyin/cytolysin (VvhA) is one of the representative exotoxins produced by Vibrio vulnificus. Cytotoxic mechanism of VvhA has been extensively studied. However, there have been controversies concerning the pathogenic significance since vvhA − mutant showed no LD 50 change in mice. In this study, we investigated whether VvhA is expressed in vivo. When V. vulnificus was cultured in the presence of normal pooled human serum, substantial amount of VvhA was detected by ELISA and the transcription of vvhA was also evidently confirmed by RT-PCR and a transcriptional reporter assay. To investigate whether VvhA is expressed in vivo, mice were infected with V. vulnificus and bacterial RNAs were harvested from the mice. In vivo vvhA transcription was observed evidently by RT-PCR. We hereby propose that VvhA is substantially produced in vivo and would contribute to the pathogenesis of V. vulnificus septicemia.

Mass value assignment of total and subclass immunoglobulin G in a human standard anthrax reference serum.

An anti-Anthrax Vaccine Adsorbed (anti-AVA) standard human reference serum pool, AVR414, has been prepared, and the total and protective antigen (PA)-specific immunoglobulin G (IgG) were quantified. AVR414 was prepared by plasmapheresis of healthy adults who had received a minimum of four subcutaneous injections of AVA. Mass values (in milligrams per milliliter) for total IgG and IgG subclasses 1 to 4 were determined by radial immunodiffusion. Anti-PA-specific IgG assignment (in micrograms per milliliter) was done by consensus of two complementary approaches: homologous enzyme-linked immunosorbent assay (ELISA) with affinity-purified anti-PA IgG as a calibrator and summation of mean PA-specific IgG subclass concentrations determined by IgG subclass-specific ELISA using the United States National Reference Preparation for Human Serum Proteins as a standard. The total IgG concentration assigned to AVR414 reference serum was 8.33 mg/ml. IgG subclass concentrations were the following: for IgG1, 4.48 mg/ml; for IgG2, 3.35 mg/ml; for IgG3, 0.37 mg/ml; and for IgG4, 0.30 mg/ml. The assigned mass value for total anti-PA-specific IgG was 141.2 microg/ml. Anti-PA-specific IgG subclass concentrations were the following: for IgG1, 79.6 microg/ml; for IgG2, 35.3 microg/ml; for IgG3, 3.2 microg/ml; and for IgG4, 25.3 microg/ml. Human reference serum pool AVR414 will have direct application in the standardization of anthrax serological assays, in reagent qualification, and as a standard for quantification of PA-specific IgG in humans who have been vaccinated with or otherwise exposed to Bacillus anthracis PA.

Different latex aeroallergen size distributions between powdered surgical and examination gloves: Significance for environmental avoidance

Background Proactive medical institutions implement latex allergen avoidance to protect workers and patients with latex allergy and to prevent latex sensitization in these groups by creating latex-safe environments that include replacement of natural rubber latex examination and surgical gloves, especially those that are powdered, with synthetic alternatives. We have hypothesized that an apparent decreasing trend in new latex allergy cases in hospitals using only synthetic examination gloves but the occasional powdered latex surgical glove might result from constitutive differences in allergen content (particle size distribution and quantity) between powdered surgical gloves and examination gloves. Objective Because aerodynamic particle size determines where inhaled airborne allergen deposits in the airway, the aim of this study was to investigate the differential particle size distribution of latex allergen released from powdered latex examination and surgical gloves. Methods Powdered and nonpowdered latex examination and surgical gloves were processed to create an aerosol in a glove box equipped with air sampling equipment capable of total particle number and mass measurements. Air particulate generated in the glove box was collected on impactors with less than 2.5-μm, 2.5- to 10-μm, and greater than 10-μm particle size limits (4 L/min for 4 hours). All filters were extracted, and latex allergen was quantified by using a latex-CAP inhibition assay with a human IgE anti-latex serum pool. Results Latex aeroallergen on powdered sterile surgical gloves resided primarily on particles greater than 10 μm in size ( P < .006). In contrast, powdered examination gloves released the highest total latex aeroallergen content, with 68% of the particles sized in the respirable 2.5- to 10-μm range and carrying 56% of the airborne latex allergen. Conclusions Significantly lower quantities and larger sizes of latex allergen–containing particles released from surgical gloves provides one potential explanation as to why an apparent decrease in new cases of latex allergy can occur in hospitals that successfully replace latex examination gloves with synthetic gloves but continue occasional use of powdered surgical gloves.

Anti-pig antibody levels in non-human primates of various origin.

Natural anti-porcine antibodies play a major role in hyperacute solid organ xenograft rejection in the pig-to-non-human primate model. Work from other groups and our experience in transplantation experiments has shown that antibody levels are highly variable between non-human primate species, and that extremely high levels can mediate hyperacute rejection even if organs from animals transgenic for human decay-accelerating factor are used. Sera were obtained from cynomolgus monkeys wild-caught in Mauritius, captive-bred in the Philippines, captive-bred in Indonesia (Indonesia-Ind), and originating from Indonesia but colony-bred in USA (Indonesia-USA), from baboons wild-caught in Kenya, and from rhesus monkeys originating from India but colony-bred in USA (10 animals in each group). Antibody levels were determined using assays for haemolytic antibody (APA), IgM and IgG class anti-Galalpha1-3Gal antibody, and IgM and IgG class anti-endothelial cell antibody. Cynomolgus monkeys from the Philippines and Indonesia-USA and rhesus monkeys showed median APA and IgM antibody levels in the same range as a pooled human serum standard, and median IgG levels well below the level in this standard. Cynomolgus monkeys from Mauritius and Indonesia-Ind showed extremely high APA levels (median seven to 10 times the human serum standard): IgM class antibodies were also higher, while IgG class antibodies were in the range of the level in the human serum standard. Antibody levels in baboons were in between these two categories. The results of the APA assay showed a highly statistically significant correlation with the assays of IgM antibody, and this was also the case for the IgM antibody assays, indicative of the assessment of the same antibodies in these assays. The same was observed for the assays for IgG antibody. Taking body weight as an indicator for age, there was no relationship between body weight and levels of antibodies. Natural antibody levels show a significant variation between various groups of non-human primates, with levels in some groups well above those in a human serum standard.

Measurement of toxaphene congeners in pooled human serum collected in three U.S. cities using high-resolution mass spectrometry.

Because human toxaphene exposure data are largely lacking, we surveyed human serum pools collected from U.S. residents to determine the feasibility of measuring toxaphene in human samples and to determine whether additional analytical requirements were needed for routine measurement of toxaphene. We report a method for quantification of toxaphene congeners in human serum using a mixed-bed gradient solid-phase extraction and analysis using gas chromatography-high-resolution mass spectrometry with electron-impact ionization. In this method, we monitored low-mass fragment ions that were common to all 22 congeners. To verify the specific congeners detected, we further analyzed the extract using negative methane chemical ionization. We used this method to measure two specific congeners, Parlar 26 and 50, at concentrations ranging from about 3 to 30 pg/ml (0.7-7 ng/g lipid) in pooled human serum collected in Atlanta, Chicago, and Cincinnati. We identified several analytical parameters that must be strengthened to routinely measure toxaphene congeners in human samples.

Gentamicin and tobramycin binding to human serum in vitro.

The binding of gentamicin and tobramycin to human serum was studied in vitro using equilibrium dialysis of pooled human serum supplemented to various concentrations of either drug. Only minimal and variable non-specific binding was noted for each drug: gentamicin, less than 15% and tobramycin, less than 30%. Conventional Scatchard analysis conducted over an array of drug concentrations failed to identify any specific binding proteins.

Depletion of specific cockroach allergens may not affect overall allergenicity determinations

Rationale Assays of allergen potency may measure specific allergens or overall allergenicity. We wanted to determine whether depletion of specific allergens in German cockroach allergen extract affects overall allergenicity by competition ELISA Methods We selectively adsorbed the allergens Bla g 1 and Bla g 2 from German cockroach extract using allergen-specific rabbit IgG on a Protein G Sepharose column. The effect of adsorption on the extracts was determined by three different methods: a two-site ELISA for Bla g 1 and Bla g 2 using monoclonal antibodies (Indoor Biotechnology, Charlottesville, Virginia), a direct ELISA for Bla g 1 and Bla g 2 using hyper-immune rabbit serum as the detection antibody, and overall allergenicity by an IgE competition ELISA using a human allergic serum pool. Results Bla g 1 was specifically reduced by 91% and Bla g 2 was specifically reduced by 87%, as measured by two-site ELISA or direct ELISA, following adsorption. However, no loss in overall allergenicity was detected by competition ELISA. Conclusions Measures of overall allergenicity may not detect the loss of specific allergens.

Standardization of immunoassays for measurement of myoglobin in serum. Phase I: Evaluation of candidate secondary reference materials

Background: Myoglobin is a low-molecular weight protein present in the cytosol of striated muscles. Its concentrations in serum can be measured by immunoassays and are used as an early indicator of myocardial necrosis. Since variability among commercial myoglobin assays exists, standardization of myoglobin assays is needed. Methods: An international collaborative study was organized with the involvement of seven companies using 12 different automated platforms for measuring myoglobin. Five candidate secondary, i.e., matrixed, reference materials were assayed in relation to linearity, imprecision, recovery rate and commutability to demonstrate a possible identity between the materials and the usual routine serum samples. Results: One lyophilized candidate material (human heart myoglobin in human serum) was selected as the most suitable secondary reference material, based on the criteria examined. Used as a calibrator a posteriori, the bias between the various myoglobin assays for a frozen human serum pool was reduced from 32% to 13%. Conclusion: This study provides the basis for the selection of an internationally recognized secondary reference material.

Retrospective Time-Trend Study of Polybrominated Diphenyl Ether and Polybrominated and Polychlorinated Biphenyl Levels in Human Serum from the United States

Six polybrominated diphenyl ethers (PBDEs), one hexabromobiphenyl [polybrominated biphenyl (PBB)], and one hexachlorobiphenyl [polychlorinated biphenyl (PCB)] were measured in 40 human serum pools collected in the southeastern United States during 1985 through 2002 and in Seattle, Washington, for 1999 through 2002. The concentrations of most of the PBDEs, which are commercially used as flame retardants in common household and commercial applications, had significant positive correlations with time of sample collection, showing that the concentrations of these compounds are increasing in serum collected in the United States. In contrast, PCB and PBB levels were negatively correlated with sample collection year, indicating that the levels of these compounds have been decreasing since their phaseout in the 1970s.

Retrospective time-trend study of polybrominated diphenyl ether and polybrominated and polychlorinated biphenyl levels in human serum from the United States.

Six polybrominated diphenyl ethers (PBDEs), one hexabromobiphenyl [polybrominated biphenyl (PBB)], and one hexachlorobiphenyl [polychlorinated biphenyl (PCB)] were measured in 40 human serum pools collected in the southeastern United States during 1985 through 2002 and in Seattle, Washington, for 1999 through 2002. The concentrations of most of the PBDEs, which are commercially used as flame retardants in common household and commercial applications, had significant positive correlations with time of sample collection, showing that the concentrations of these compounds are increasing in serum collected in the United States. In contrast, PCB and PBB levels were negatively correlated with sample collection year, indicating that the levels of these compounds have been decreasing since their phaseout in the 1970s.

G-CSF modulates LPS-induced apoptosis and IL-8 in human microvascular endothelial cells: involvement of calcium signaling.

Microvascular endothelial cells (mECs) circulate at higher numbers in patients with severe sepsis and hemophagocytic syndromes. Although these blood mECs might stem from damaged microvasculature, they are perfectly viable and lead to the establishment of cell lines. Such mECs were cultured in low-dose human serum pools (0.5%) and MEM-alpha medium. Antigenic profiling revealed the expression of CD36, factor VIIIa, CD95-ligand, and CD44, but also CD146. We studied the antioxidative effect of the hematopoietic growth factor G-CSF(1) after in vitro stimulation with LPS from E. coli 0111:B4; the growth factor appeared to exhibit a protective effect on organ function in patients with SIRS. mECs were stimulated with 1 micro g/mL of LPS for 24 h and 48 h with and without G-CSF (3x10(3) U/mL) preincubation. After 24 h, supernatants of the stimulated mEC were tested for IL-8 by ELISA, and cells were tested for hemoxygenase-1 (HO-1, Hsp32) by immunohistochemistry and flow cytometry using OSA110 (mAb, Stressgene). Stimulation with LPS upregulated IL-8 by a factor of 2 to 10 in mEC. Preincubation with G-CSF markedly downregulated the LPS-induced IL-8 secretion (20-50%), but IL-6 production was not affected. Upon 48 h of LPS stimulation, mECs developed massive signs of apoptosis and concomitant caspase 3 activation. Caspase 3 activity induced by LPS (24 h) or by staurosporin (6 h) was found to be dramatically downregulated by the G-CSF preincubation protocol.

Identification of Oilseed Rape (Brassica napus) Pollen Profilin as a Cross-Reactive Allergen

Background: Major allergens of oilseed rape (OSR) pollen with molecular weights of 6/8, 14 and between 27 and 69 kD have been described. The aim of the present study was to further characterize the 14-kD allergen. Methods: The 14-kD protein was purified from OSR pollen extracts by poly-(L-proline) (PLP)-Sepharose affinity chromatography and characterized immunologically by means of allergic patients’ IgE antibodies, profilin-specific rabbit antisera, Western blot and ELISA inhibition using recombinant birch profilin (rBet v 2), and skin prick testing. Results: By PLP affinity chromatography, OSR pollen profilin was purified as a single protein of 14.5 kD and further identified as a profilin by three polyclonal rabbit antisera raised against ragweed and tobacco pollen profilin and the C-terminus of birch profilin. IgE binding of a human serum pool (n = 15) and four profilin-reactive sera to nitrocellulose-blotted OSR profilin was completely inhibited by 1 µg/ml rBet v 2 (birch profilin). Reciprocal ELISA inhibition using increasing concentrations of rBet v 2 and purified OSR profilin, respectively, showed that rBet v 2 strongly inhibits antibody binding to OSR profilin, whereas almost 100 times the amount of OSR profilin was needed to inhibit IgE binding to rBet v 2. Skin prick tests were positive (wheal ≧3 mm) with 5 µg/ml rBet v 2 in all three patients tested, and with OSR profilin in two patients at a concentration of 50 µg/ml. Conclusions: OSR pollen profilin shares IgE and IgG epitopes with Bet v 2 and other plant profilins and may represent a potentially relevant allergen for profilin-sensitized patients.

Citrobacter koseri brain abscess in the neonatal rat: survival and replication within human and rat macrophages.

A unique feature of Citrobacter koseri is the extremely high propensity to initiate brain abscesses during neonatal meningitis. Previous clinical reports and studies on infant rats have documented many Citrobacter-filled macrophages within the ventricles and brain abscesses. It has been hypothesized that intracellular survival and replication within macrophages may be a mechanism by which C. koseri subverts the host response and elicits chronic infection, resulting in brain abscess formation. In this study, we showed that C. koseri causes meningitis and brain abscesses in the neonatal rat model, and we utilized histology and magnetic resonance imaging technology to visualize brain abscess formation. Histology and electron microscopy (EM) revealed that macrophages (and not fibroblasts, astrocytes, oligodendrocytes, or neurons) were the primary target for long-term C. koseri infection. To better understand C. koseri pathogenesis, we have characterized the interactions of C. koseri with human macrophages. We found that C. koseri survives and replicates within macrophages in vitro and that uptake of C. koseri increases in the presence of human pooled serum in a dose-dependent manner. EM studies lend support to the hypothesis that C. koseri uses morphologically different methods of uptake to enter macrophages. FcgammaRI blocking experiments show that this receptor primarily facilitates the entry of opsonized C. koseri into macrophages. Further, confocal fluorescence microscopy demonstrates that C. koseri survives phagolysosomal fusion and that more than 90% of intracellular C. koseri organisms are colocalized within phagolysosomes. The ability of C. koseri to survive phagolysosome fusion and replicate within macrophages may contribute to the establishment of chronic central nervous system infection including brain abscesses.

DNA immunization with the cysteine-rich interdomain region 1 of the Plasmodium falciparum variant antigen elicits limited cross-reactive antibody responses.

The variant surface antigens of Plasmodium falciparum are an important component of naturally acquired immunity and an important vaccine target. However, these proteins appear to elicit primarily variant-specific antibodies. We tested if naked DNA immunization can elicit more cross-reactive antibody responses and allow simultaneous immunization with several variant constructs. Mice immunized with plasmid DNA expressing variant cysteine-rich interdomain region 1 (CIDR1) domains of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) developed antibodies that were reactive to the corresponding PfEMP1s as measured by an enzyme-linked immunosorbent assay, flow cytometry, and agglutination of parasitized erythrocytes (PEs). We observed some cross-reactive immune responses; for example, sera from mice immunized with one domain agglutinated PEs of various lines and recognized heterologous domains expressed on the surface of Chinese hamster ovary (CHO) cells. We found no significant antigenic competition when animals were immunized with a mixture of plasmids or immunized sequentially with individual constructs. Moreover, mixed or sequential immunizations resulted in greater cross-reactive agglutination responses than immunization with a single domain. Recombinant protein (Sc y179) immunization after priming with DNA (prime-boost regimen) increased antibody titers to the homologous domain substantially but seemed to diminish the cross-reactive responses somewhat. The titer of agglutinating antibodies was previously shown to correlate with protection. Surprisingly, the agglutination titers of sera from DNA immunization were high, similar to those of pooled human hyperimmune sera. These sera also appeared to give limited low-titer variant transcending agglutination. Thus, DNA immunization appears to be a very useful tool for developing variant antigen vaccines.

Starvation survival, growth and recovery of Enterococcus faecalis in human serum.

The ability of Enterococcus faecalis to survive starvation for long periods in the obturated root canal is likely to be an important factor in the pathogenesis and maintenance of a persistent infection after endodontic treatment. The response of E. faecalis to starvation survival in water and glucose-, phosphate- or amino acid-limited chemically defined medium was studied, along with the capacity for growth and recovery of starved cells of E. faecalis in pooled human serum. After an initial rapid fall in cell numbers, a small remaining population of E. faecalis was able to survive in water for over 4 months and in nutrient-limited media for extended periods. A high cell density at the onset of starvation was critical for the ability of E. faecalis to endure prolonged nutrient limitation. Upon starvation, a static population of starved cells developed and were apparently in a minimal metabolic state, since blocking cell wall synthesis with penicillin G or inhibiting DNA synthesis with norfloxacin during starvation resulted in limited change in the rate of loss of viable cells. In 50% serum, E. faecalis grew, then stabilized at a relatively constant population of 106 colony-forming units/ml for 4 months, irrespective of the initial cell density. In summary, E. faecalis is capable of withstanding prolonged periods of starvation in a minimal metabolic state provided that there is a high cell density at the onset of starvation. Starved cells were capable of recovery upon addition of human serum.

Hevein-specific recombinant IgE antibodies from human single-chain antibody phage display libraries

IgE antibodies distinctively recognising allergenic epitopes would be ideal reagents in immunodiagnostics to detect and quantify allergens, as well as for the development of allergy diagnostics and therapeutics. We have isolated recombinant human IgE antibodies specific for the major latex allergen, hevein, from antibody phage display libraries using a green fluorescent protein (GFP)–hevein fusion as a selection antigen. Human IgE phage display libraries were constructed by combining the IgE heavy chain genes to kappa and lambda light-chain genes which were isolated from lymphocytes of a latex allergic patient. The screening of antibody libraries resulted in the enrichment of two hevein-binding scFvs designated as 1A4 and 1C2. Both antibodies showed specific binding to the hevein that could be inhibited by both the recombinant GFP–hevein and native hevein isolated from latex examination gloves. The scFvs were prone to aggregate and, thus, for further characterisation, they were converted to Fab fragments with human IgG1 or IgE isotype. Similar hevein-binding properties of the 1A4 and 1C2 Fab fragments and human IgE serum pool, conventionally used in the detection of latex allergens, demonstrate the potential utility of these recombinant antibodies for the analysis of latex allergen.

Uremia induces the osteoblast differentiation factor Cbfa1 in human blood vessels

Bone matrix proteins are expressed in calcified arteries from dialysis patients, suggesting that vascular smooth muscle cells (VSMCs) may transform to osteoblast-like cells. One of the key transcriptional regulators of osteoblast differentiation is Cbfa1. Thus, we hypothesized that this may be a key factor in arterial calcification. To test this hypothesis, we examined sections of the inferior epigastric artery from uremic patients for the presence of Cbfa1 and type I collagen and osteopontin by in situ hybridization and immunostaining. We also examined the effect of pooled uremic sera from dialysis patients on the expression of Cbfa1 by reverse transcription-polymerase chain reaction (RT-PCR) in bovine VSMCs in vitro. Cbfa1 and osteopontin were expressed in both the media and the intima in vessels that were calcified, but there was only minimal staining in non-calcified vessels. In vitro studies demonstrated that pooled uremic serum, compared to pooled control human serum induced the expression of Cbfa1 by RT-PCR in bovine VSMCs in a time-dependent, nonphosphorus-mediated mechanism. These results support that Cbfa1 is a key regulatory factor in the vascular calcification observed in dialysis patients and is up-regulated in response to many uremic toxins.

Inhibition of human serum arylesterase by metal chlorides

The inhibition of arylesterase (paraoxonase, EC 3.1.8.1) by metal chlorides was studied with both pooled human serum (A phenotype) and purified enzyme, using phenyl acetate as substrate. Inhibition data were analysed with the Hill equation. Results obtained with whole serum and purified enzyme were very similar. On the basis of the Hill coefficient, n H, three groups of inhibitors were distinguished: (1) Cu 2+ and Hg 2+ for which n H=1, suggesting a single binding site (probably the free cysteine at position 283); these metals were mixed inhibitors, with more affinity for the free enzyme than for the enzyme–substrate complex; (2) Mn 2+, Co 2+, Ni 2+, Zn 2+, and Cd 2+ for which n H>1, suggesting several cooperative binding sites; (3) La 3+, for which n H<1. Within groups (1) and (2) the inhibiting potency followed the order of the periodic table. For the 3d elements the inhibiting order followed the Irving–Williams series, with the classical exception of Cu 2+. Only Zn 2+ was inhibitory at its physiological concentration.

Pretreatment of serum containing hemoglobin vesicles (oxygen carriers) to prevent their interference in laboratory tests.

Hemoglobin vesicles (HbV, diameter: 251 +/- 81 nm) are artificial oxygen (O2) carriers encapsulating concentrated hemoglobin (Hb) solution with phospholipid bilayer membrane, and their O2 transporting ability in vivo has been extensively studied. It is important to clarify the interference of the HbV suspension in clinical laboratory tests performed on serum and to establish a pretreatment method to avoid such an interference. The HbV suspension, acellular Hb solution ([Hb] = 10 g/dl) or saline, was mixed with a pooled human serum at various ratios up to 50 vol% ([Hb] = 5 g/dl), and the magnitude of the interference effect of HbV and Hb on 30 analytes was studied. The mixture of the HbV suspension and serum was ultracentrifuged (50,000 g, 20 min) to remove the HbV particles as precipitate, and the supernatant was analyzed and compared with the saline control group. The HbV particles were also removed by centrifugation (2,700 g, 30 min) in the presence of dextran (Mw 200 kDa). The HbV suspension showed considerable interference effects in most analytes. The majority of these effects was more serious than those of the acellular Hb solution. These findings are thought to be due to the light absorption of Hb in HbV and/or the light scattering generated in the suspension that interferes with the colorimetric and turbidimetric measurements. The components of HbV may also interfere with the chemical reactions of the studied assays. However, removal of the HbV from the supernatant diminished the interference in most of the assays: this is an advantage of HbV in comparison with acellular chemically modified Hb solutions.

One Step Enzyme Linked Immunosorbent Assay for Direct Estimation of Serum Testosterone

One step competitive enzyme linked immunosorbent assay (ELISA) for direct estimation of testosterone in human serum is described. Testosterone-3-O-carboxymethyl-oxime-bovine serum albumin (testosterone-3-O-CMO-BSA), was used as immunogen and testosterone-3-O-carboxymethyl-oxime-adipic-acid dihydrazide-horseradish peroxidase (testosterone-3-O-CMO-ADH-HRP) was used as tracer. To the testosterone antibody coated microtiter wells, standard or serum samples (100 µL), along with testosterone-3-O-CMO-ADH-HRP conjugate (100 µL) were incubated for 1 h at 37°C. Bound enzyme activity was measured by using tetra methyl benzidine/hydrogen peroxide (TMB/H2O2) as a substrate. In this new strategy, charcoal stripped pooled human serum spiked with non-cross reactive C18, C19, C21, and C27 steroids, used for preparing the standards and blocking the sex hormone binding globulin (SHBG)/and other steroid binding globulins (SBG). The sensitivity of the assay was 0.015 ng/mL. The intra-assay and inter-assay coefficients of variation (CVs) were ranged from 7.8 to 11.8 and 4.8 to 10.4, respectively. The serum testosterone values, obtained by this method, were correlated well with those obtained by radioimmunoassay r = .98 (n = 100).