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Background The intra‐individual reference range is generally narrower than the commonly used reference range. Consequently, close monitoring of changes in the laboratory test results of individuals based on the inter‐individual reference range remains challenging. Methods We examined the determination of individual reference ranges using four indicators of nutritional conditions: transferrin (TRF), albumin (ALB), retinol‐binding protein (RBP), and transthyretin (TTR). The subjects comprised 20 healthy individuals and blood samples were collected and tested five times at 2‐week intervals. We used the measurement results for the four indicators and examined individual reference ranges using four methods, including calculation methods based on the reference change value and Bayesian inference. Results The resulting intra‐individual reference ranges were narrower than the currently used inter‐individual reference range for all measurements using four methods. Furthermore, the intra‐individual coefficient of variation [CV (intra)] was smaller than the inter‐individual coefficient of variation [CV (inter)] for TRF, RBP, and TTR for all 20 subjects. The means CV (intra) for the four indicators were also lower than the corresponding CV (inter). Conclusions The intra‐individual reference range can be used to validate the standard deviation and coefficient of variation for currently used indicators. Moreover, Bayesian methods are speculated to be the most versatile., In our paper, we demonstrate a novel method of diagnostic threshold estimation using the test results of the subjects from this study. In current clinical practice, the inter‐individual reference range of analytes, such as transferrin, is used for patient diagnosis in routine clinical settings. However, in some subjects, even if the variation of the measured analyte is small, one or more of its values may surpass the normal range and be sent for reanalysis (upper diagram). In addition to two previously studied methods for constructing intra‐individual reference ranges, our current study reports on intra‐individual reference ranges that we constructed using the Bayesian estimation method (lower diagram). This new method uses the standard deviation of an individual’s daily test results taken prior to the initial diagnostic assessment to calculate the reference standard range to be used when the subject takes the diagnostic test for the first time; the reference standard range is recalculated using Bayesian estimation after the second test. It is a method to refer to the reference range obtained by the method. Clinicians will be able to diagnose patients based on the currently used inter‐individual reference range, as well as the reference range obtained by the Bayesian estimation method

This study discusses the creep-fatigue life prediction of eutectic Sn37Pb solder. Creep-fatigue lives were experimentally obtained at 313 K using fast-fast, slow-slow, slow-fast, fast-slow, and strain-hold waveforms. Conventional four creep-fatigue life prediction rules, time fraction rule, strain range partitioning method, frequency modified fatigue life, and ductility exhaustion model was used to predict the creep-fatigue lives. However, not all the rules were able to properly estimate them. Since the grain boundary sliding rates were closely related to creep-fatigue lives, a new life prediction method based on grain boundary sliding rates was proposed.

It has been proposed that α-fetoprotein (AFP) is a new member of the intracellular signaling molecule family of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway via interaction with the phosphatase and tensin homolog (PTEN). In this study, the effects of anti-human AFP antibody on the functions of PTEN were examined using an AFP-producing human hepatoma cell line. The antibody caused significant inhibition of cell growth, compared to a normal IgG control, with the accumulation of intracellular immune complexes followed by significant reduction of cytosolic functional AFP. Decrease in the amount of AKT phosphorylated on serine (S) 473 indicated that PI3K/AKT signaling was suppressed in the cells. S380-phosphorylated PTEN increased markedly by the second day after antibody treatment, with slight but significant increase in the PTEN protein level. Since phosphorylation at S380 is critical for PTEN stability, the increase in S380-phosphorylated PTEN indicated maintenance of the number of PTEN molecules and the related potential to control PI3K/AKT signaling. p53 protein (P53) significantly, but slightly increased during antibody treatment, because PTEN expression increased the stability and function of P53 via both molecular interactions. P53 phosphorylated at S20 or at S392 dramatically increased, suggesting an increase in the stability, accumulation and activation of P53. Glucose transporter 1 (GLUT1) increased immediately after antibody treatment, pointing to a deficiency of glucose in the cells. Immunofluorescence cytology revealed that antibody-treatment re-distributed GLUT1 molecules throughout the cytoplasm with a reduction of their patchy localization on the cell surface. This suggested that translocation of GLUT1 depends on the PI3K/AKT pathway, in particular on PTEN expression. Antibody therapy targeted at AFP-producing tumor cells showed an inhibitory effect on the PI3K/AKT pathway via the liberation, restoration and functional stabilization of PTEN. PTEN simultaneously induced both P53 activation and intracellular translocation of GLUT1, since these are closely associated with PTEN.

This paper describes a creep–fatigue life of Sn–8Zn–3Bi solder under multiaxial loading. A push–pull and a reversed torsion tests were carried out using seven types of strain waveforms, which are a fast–fast, a fast–slow, a slow–fast and a slow–slow waveforms and three types trapezoidal strain waveforms with different strain holding times. The strain waveforms had a significant effect on creep–fatigue life and the shortest creep–fatigue life was found in the slow–fast strain waveform while the longest life in the slow–slow waveform in the push–pull and the reversed torsion tests. Creep–fatigue life in the reversed torsion test was approximately twice longer than that in the push–pull test at each strain waveform. Applicability of common used creep–fatigue damage models for life evaluation was discussed based on the obtained experimental results and only a grain boundary sliding model could evaluate the lives within a small scatter.

Electromigration behavior of SAC flip-chip joints with respect to the crystallographic orientation of Sn grains was investigated. The test sample had direct contact of Sn-3.0wt% Ag-0.5wt% Cu on Cu-OSP (organic surface preservative) and the applied current density was 15 kA/cm2 at 160 °C. Without current stressing, no microstructural change depending on the crystallographic orientation of Sn was observed. With current stressing, however, the bumps showed the substantial microstructural changes with respect to the crystallographic orientation of Sn. When the orientation of the current flow was parallel to the c-axis, fast failure of the bumps occurred due to the massive dissolution of the Cu electrode on the cathode side caused by the fast diffusion of Cu atoms along the c-axis of Sn grains while only a slight microstructural change was observed when the c-axis of Sn grains was perpendicular to the electron flow.

This work studies the low cycle fatigue crack propagation for three types of notched specimens of Sn–3.5Ag lead free solder. Low cycle fatigue tests were carried out using circumferential notched specimens. The fatigue life for crack initiation and propagation were experimentally obtained. The applicability of stress intensity factor range, J-integral range and strain intensity factor range for correlating the crack propagation rates was discussed. It was observed that the three fracture mechanics parameters did not correlate the crack propagation rates in a whole propagation stage with a small scatter but the stress intensity factor and J-integral ranges correlated the saturated crack propagation rates with a small scatter.

The propagation phenomenon of a detonation wave is particularly interesting, because the detonation wave is composed of a 3D shock wave system accompanied by a reaction front. Thus, the passage of a detonation wave draws cellular patterns on a soot-covered plate. The pressure and temperature behind the detonation wave are extremely high and may cause serious damages around the wave. Therefore, it is of great significance from a safety-engineering point of view to decay the detonation wave with a short distance from the origin. In the present study, experiments using high-speed schlieren photography are conducted in order to investigate the behaviors of the detonation wave diffracting from two slits. The detonation wave produced in a stoichiometric mixture of hydrogen and oxygen is propagated through the slits, and the behaviors behind the slit-plate are investigated experimentally. When a detonation wave diffracts from the slits, a shock wave is decoupled with a reaction front. Since the two shock waves propagate from the slits interact with each other at the center behind the plate, the detonation wave is reinitiated by generating a hot-spot sufficient to cause local explosions. Furthermore, it is clarified that the shock wave reflected from a tube-wall is also capable of reinitiating the detonation wave. The reinitiation distance of the detonation wave from the slit-plate is correlated using a number of cells emerged from each slit.

Electromigration in the Cu/In/Cu flip chip joint system was investigated at a temperature of 398 K with a current density of 20 kA/cm2. Indium was transported towards the anode, and a large void nucleated by electromigration at the In/Cu11In9 interface in the highest current density region near the cathode. It was found that a unique solder bump deformation towards the cathode occurred in the early stage of the electromigration test, when no underfill material was used. When the underfill material was used to fill the space between the chip and the substrate, the electromigration lifetime in the joint was prolonged by more than ten folds in comparison with that of the joint without underfill. This was a result from that indium transport was inhibited due to a larger backstress induced by the constraint of the underfill.

Electromigration in Cu/In/Cu flip chip joint system has been studied at 398 K with a current of 20 kA/cm 2 . The void nucleation and growth process was investigated with the experiment and the 3D simulation. In was transported towards the anode and a void nucleated at the highest current density point near the cathode. The void nucleated at the In/Cu 11 In 9 interface and grew along the cathode side. The unique solder bump deformation towards the cathode occurred in the early stage of the electromigration test. The temperature of the solder joint during the electromigration test was measured successfully both by a thermo-couple and by the resistance change of the junction line between two flip chip joints.

A propagation of detonation wave shows particularly interesting phenomena, since the detonation wave is constituted from a three-dimensional shock wave system accompanied with a reaction front. Thus, the passage of a detonation wave draws cellular patterns on a soot-covered plate. Pressure and temperature behind the detonation wave are extremely high and have a potential to cause serious damages around it. Therefore, it is necessary from safety engineering point of view to quench the detonation wave with short distance from the origin. In this study, experiments applied high-speed schlieren photography are conducted to investigate behaviors of the detonation wave diffracting from two pieces of slits. The detonation wave produced in a stoichiometric mixture of hydrogen and oxygen is propagated through the slits and behaviors behind slit-plate are experimentally investigated. As a result, when the detonation wave diffracted from the slits, a shock wave is decoupled with a reaction front. Since two shock waves progagated from the slits interact each other at centre behind the plate, the detonation wave is re-initiated by generating a hot-spot enough to cause local explosion. It is also clarified that the shock wave reflected from a tube-wall induces detonation re-initiation.

Pressure and temperature behind a detonation wave are extremely high and have a potential to cause serious damages around it. Therefore, it is necessary from safety engineering point of view to decay the detonation wave with short distance from a generation of it. In this study, experiments are conducted in order to investigate behaviors of the detonation wave propagating into two pieces of slits, since the detonation wave might be quenched behind the slits by expansion waves generated at a corner of the slits and this behavior might be applicable to a technique of detonation-arrester. The detonation wave produced in a stoichiometric mixture of hydrogen and oxygen is propagated through the slits and behaviors of it are experimentally investigated by using a technique of pressure measurement, soot track record and high-speed schlieren photography. As a result, when the detonation wave propagated through the slits, a shock wave is decoupled with a reaction front. Two shock waves diffracted from the slits interact each other at center behind the slits, then this shock wave interaction induces a hot-spot enough to cause local explosion. Since, the shock wave is reflected from a tube-wall eventually, the detonation wave is re-initiated by mechanisms of shock-shock interaction or shock-wall interactions.

The electromigration effect on solder bump and interfacial reactions in the Cu/Sn–3Ag–0.5Cu/Cu flip chip joint was investigated at 453 K with 10 kA/cm2. Cu was transported towards the anode and avoid nucleated at the highest current density point near the cathode. The void nucleated at the Sn/Cu6Sn5 interface and grew along the cathode side. It was observed that the unique solder bump deformation towards the cathode started in the early stage of the electromigration test.

Experiments were conducted in order to investigate behaviors of a detonation wave propagating into two pieces of slit, since the detonation wave might be quenched behind a slit-plate by expansion waves generated at a corner of the plate and this technique might be applicable to a detonation arrestor. The detonation wave produced in a stoichiometric mixture of hydrogen and oxygen was propagated through the slits and behaviors of the detonation wave were experimentally investigated by using a technique of pressure measurement, soot track record and high-speed schlieren photography. As a result, when the detonation wave propagated through the slits, it was quenched and a schock wave was decoupled with a reaction front. Two shock waves diffracted from the slits interacted each other at centre of the tube, then this shock wave interaction induced a hot-spot enough to cause local explosions behind the slit-plate. Since the shock wave was reflected from the wall and interacted at centre of the tube, the detonation wave was re-initiated behind the slits.

A detonation wave produced in a combustible gaseous mixture might cause serious damages by interacting with an artificial structure or human bodies because of an extremely high-pressure and high-temperature behind this wave. Therefore, the detonation wave produced in the gaseous mixture and propagated into a circumstance by accident should be attenuated or quenched within a short distance from its origin. Experiments were conducted in order to investigate behaviors of the detonation wave passing through narrow grooves, since the detonation wave was accompained with a cellular structure and no detonation wave could be propagated. In this study, the detonation wave produced in a gaseous mixture of hydrogen and oxygen was propagated through a grooved block and behaviors of the detonation wave were experimentally investigated by using techniques of pressure measurement and soot track record. As a result, the behavior of detonation wave propagating through the grooved block was classified into two categories, i. e. (i) the detonation wave was quenched, (ii) the detonation wave was once quenched behind the block but re-initiated again by focusing mechanisms of a reflected shock wave on a central axis.

This paper studies the creep-fatigue life evaluation of Sn-3.5Ag solder under push-pull loading using fast-fast, fast-slow, slow-fast, slow-slow, and strain-hold strain waves. Extensive creep-fatigue data were generated using these strain waves and the applicability of four conventional creep-fatigue damage rules, the linear damage rule, the frequency modified fatigue life, the ductility exhaustion model, and the strain range partitioning method, was examined. No conventional damage rules evaluated creep-fatigue lives accurately. Only the grain boundary sliding model, developed recently for solders, predicted creep-fatigue lives with a small scatter.

This paper studies creep-fatigue life prediction under shear loading by making extensive torsion creep-fatigue experiments using four kinds of strain waves. The linear damage rule, strain range partitioning method, frequency modified fatigue life, and ductility exhaustion model were applied to the experimental data, but no methods accurately predicted the creep-fatigue life. A new method based on the strain rate ratio, which predicted the creep-fatigue life within a factor of 4 scatter band, was developed.

An introduced new substrate technology for semiconductor packaging by flip chip bonding has featured with following design deliverables. The conductor line width is 25 μm and the space between the lines is 25 μm minimum at an escape point between flip chip pads. The thickness of dielectric layer is adjusted to obtain characteristic impedance with 50 Ω. A film resin for dielectric is applied to a base core sequentially with conductor plane alternately and laser-drilled micro-via hole is formed in the resin to connect conductor planes. UV-laser drill is employed to provide micro-via hole with 48 μm as drilled. The conductor layers are formed with copper pulse-pattern plating after electroless seed layer plating. The substrate passed JEDEC level-3 stress test and 10 GHz clock frequency possibility was demonstrated.

This paper describes a new miniature creep tester and the size effect of creep and creep rupture times for solders. The new miniature creep tester was developed and creep rupture tests were carried out using five types of Sn-37 Pb specimens with different gage diameters. Creep rupture times of miniature specimens did not differ from those of bulk specimens at a higher stress but they were scattered around the bulk data at lower stresses. Creep rates of miniature specimens were slower than those of bulk specimens and creep ductility of miniature specimens was smaller than that of bulk specimens.

This paper studies the creep and creep rupture properties of Sn-95 Pb solder. Creep and creep rupture tests were carried out using Sn-95 Pb solder specimens at 313 K, 353 K and 398 K to develop a creep constitutive equation and rupture time equation. There were clear short primary, long tertiary and steady creep stages in Sn-95 Pb solder at the three temperatures. A creep constitutive equation was proposed as a function of stress and temperature. The proposed equation predicted the experimental creep strain within a factor of 1.25. A new equation for predicting creep rupture time was also proposed. The equation predicted the experimental rupture time within a factor of two. These results were compared with those of Sn-37 Pb solder to discuss differences resulted from melting temperature.

This paper describes the life assessment of flip chip joints. Flip chip joints of 63Sn-37Pb and 5Sn-95Pb solders on a printed circuit board were stressed thermally for fatigue. Fatigue lives of the joints were determined by an electrical potential drop method and the effect of encapsulation on fatigue life was discussed. The encapsulation had a significant effect of prolonging the fatigue life of the joints. Thermo-mechanical finite element analyses proved that the encapsulation lowered the strain amplitude of the joints by distributing the strain over a whole package and bending effect. Cracking location was also discussed in relation with the strain concentration in the joints. Fatigue lives of the flip chip joints were compared with those of bulk round bar specimens and the difference in fatigue life between two types of specimens was discussed from the specimen dimensions and ratchet effect. [S1043-7398(00)00203-6]

Advanced Glycation End products (AGEs) は, メイラード反応後期生成物とも呼ばれ, 糖尿病性合併症の原因物質の一つではないかと考えられている. われわれは, 血清中AGE測定系を確立し, 糖尿病さらにその合併症進展と血中AGE量との関係等について検討を行ってきた. 今回, 腹膜透析液を用い, ゲル濾過, affinity chromatography, western blotting 等の手法により, AGEの血中存在様式について検討を行った. その結果, AGE構造の大部分は, 血清蛋白と結合していることが明らかとなった. さらに, AGE化されている蛋白成分としてIgG, アルブミン, β2-マイクログロブリンを同定することができた.

This paper describes the tensile property of six kinds of Sn-Pb solders. Static tension tests were carried out at strain rates ranging from 0.001%/sec to 2.0% / sec using 5Sn-95Pb, 10Sn-90Pb, 40Sn-60Pb, 60Sn-40Pb, 63Sn-37Pb, and 62Sn-36Pb-2Ag solders at 313-398 K. Strain rates faster than 2.0%/sec were required to obtain time-independent Young's modulus and yield stress of the solders. In tests at the strain rates slower than 2.0%/sec, creep deformation was included in the stress-strain relationship, and Young's modulus and yield stress which do not depend on time could not be obtained. A new equation which can predict tensile strength of Sn-Pb solders as functions of temperature, strain rate and chemical composition were proposed. New equations were also proposed for predicting Young's modulus and yield stress as functions of temperature and chemical composition. These equations predict Young's modules and yield stress within a factor of 1.25 and 1.15, respectively. Low cycle fatigue lives were discussed based on the universal slope method.

We determined serum advanced glycation end-products (AGE) levels in patients with NIDDM and evaluated the relationship between these levels and diabetic complications. The subjects consisted of 125 patients (mean age, 59.2 +/- 11.1 years, duration of diabetes 11.6 +/- 8.9 years, mean HbA1c, 6.8 +/- 1.0%) with stable blood sugar control. Sixty-three healthy volunteers (mean age, 58.3 +/- 12.7 years) served as controls. Serum AGE were measured by a newly developed ELISA method. Serum AGE levels were significantly higher in the diabetic group compared with the normal control group (7.2 +/- 14.6 vs. 3.3 +/- 1.0 mU/ml, P0.05). Significant correlations were seen between serum AGE and the degree of diabetic nephropathy. Serum AGE levels of diabetic patients with proliferative retinopathy were significantly higher than those of patients without proliferative retinopathy (5.7 +/- 1.8 vs. 3.1 +/- 1.0 mU/ml, P0.025) in the patient groups whose serum creatinine levels were between 2.0 and 3.9 mg/dl, although serum creatinine levels of both groups were not significantly different. Serum AGE levels reflected the severity of diabetic complications, including nephropathy and retinopathy.

Background: We examined the utility for prostate cancer staging of nested reverse transcription polymerase chain reaction (RT-PCR) using either prostate specific antigen (PSA) or prostate specific membrane antigen (PSM) as primer. Methods: LNCaP cells were used for the in vitro quantification of RT-PCR. RT-PCR was performed on the peripheral blood of 105 control subjects and 63 patients with prostate cancer (32 who eventually underwent radical prostatectomy and 31 with clinical stage D2 cancer). Results: The nested RT-PCR for the PSA and PSM primers was able to detect 1 LNCaP cell per 106 leukemia (K562) cells. Noneof the control subjects was found positive for the presence of prostate cancer cells by nested RT-PCR. In the 32-patient surgery group, the results of nested RT-PCR were significantly correlated with the pathologic stage of the cancer when using PSM primers (P=2.00times10-3 by Kendall's correlation test) but not when using PSA primers (P=0.06). Extraprostatic extension was significantly more closely correlated with positive PSM nested RT-PCR results (P=0.012 by Fisher's exact probability test) than with positive results of PSA, nested RT-PCR, digital rectal examination, CT imaging, level of serum PSA or Cleason score. In the untreated stage D2 patients, the positive result rate of PSM nested RT-PCR was significantly higher than that of PSA nested RT-PCR (P=0.025 by McNemar test). Conclusion: Nested RT-PCR using PSM primers appears to predict the prostate cancer stage more accurately than does nested RT-PCR using PSA primers or conventional clinical staging modalities.

This paper describes the multiaxial creep-fatigue of 63Sn-37Pb solder solid and tube specimens in push-pull and reversed torsion using fast-fast, slow-fast, fast-slow and slow-slow strain waves. The strain rate of fast straining was 0.5%/s and that of slow straining was 0.005%/s. The suitability of multiaxial strain parameters for correlating multiaxial creep-fatigue lives was discussed. Mises' strain and maximum shear strain were an appropriate parameter for the data correlation. The largest fatigue lives were found in fast-fast strain wave test and the smallest ones in slow-fast test. The fatigue lives in slow-slow test were somewhat smaller than those in fast-fast test, but there was no large difference in fatigue life between the two strain waves. Main cracks of which length was more than 1mm were initiated and propagated in fast-fast and slow-slow tests but many small cracks whose length was less than 100 micron meter were initiated and no long main cracks were propagated in slow-fast and fast-slow tests. These crack behaviors corresponded with the stress change with cycles. Stress amplitude rapidly decreased with cycles in the final stage of life in fast-fast and slow-slow tests due to the main crack extension but it gradually decreased with cycles in slow-fast test resulted by the extension of many small cracks. Deformation of grains in slow-fast and fast-slow tests was discussed in relation with the micro rachet deformation of grain boundaries.

Free ubiquitin (mainly monoubiquitin) and multi-ubiquitin chains coexist in eukaryote cells and serve distinct cellular roles. However, any immunoassay systems established previously have not been proved to be applicable for measuring the former without cross-reactive responses with the latter. For this purpose, we developed a radioimmunoassay specific to monoubiquitin by employing antiserum US- I against ubiquitin. In this assay, ubiquitin-protein conjugates, prepared by a reticulocyte lysate fraction 11 and fractionated on Moro Q and Superdex 200 columns, exhibited practically no cross-reactivity. The cross-reactivity of fractionated ubiquitin-lysozyme conjugates was also analyzed as a function of their multi-ubiquitin chain size. As a result, the larger the conjugates were found to be, the weaker were the cross-reactive responses they showed, and the multi-ubiquitin chains (n > approx. 20) were substantially unreactive in the radioimmunoassay. By using the radioimmunoassay, heat-shock-induced decrease in the level of cellular free (mono)ubiquitin was detected. In addition, the standard preparation of multi-ubiquitin chains was not cross-reactive in all other five radioimmunoassays employing distinct antibodies to ubiquitin (four antisera and a monoclonal antibody). These data suggest that radioimmunoassays employing ubiquitin antibodies raised by the general methods can discriminate between monoubiquitin and multi-ubiquitin chains and quantitate cellular free ubiquitin.

The isolation and identification of the 87A strain of hepatitis E virus (HEV) by means of cell culture have been described previously. This paper reports the nucleotide sequence of a portion of this HEV strain. The RNA extracted from the supernatants of the different passages of the 87A strain cultured in the A549 cell line was reverse-transcribed (RT) to cDNA, and then the polymerase chain reaction (PCR) amplification was carried out using the primers of HEV ET1.1 region. The PCR products from 1) the supernatant of the infected cells at the fourth passage, 2) the virus concentrated by polyethylene glycol (PEG) precipitation at the tenth passage, and 3) the virus purified by a sucrose gradient at the tenth passage were sequenced. In addition, three other PCR products obtained from sera of acute hepatitis E patients in Beijing (B-9) and Guangzhou (G-9 and G-20) were also sequenced. The nucleotide sequences of the above four strains of HEV (located in the genome from positions 4545-4754) were compared to those of some reported HEV strains. The nucleotide sequences of the B-9 strain and the 87A strain were similar to the Burmese strain and may belong to the same branch of HEV. The nucleotide sequences of the G-9 strain and the G-20 strain were a novel and unique branch. The Chinese HEV strains are multiplex and variable in gene structure.

A novel high-molecular-weight (MW) form of immunoreactive TSH [35,000 Da on Sephacryl S-200HR gel chromatography (S-200 chromatography)] was documented in a 32-year-old healthy woman who delivered two neonates with transient hyperthyrotropinemia. Her TSH levels ranged from 21.2 to 53.9 mU/L on different days or from 11.0 to 48.1 mU/L by the different immunoradiometric assay methods. The IgG fractions showed specific 125I-labeled hTSH binding and inhibited in vitro cAMP increase induced by hTSH but not that induced by bTSH. On protein G Superose HR affinity chromatography (protein G chromatography) equilibrated with 10 mmol/L sodium/potassium phosphate buffer (PB) followed by elution with 0.1mol/L glycine buffer, 95-99% of her TSH immunoreactivity eluted in the latter (bound) fraction while almost all was in the former (unbound) fraction in the control serum containing authentic hTSH. However, after dialysis of this bound fraction overnight with PB adding 0.5 mol/L NaCl (PB/NaCl), which exhibited greater ionic strength than PB, almost all TSH immunoreactivity changed from the bound fraction into the unbound fraction on the protein G chromatography equilibrated with PB/NaCl. These data indicate that the novel immunoreactive TSH was due to hTSH and hTSH-specific antibody complex, and dissociation of the complex may be incomplete on direct S-200. The immunoreactive TSH showed high MW form (35,000 Da). The dissociation may be almost complete during dialysis with greater ionic strength; the native TSH then appeared to be of formal size.

To establish a rapid, specific, sensitive and simple assay method for doxorubicin (DXR) in body fluid, monoclonal antibodies (MAbs) against DXR were generated by immunizing mice with keyhole limpet hemocyanin-DXR conjugate, cell fusion, and a one step, time saving screening ELISA method using aminoplate-coupled DXR via a glutaraldehyde bridge as solid phase antigen. Inhibition ELISA for DXR-immunoassay was established using anti-DXR MAb of the best producer (2E9) and aminoplate-coupled DXR as antigen and DXR ranging from 50 pg to 50 ng in the body fluid or in the cell extract could be detected. MAb 2E9 cross-reacted to various degrees to anthracycline compounds, such as some DXR analogues and derivatives, but did not recognize anthracene and anthraquinone structures.