Kouichi Hirota

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.

WHOLE SALIVA DRIED ON FILTER PAPER FOR DIAGNOSIS OF HIV-1 INFECTION BY DETECTION OF ANTIBODY IGG TO HIV-1 WITH ULTRASENSITIVE ENZYME IMMUNOASSAY USING RECOMBINANT REVERSE TRANSCRIPTASE AS ANTIGEN

Whole saliva samples collected from HIV‐1 seropositive subjects by simple spitting without using any devices were dried on filter paper strips, from which filter paper discs of 3‐mm diameter were punched out. The eluates of the discs were subjected to the immune complex transfer enzyme immunoassay for antibody IgG to HIV‐1 using recombinant reverse transcriptase of HIV‐1 as antigen and a two‐site enzyme immunoassay for whole IgG. The signals for antibody IgG to HIV‐1 and the amounts of whole IgG obtained with one disc per assay tube were 126–290% of those obtained with 1 μl of whole saliva samples, provided that filter paper strips were treated with nonspecific rabbit serum prior to drying whole saliva samples and that filter paper discs were tested within a few days after drying whole saliva samples. From these results, diagnosis of HIV‐1 infection was indicated to be possible with whole saliva samples dried on filter papers, since the diagnosis was previously shown to be possible with 1 μl of whole saliva samples. The test for HIV‐1 infection with whole saliva samples dried on filter papers was suggested to be useful for various purposes.

Salivary indicators of protein nutritional status in the elderly

Abstract Protein-energy malnutrition (PEM) is a serious nutritional problem in hospitalized elderly patients. Serum albumin, transthyretin (prealbumin), and total protein have been used as biochemical indicators of protein nutritional status, but taking blood from the elderly is difficult and invasive. We therefore assessed the possibility of using saliva or urine as noninvasive materials to estimate serum concentrations of albumin, transthyretin and total protein. Blood, saliva and urine were collected from 32 hospitalized elderly (aged 68–97 y) and the correlation between the concentrations of albumin, transthyretin and total protein in serum and those in saliva or urine were evaluated. There was no correlation among them, but the concentrations of albumin and transthyretin adjusted for the concentration of IgG in saliva were significantly correlated with the concentrations of albumin and transthyretin in serum (albumin: R 2 = 0.308, p=0.0010, transthyretin: R 2 = 0.433, p

Commutability of National Institute of Standards and Technology standard reference material 1955 homocysteine and folate in frozen human serum for total folate with automated assays

Background The aim of the present study was to evaluate standard reference material (SRM) 1955 commutability as a reference material for serum folate using automated methods. We also designed so as to reduce the intermethod variability present in different automated methods. Methods Using a microbiological assay related to the ‘information value’ of SRM 1955 as a comparison method, we investigated the possibility of standardization for the assay values of serum folate as measured by the automated methods (Access, Centaur and Elecsys). In the assay of 50 patient sera by these automated methods, we corrected observed values by the SRM 1955 and compared with comparison values. Results The observed values of SRM 1955 Levels I, II and III were within or outside (but near) a 95% prediction interval obtained from patient sera by the automated methods. The normalized residuals obtained from SRM 1955 were within ±3.0 (in SD units), which enabled us to conclude that the SRM 1955 had a physicochemical characterization similar to native serum. Twelve patients were assessed as hypofolataemia (<6.0 ng/mL) and 38 patients as normal (≥6.0 ng/mL). Before correction, folate levels in six of 12 patients were lower than 6.0 ng/mL, and those in seven of 38 patients were higher than 6.0 ng/mL with the automated methods. After correction, low levels were found in four of 12 patients, and normal levels were found in 33 of 38 patients. Conclusions The use of SRM 1955 would help to reduce the intermethod variability present in different automated methods for serum folate measurement.

Detection of Anti-Thyroglobulin Immunoglobulin G in Urine by Sensitive Enzyme Immunoassay (Immune Complex Transfer Enzyme Immunoassay) as a Diagnostic Aid for Autoimmune Thyroid Diseases

Abstract Urine samples of patients with Graves′ and Hashimoto′s diseases were dialysed and subjected to a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-thyroglobulin IgG. Anti-thyroglobulin IgG in urine was reacted simultaneously with 2,4-dinitrophenylated thyroglobulin and thyroglobulin-β-D-galactosidase conjugate. The immune complex formed consisting of the three components was trapped onto polystyrene balls coated with (anti-2,4-dinitrophenyl group) IgG, eluted with eN-2,4-dinitrophenyl-L-lysine and transferred onto polystyrene balls coated with (anti-human IgG γ-chain) IgG. β-D-Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. Anti-thyroglobulin IgG was detected in most of the patients. The detection of anti-thyroglobulin IgG in urine by the immune complex transfer enzyme immunoassay was suggested to be useful as a diagnostic aid for autoimmune thyroid diseases.

Surveillance evaluation of the standardization of assay values for serum total 25-hydroxyvitamin D concentration in Japan:

Background To assess the vitamin D nutritional status, serum total 25-hydroxyvitamin D (25(OH)D) concentration is measured. We used six automated 25(OH)D immunoassays (AIAs) available in Japan and certified by the Vitamin D Standardization Program (VDSP) at the U.S. Center for Disease Control and Prevention to assess the concordance of the assay results. Methods Serum total 25(OH)D concentrations in SRM 972a and 20 serum samples from patients were determined using three liquid chromatography-tandem mass spectrometry (LC-MS/MS) and six AIAs (pilot study), and an additional 110 serum samples were assessed by the six AIAs (surveillance study). The assay bias from the results of LC-MS/MS by Chiba University or consensus values (i.e. average of six AIAs) was estimated using the procedure described in CLSI document EP09-A3. Results LC-MS/MS at Chiba University could completely separate 25(OH)D2, 25(OH)D3 and 3-epi-25(OH)D3, and the observed values including total 25(OH)D in SRM 972a were all within ±1·SD of the assigned values. All AIAs produced results greater than ±3·SD. In the pilot study, four of the six AIAs had an average percentage bias, as estimated by confidence interval (CI), larger than ±5% (acceptance criterion in CLSI); the bias converged from −6.5% to 3.2% after adjustment by LC-MS/MS. In the surveillance study, 25(OH)D concentrations in AIAs all adjusted to LC-MS/MS converged within ±5% from consensus values. However, some AIAs showed negative or positive bias from the consensus values. Conclusions Current AIAs in Japan continue to lack standardization. Manufacturers should implement quality assurance strategies so that their values more closely align to those of standard reference material 972a.

Immune complex transfer enzyme immunoassay for anti-ovalbumin IgA in serum

Background: An immune complex transfer enzyme immunoassay for antiovalbumin immunoglobulin A (IgA) was developed. Methods: Serum-specific antibody was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-ovalbumin conjugate and ovalbumin-β-D-galactosidase conjugate. The complex formed from the three components was trapped onto polystyrene balls coated with anti-2,4-dinitrophenyl group immunoglobulin G, eluted with εN-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with anti-human IgA α-chain. Bound β-D-galactosidase activity was determined by fluorometry. Results & Conclusions: The detection limit of this method for the measurement of specific anti-ovalbumin IgA was 9 fmol/tube, which was 20-fold lower than that of the enzyme-linked immunosorbent assay (ELISA). Because serum interference with this method was lower than that with the ELISA, the detection limit of this method was 300-fold lower than that by the ELISA. Anti-ovalbumin IgA was detected in 100% of healthy subjects, which was confirmed by pre-incubation with an excess amount of ovalbumin.

Sensitive Enzyme Immunoassay for Anti-β-Lactoglobulin IgG in Serum

A sensitive enzyme immunoassay for anti-β-lactoglobulin immunoglobulin G (IgG) in serum is described. Serum containing anti-β-lactoglobulin IgG was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-β-lactoglobulin conjugate and β-lactoglobulin-peroxidase conjugate. The complex formed from the three components was trapped onto polystyrene balls coated with anti-2,4-dinitrophenyl group IgG, eluted with ε-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with anti-human IgG-γ-chain IgG. Bound peroxidase activity was determined by fluorometry. This enzyme immunoassay was 100- to 1000-fold more sensitive and more reliable than the enzyme-linked immunosorbent assay (ELISA). Anti-β-lactoglobulin IgG was detected in 91% of healthy subjects using this method.

Immune Complex Transfer Enzyme Immunoassay for Anti-Thyroglobulin IgG Using 2,4-Dinitrophenyl-thyroglobulin, Biotinyl-thyroglobulin and Streptavidin-β-D-galactosidase Conjugate

Abstract A sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-thyroglobulin IgG in serum is described. Anti-thyroglobulin IgG in serum was reacted simultaneously with 2,4-dinitrophenyl-thyroglobulin and biotinyl-thyroglobulin. The immune complex formed of the three components was trapped onto polystyrene balls coated with (anti-2,4-dinitrophenyl group) IgG and, after washing, reacted with streptavidin-β-D-galactosidase conjugate. After washing, the immune complex was eluted from the polystyrene balls with eN-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with (anti-human IgG γ-chain) IgG. β-D-Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. Biotinylthyroglobulin and streptavidin-β-D-galactosidase conjugate could be prepared more easily than thyroglobulin-β-D-galactosidase conjugate used in the previous immunoassay. Inactive β-D-galactosidase, used to eliminate interference by anti-β-D-galactosidase antibodies in ...