A method for measuring specific IgE in sera by direct ELISA without interference by IgG competition or IgG autoantibodies to IgE.

BACKGROUND: In measuring specific IgE levels in sera by direct ELISA, competition with coexisting IgG often impedes an exact IgE determination; additionally, IgG autoantibodies to IgE (IgG-IgE) in sera affect the assay. In this paper, we attempt to determine accurate specific IgE levels by selective removal of IgG with a protein G-immobilized gel (PG) and by acid treatment of the PG to compensate for the unintended removal of IgE, probably due to the PG binding IgG-IgE. METHODS: IgG in sera was removed using PG at pH 7.0. Then, the PG was treated with citrate buffer at pH 3.0 for 5 min to liberate IgE from IgG-IgE complexes, after IgG-binding sites on the PG were saturated with bovine IgG, since PG came to bind IgE at acidic pHs. IgE levels were then measured by ELISA. RESULTS: The PG treatment of sera removed the effect of inhibitory competition by coexisting IgG, especially at higher concentrations of sera, to improve specific IgE detection by direct ELISA. However, PG treatment alone sometimes reduced IgE levels (39% of sera tested), even though PG does not bind IgE at pH 7.0, which indicated the presence of IgG-IgE complexes. The reduction in IgE returned almost to their original levels in the sera by acid treatment of the PG. By combining the PG treatment with acid treatment, specific IgE measurement in sera was improved significantly (p < 0.01, Wilcoxon signed rank test). CONCLUSION: Measurement of specific IgE in sera by direct ELISA was improved by using the PG and acid treatment technique.

Synthetic polymer sulphonated polyisoprene as a universal anticoagulant for laboratory testing.

To evaluate the feasibility of a synthetic polymer, sulphonated polyisoprene (SPIP), to be used as a new laboratory anticoagulant, hematological items were compared between blood samples anticoagulated with EDTA and SPIP, as were biochemical and electrolyte items between serum samples and SPIP-anticoagulated blood. Among hematological items, the average platelet count of the SPIP group was significantly lower than that of the EDTA group, due to platelet aggregation in the SPIP group. Addition of kanamycin to SPIP, but not of aspirin or prostaglandin E1, restored platelet count to about 80% of that in the EDTA group, and increased coefficients of correlation with the EDTA group for most hematological items examined. For biochemical and electrolyte items, high (> 0.95) correlation coefficients were obtained for 15 of 18 items between the serum and SPIP-anticoagulated samples. However, for some of these items, the addition of kanamycin decreased coefficients of correlation. Thus, SPIPs are excellent candidates for new anticoagulants that can be used for evaluation of hematological, biochemical, and electrolyte items with a single test tube in routine laboratory work. However, some improvements are required for use of kanamycin as an additive to inhibit platelet aggregation induced by SPIP, and in maintaining reliability of biochemical and electrolyte measurements.

One-step enzyme-linked immunosorbent assay (ELISA) for measurement of serum free leptin.

In man, circulating leptin levels are increased with obesity and are regulated by a complex of hormonal, feeding and body-weight changes. Accurate and precise methods to quantitate circulating serum free leptin (f-leptin) concentrations are needed for physiological and clinical studies. We developed a one-step enzyme immunoassay to measure human f-leptin in serum. The detection limit was 0.40 ng/ml. The recovery of leptin added to serum was 90.8-102.8%. The within-run and between-day coefficients of variation (C.V.) ranged from 2.8 to 7.7 and 5.7 to 9.7%, respectively, and the immunoassay had an overall recovery rate for serial dilution in the range of 94. 0-109.9%. Measured serum f-leptin concentrations in 201 adults correlated (r=0.449, P<0.001) directly with body mass index (BMI kg/m(2)), particularly when results were separated by gender (r=0. 709 for male, P<0.001; r=0.643 for female, P<0.001). We conclude that this one-step enzyme immunoassay is accurate for measuring f-leptin in human serum.

ELISA for a complexed antigen with a monoclonal antibody blocking reaction with the free antigen-assay-specific for complexed prostate-specific antigen.

We have developed an enzyme-linked immunoassay (ELISA) for serum complexed-antigen (prostate-specific antigen; PSA)(c-PSA) with simultaneous blocking of free-antigen (PSA)(f-PSA). The assay utilizes three different monoclonal antibodies (MAbs) recognising three distinct PSA epitopes. The detection limit was established as 0.19 microg/l (n=20, mean of zero standard+2S.D.) and the average recovery of f-PSA was 98-100%. The within-run and between-day coefficients of variation (CV) ranged from 2.1% to 3.2% and 2.8% to 6.3%, respectively. There was a good correlation between serum c-PSA measured by the present ELISA and PSA-alpha(1)-antichymotrypsin complex (PSA-ACT) concentrations (r=0.991). This method should provide a better tool for discriminating between benign and malignant prostatic disease.

[Non-hemophilic patient with inhibitor against factor VIII produced after the second delivery].

A 33-year-old woman who had been healthy and had no history of abnormal bleeding developed widespread ecchymoses and intramuscular bleeding 4 months after her second delivery. On admission, laboratory examination data revealed that factor VIII activity was markedly reduced (4%) and APTT was prolonged (119.7s). Factor VIII inhibitor titer was high, at 19 Bethesda units. Her chemical and serological data were normal. No antinuclear antibodies were detected. We concluded that factor VIII inhibitor had been spontaneously produced after the second delivery and was responsible for her bleeding tendency. Prednisolone (60 mg/day) and factor VIII concentrates were administered to stop the bleeding, but factor VIII activity did not increase while factor VIII inhibitor titer increased to 29 Bethesda units. Therefore, treatment with factor VIII concentrate (was stopped while prednisolone was continued resulting in reduction of factor VIII inhibitor titer and improvement of her bleeding tendency. At 8 months after admission, factor VIII inhibitor titer was not detected by the Bethesda method and factor VIII activity and APTT were normal.

[A consideration for the situation of the commercial laboratories and their connection with the medical administration in Japan].

The commercial laboratories in Japan have been serving all physicians to be able to use all laboratory tests even if they are newly developed. Though the commercial laboratories have functioned very well for clinically, for economically they have made a great margin between the official price which is decided by medical insurance and the business price which is dealt with hospitals. The margin and the margin from drugs have caused excess clinical testing and drug prescription, subsequent increase of total medical cost in Japan. We are going to revise our medical insurance system to improve various inconvenience in the present and future status. For the Japan Registered Clinical Laboratories Association which is the representative of the commercial laboratories, it's a time to cooperate with the other clinical laboratory tests-related organizations (ex. Japan Association of Medical Technologists, Japan Association of Clinical Laboratory Physicians, etc.) and to built new acceptable circumstances of the clinical testing corresponding to medical administration.

A highly sensitive enzyme-linked immunoassay for serum free prostate specific antigen (f-PSA).

Monoclonal antibodies (MAbs) were generated against human prostate specific antigen (PSA) to allow development of a sensitive free-PSA (f-PSA) assay. Of a total of 211, 12 could detect only f-PSA, the other 199 MAbs binding to both f-PSA and complex-PSA. In the present study, one MAb (no. 5C6) specific for f-PSA and another (no. 79) reacting with both forms were used to develop an enzyme-linked immunoassay (ELISA) for serum f-PSA. The detection limit was established as 0.008 microg/l (n = 20, mean of zero standard + 3 S.D.) and the average recovery of f-PSA was 93-102%. The within-run and between-day coefficient of variation (CV) varied from 5.4-7.4% and 4.8-6.5%, respectively. The cross-reactivity of the assay to PSA-alpha1-antichymotrypsin complex was determined to be < 0.4%.

[Enzyme-linked immunosorbent assay for serum prostate specific antigen (PSA) using monoclonal antibodies].

Prostate specific antigen (PSA) is a widely used marker for screening and monitoring prostate cancer. However, different commercial immunoassays, often give different PSA values for the same patient sample. We produced 59 monoclonal antibodies against human PSA. Two monoclonal antibodies (No. 54 and No. 47) against PSA were used to develop one-step ELISA for serum PSA. In the combination of these two antibodies, incubation for more than 30 minutes gave an equimolar response to both free-PSA and PSA complexed to alpha 1-antichymotrypsin. The procedure is simple, and the method is specific and reproducible. The average recovery for serum sample was 90-107%.

[Consultation services at the commercial laboratories].

Only few customers consult with their using commercial laboratories about how to interpret the laboratory examination results, or how to proceed the laboratory diagnosis. But there are many questions about the test item, methodology, time course, and how to collect a specimen, and also there are many complaints about the test results, methods, reference values and reporting styles. Therefore, most of the commercial laboratories set up the information desk instead of the consultation desk. In our laboratory, Japan clinical laboratories, Inc., information services are done by not only the information telephone desk, but also the persons who go to the customers office(clinics or hospitals) for sales of the tests or collection and delivery of the samples. In this report, our information services were analyzed with the questionnaire and hearing to the sales and laboratory division, and with the records of the transaction of complaints(past 3 years) and the information service daily reports(past 2 months). From the results of the above analyses, it was observed that the commercial laboratory customers, e.g. clinicians, nurses, hospital clerks and medical technologists, have many various questions and complaints to the tests and the laboratories. And it was revealed that the education of the both staff of sales and laboratory division in the commercial laboratory is one of the most important subjects. For more effective use of the laboratory tests, clinical pathologists and medical technologists have to guide and supervise the commercial laboratories more adequately.

Association between Helicobacter pylori infection and serum pepsinogen concentrations in gastroduodenal disease.

AIM: To investigate the association between Helicobacter pylori infection and serum pepsinogen (PG) 1 and 2 concentrations in various gastroduodenal diseases. METHODS: Serum PG1 and 2 concentrations and antibodies to H pylori were measured by enzyme linked immunosorbent assay (ELISA); gastric mucosal pH was assessed and urease activity in biopsy tissue was determined. A comparison of the ELISA and urease test results permitted division of the cases into positive, false positive, false negative and negative categories for control, gastritis, and ulcer groups. RESULTS: The gastric mucosal pH and serum PG2 in cases positive for H pylori were significantly increased in ulcer and gastritis cases compared with H pylori negative cases. Similar tendencies were observed for the false positive and false negative categories. CONCLUSIONS: A positive ELISA reaction for antibodies and an increased serum PG2 concentration are reliable indicators of H pylori infection.

[The clinical evaluation of hepatic arterial infusion chemotherapy in patients with liver metastases from colorectal cancer employing an implanted port system].

Between 1986 and 1994, 66 patients with unresectable liver metastases from colorectal cancer were treated by repeated hepatic arterial infusion chemotherapy employing an implantable port system. In this study, 44 patients who received intermittent hepatic arterial infusion of high dose 5-FU (1000mg/m2) were analysed according to a response rate, survival rates, developments of extrahepatic lesions, periods of hepatic arterial infusion, and the rates at home. Two cases (4.5%) achieved a complete response (CR) and 27 cases (61.4%) a partial response (PR). The overall one-year and two-year survival rates were 56.7% and 37.8% respectively. The 50% survival time for all patients was 17.2 months. During the course of observation, extrahepatic lesions developed in 16 of the 39 patients (41.0%) and many responders died due to deterioration of extrahepatic lesions. The over all rates at home were more than 85%. Hepatic arterial infusion of the 5-FU at 1000mg/m2 every week showed a high response rate in colorectal cancer patients with hepatic metastases, and the responders showed a low rate of death due to the hepatic metastases. However, in many patients the prognosis-determining-factor was extrahepatic lesions. Thus, countermeasures were necessary for extrahepatic lesions.

[The pharmacokinetics of the glycyrrhizin and glycyrrhetic acid after intravenous administration of glycyrrhizin for the patients with chronic liver disease caused by type C hepatitis virus].

Glycyrrhizin had been used widely for the patients with chronic liver disease. We examined the pharmacokinetics of the glycyrrhizin and glycyrrhetic acid in the blood stream after intra-venous administration of glycyrrhizin. The stream concentration of glycyrrhizin in the patients of liver cirrhosis tend to be kept higher than that of chronic hepatitis but there were no significant difference between them except for after a half hour from the administration. There was negative correlation between ICG R15 and the speed of excretion of glycyrrhizin from the serum. On the other hand, the concentration of the glycyrrhetic acid was kept higher in the patients with liver cirrhosis than that of chronic hepatitis, but there were no significant difference between them except for after a half hour from the administration. These findings suggested that the accumulation of glycyrrhizin and glycyrrhetic acid in the patients of liver cirrhosis can be seen by long term administration.

[Newly developed detection system for serum Helicobacter pylori antibodies using ELISA and its clinical significance].

Using ELISA, a specific and sensitive system that detects serum immunoglobulin G antibodies against all soluble antigens of Helicobacter pylori (Hp) has been developed. This system is used at three different concentrations of Hp antigens coated on the solid phase of the ELISA. Anti-Hp antibodies were judged positive when the difference in the ELISA values between high and middle or middle and low concentrations of antigen were over the specific values. For the ELISA system, called the three point antigen method, sensitivity and specificity of urease activity were 86.9% and 70.8%, respectively. Positive rate for antibodies to serum Helicobacter pylori in dyspeptic patients was higher than that in healthy subjects.

[Blood coagulation factors in hepatic disorders].

The major factors related to the coagulo-fibrinolytic reactions are synthesized in the hepatic cells. As a reticuloendothelial organ, the liver also removes the activated factors and metabolic products in the coagulo-fibrinolytic process from the plasma. Therefore, many investigators have been trying to use measurements of the coagulo-fibrinolytic factors for the evaluation of the type and degree of a hepatic disorder and for the determination of therapeutic effects or prognosis. On the other hand, the recent advances in the blood coagulation study show that the hemostatic mechanism is a kind of maintenance mechanism of homeostasis for blood-flow. The functions of many factors in and from the platelets and vascular endothelial cells as well as those produced by the liver have been identified. Recently, some hemostatic molecular markers and complexes of activated factors with their inhibitors can also be measured. In this article, the recent concept of hemostatic mechanism and the relation between various hemostatic factors and hepatic disorders are presented.

[Clinical significance of the measurement of serum pepsinogen group I and II by enzyme-linked immunosorbent assay].

We have developed enzyme-linked immunosorbent assay (ELISA) for pepsinogen group I and II (PG I.II) in human serum, and clinical significance of serum pepsinogen measurement was evaluated. Serum PG I.II levels in patients with gastric and duodenal ulcer were higher than those in normal healthy subjects. On the other hand, serum PG I levels in patients with pernicious anemia were significantly low levels. In both gastric ulcer and duodenal ulcer, serum PG I.II levels at active stage were higher than healing stage. These results suggested that the measurement of PG I.II levels was useful for screening or monitoring test for the injury of gastric and duodenal mucosa.