Seiichi Hashida

Enzyme-Labeling of Antibodies and Their Fragments for Enzyme Immunoassay and Immunohistochemical Staining

Abstract The use of an enzyme as a label has a number of advantages over the use of other labels in both immunohistochemistry and immunoassay. Immunofluorescence techniques are not suitable for ultrastructural research on cells, and ferritin-labeled antibodies allow only electronmicroscopic studies. By contrast, enzyme-labeled antibodies permit localization of cellular antigens in relation to tissue structures under light microscope and also demonstration of cellular antigens at an ultrastructural level by electronmicroscopy. Antibody or antibody fragments labeled with enzymes of small molecular weight can more readily permeate cells of tissue sections than ferritin-labeled antibodies. The color of tissue sections prepared by immunoenzymatic techniques is stable for years, while immunofluorescence of tissue sections decreases rapidly when exposed to light. Radioisotope-labeled reagents decay with time; there are health hazards due to radio isotopes; and disposal of radioactive waste is becoming increasing...

The Clostridium cellulovorans Cellulosome

The Clostridium cellulovorans cellulosome is comprised of a large, nonenzymatic scaffolding protein called the cellulose binding protein A (CbpA) and a number of endoglucanases/xylanases. The CbpA contains several functional domains, including a signal peptide, a cellulose binding domain (CBD), a hydrophilic domain (HLD) present four times, and a hydrophobic domain (HBD) present nine times. The functions of the domains were studied by the construction of minigenes containing the putative functional domains and by expression of the minigenes in Escherichia coli. The purified product of the CBD was able to bind to various crystalline forms of cellulose and chitin with a Kd of 1 microM. The binding capacity for CBD was a function of the crystallinity of the cellulose sample. Furthermore, the binding of CBD to Avicel was not inhibited by cellobiose or carboxymethylcellulose, suggesting that the CBD binding target was a three-dimensional structure found only in crystalline forms of cellulose. The HBD was tested for its ability to bind endoglucanases by an interaction Western as well as a sandwich enzyme immunoassay technique. The HBD was able to bind both EngB and EngD, indicating that the HBD contained an endoglucanase binding domain (EBD). Because there are nine EBD domains, it is possible that CbpA can bind up to nine endoglucanases. The role of the HLDs remains elusive. The data indicate that the cellulosome is a complex enzyme containing a scaffolding protein (CbpA) to which is attached a number of endoglucanase molecules. This arrangement allows the complex to bind and degrade crystalline cellulose, which resists degradation by the free forms of cellulosomal endoglucanases.

Demonstration of Human Growth Hormone in Normal Urine by a Highly Specific and Sensitive Sandwich Enzyme Immunoassay

Abstract Human growth hormone (hGH) was demonstrated in normal urine by a highly specific and sensitive sandwich enzyme immunoassay. The molecular weight of hGH in normal urine was shown to be 22,000 by gel filtration, and levels of urine hGH were 15–64 ng/1 or 50–83 ng/g of creatinine in normal subjects aged 1.2–5.9 yr.

The 24‐hour Rhythms in Plasma Growth Hormone, Prolactin and Thyroid Stimulating Hormone: Effect of Sleep Deprivation

Twenty‐four hour secretory rhythms of growth hormone (GH), prolactin (PRL) and thyroid stimulating hormone (TSH) were investigated in 9 normal adult men by means of serial blood sampling at 30 min intervals. The profiles of pituitary hormones were compared in 6 subjects between in normal nocturnal sleep condition and in delayed sleep condition. Plasma GH was measured with use of highly sensitive enzyme immunoassay (EIA) recently developed. Plasma TSH was also evaluated by highly sensitive time‐resolved fluorometric immunoassay (TR‐FIA). Time series analysis of plasma GH and PRL was performed by auto‐ and cross‐ correlation and spectral analysis.

A micro-scale affinity-purification of Fab'-horseradish peroxidase conjugates and its use for sandwich enzyme immunoassay of insulin in human serum.

We describe methods for the conjugation of Fab’ to horseradish peroxidase through thiol groups in the hinge of Fab’ using reaction with maleimide groups or the thiol-disulfide exchange reaction [l]. The Fab’-horseradish peroxidase conjugates prepared by these methods were demonstrated to be superior in both sandwich enzyme immunoassay and immunohistochemical staining to conjugates prepared by methods in which the enzyme was conjugated through Fab’ amino groups [l], with detection limits in the attomole range [2]. Hinge conjugation is, however, not suitable for small amounts of affinity-purified Fab’, due to loss of thiol groups during gel filtration and concentration. We describe a micro-scale method for affinity purification after Fab’-peroxidase conjugation and its application to a sandwich enzyme immunoassay of insulin in human serum. The non-specific binding of the affinity-purified conjugate was lowered by gel filtration, and the sensitivity for insulin was 1 nU/tube and 100 nU/ml of serum.

A Highly Sensitive Sandwich Enzyme Immunoassay of Human Growth Hormone in Serum Using Affinity-Purified Anti-Human Growth Hormone Fab′-Horseradish Peroxidase Conjugate

Abstract A highly sensitive sandwich enzyme immunoassay (EIA) for human growth hormone (hGH) was developed. hGH to be assayed was incubated with an anti-hGH IgG-coated polystyrene ball, and then the polystyrene ball after washing was incubated with anti-hGH Fab1 -peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was correlated to the amount of hGH to be assayed. Polystyrene balls were coated by physical adsorption with rabbit anti-hGH IgG which was not affinity-purified. Rabbit anti-hGH Fab1 which had been affinity-purified was coajugated with horseradish peroxidase using N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate. The sensitivity was 0.06 pg of hGH per tube or 6 pg/ml of serum when 0.01 ml of serum samples was used. No significant interference was observed in the presence of 8 ng/tube of human prolactin, 8 μU/tube of human thyroid-stimulating hormone, 50 mU/tube of human luteinizing hormone, 10 mU/tube of human follicle-stimulating hormone, or 5 U/tube of human c...

Development of ultrasensitive enzyme immunoassay reviewed with emphasis on factors which limit the sensitivity.

Development of ultrasensitive enzyme immunoassays for antigens, haptens and antibodies is reviewed with emphasis on factors which limit the sensitivity. One of the most important conditions for ultrasensitive immunoassays is the use of non-competitive solid phase assay systems rather than competitive ones. Although non-competitive immunoassays are available for antigens and antibodies, there are only competitive immunoassays for hapten molecules which can not be bound simultaneously by two different antibodies. In order to overcome this difficulty, methods have been developed to derivatize haptens with amino groups so that the derivatized haptens may be measured by two-site assays. The other condition for ultrasensitive immunoassays is to minimize the non-specific binding of labelled reactants. This has been achieved by developing methods to transfer the complex of analytes and labelled reactants from solid phase to solid phase without dissociation. Thus, the sensitivity for antigens, haptens and antibodies has been markedly improved. However, the sensitivity for the detection of label enzymes remains to be improved.

Shortening of the window period in diagnosis of HIV-1 infection by simultaneous detection of p24 antigen and antibody IgG to p17 and reverse transcriptase in serum with ultrasensitive enzyme immunoassay

Following HIV infection, there is a window period of 6-8 weeks, during which HIV antibodies are not detectable and the infection cannot be diagnosed by methods for detecting HIV antibodies. However, HIV antigens are detectable in the latter part of the window period, although the level of HIV antigens declines as the level of HIV antibodies increases. We developed an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for the simultaneous detection of both p24 antigen of HIV-1 and antibody IgGs to p17 and reverse transcriptase of HIV-1 in a single assay tube and tested 11 HIV-1 seroconversion serum panels and serum samples randomly collected from 79 HIV-1 seropositive subjects and 100 HIV-1 seronegative subjects. The simultaneous detection was shown not only to shorten the window period significantly as compared with conventional methods for HIV-1 antibody detection but also to make possible a reliable diagnosis of HIV-1 infection from the time of seroconversion until late stages of the infection.

Diagnosis of HIV-1 infection with whole saliva by detection of antibody IgG to HIV-1 with ultrasensitive enzyme immunoassay using recombinant reverse transcriptase as antigen.

Whole-saliva samples were collected from 45 asymptomatic carriers, 18 patients with AIDS-related complex (ARC) or AIDS, and 76 medical students by simple spitting with no stimulation and tested by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-HIV-1 IgG using recombinant reverse transcriptase as antigen and beta-D-galactosidase as label. With as little as 1 microliter of whole saliva, the lowest signals among the 45 asymptomatic carriers, 8 patients with ARC, and 10 patients with AIDS were 38-, 78-, and 3-fold, respectively, higher than the highest signal among the medical students. When the volume of whole saliva for test was increased up to 100 microliters, no significant effect was observed on signals for seropositive cases and signals for the medical students increased only very slightly. Therefore, whole-saliva samples containing extremely low levels of anti-HIV-1 IgG, even 2,000-fold lower than the lowest level among the 45 asymptomatic carriers tested, were considered to be discriminated from those of seronegative individuals. Thus, the sensitivity and specificity were expected to be both 100% with whole saliva even for a larger number of samples, although the number of samples tested was limited.

Soluble insulin receptor ectodomain is elevated in the plasma of patients with diabetes

OBJECTIVE—Insulin binds to the α-subunit of the insulin receptor (IRα) and subsequently exerts its effects in the cells. The soluble ectodomains of several receptors have been found to circulate in the plasma. Therefore, we hypothesized that soluble human insulin receptor (hIR) ectodomain (α-subunit and a part of β-subunit) may exist in the plasma of diabetic patients. RESEARCH DESIGN AND METHODS—We identified soluble hIR ectodomain in human plasma by a two-step purification followed by immunoblotting and gel-filtration chromatography. Furthermore, we established an hIRα-specific enzyme-linked immunosorbent assay and measured the plasma IRα levels in patients with diabetes. We also investigated this phenomenon in streptozotocin-induced diabetic hIR transgenic mice. RESULTS—Soluble hIRα, but not intact hIRβ or whole hIR, exists in human plasma. The plasma IRα levels were significantly higher in type 1 (2.00 ± 0.60 ng/ml; n = 53) and type 2 (2.26 ± 0.80; n = 473) diabetic patients than in control subjects (1.59 ± 0.40 ng/ml; n = 123 (P < 0.001 vs. control). Plasma IRα level was positively correlated with blood glucose level, and 10–20% of the insulin in plasma bound to hIRα. In the in vivo experiments using diabetic hIR transgenic mice, hyperglycemia was confirmed to increase the plasma hIRα level and the half-life estimated to be ∼6 h. CONCLUSIONS—We propose that the increased soluble IR ectodomain level appears to be a more rapid glycemic marker than A1C or glycoalbumin.