[Significance of the confirmatory test of enzyme immunoassay and western blotting method in detection of anti-HTLV-1 in sera].

In this study, we directly compared the sensitivity of the detection of anti-human T-cell leukemia virus type-1 (anti-HTLV-1) antibody in serum by enzyme immunoassay (EIA), gelatin particle agglutination method (PA), and Western blotting method (WB). Of 69 cases studied, 6 cases positive by WB were negative by both PA and EIA methods. Of these 6 cases, 5 cases were interpreted to be positive by the confirmatory test of EIA (inhibitory rates of greater than 50%). Thus, the results of the confirmatory test were most consistent with those of WB methods. We also determined the levels of anti-HTLV-1 antibody in sera from the babies born of anti-HTLV-1 positive mothers at various months of age by the above three methods. As ages progressed, a parallel decline in the levels of anti-HTLV-1 antibody was shown by the three methods. However, at the time when sera became negative by both PA and EIA methods, the antibody was still detected by WB method. The study concludes that both WB and confirmatory test of EIA are more sensitive indicators of HTLV-1 infection.

[Development of highly sensitive assay for detection of low serum level of PIVKA-II and its clinical usefulness].

PIVKA-II is well known as a tumor marker of hepatocellular carcinoma. We describe the adaptations to render a commercially available PIVKA-II immunoassay kit (Eitest MONO P-II: Eisai Co., Ltd, Tokyo) more sensitive and evaluated clinical usefulness of this highly sensitive assay for early diagnosis of hepatocellular carcinoma. We measured serum PIVKA-II concentrations with the Eitest kit as described for the kit. To measure serum PIVKA-II concentrations below the sensitivity limit of the Eitest kit (0.0625AU/ml), linearity was determined using a series of dilutions of standard antigen diluted in standard diluent, including PIVKA-II concentrations of 0.002, 0.004, 0.008, 0.016, 0.031, 0.0625AU/ml. The assay was found to be liner from 0.004AU/ml. In this way, one can detect as low PIVKA-II concentrations as 0.004AU/ml. The repeatability-assay coefficients of variation (CV%) obtained from sixteen repeated assays of four sera were distributed between 3.63 and 18.75% and the reproducibility-assay coefficients of variation (CV%) obtained from ten repeated assays of four sera were distributed between 6.27 and 14.04%. We determined the serial changes in serum PIVKA-II levels of two patients with hepatocellular carcinoma after the resection of hepatic tumor. In these patients, elevation of serum PIVKA-II level determined by the highly sensitive assay preceded the detection of relapse by imaging diagnostic procedures. In summary, the assay system we developed has good accuracy and reproducibility for assaying low concentrations of PIVKA-II in serum and is suitable for detecting small increases in PIVKA-II concentrations. This assay system may be useful to earlier detect a relapse of hepatocellular carcinoma.

[Clinical usefulness of serum PIVKA-II levels determined by ECLIA system as a tumor maker for hepatocellular carcinoma].

PIVKA-II is well known as a tumor maker of hepatocellular carcinoma (HCC). We measured serum PIVKA-II concentrations with a commercially available PIVKA-II immunoassay kit (Picolumi PIVKA-II: Eisai Co., Ltd., Tokyo) using Electrochemiluminescence Immunoassay (ECLIA). ECLIA system is a novel immunoassay system using a Ruthenium (II) Tris (bipyridyl) luminesced by electric energy. Cut off value was 40 mAU/ml by receiver-operating characteristic curves as a tumor maker for HCC. Eighty-nine out of 142 (62.2%) patients with HCC had elevated serum PIVKA-II levels and seventeen out of 36 (47.2%) patients whose tumor size was below 2 cm in diameter showed high PIVKA-II levels. We determined the serial changes in serum PIVKA-II levels of two patients with HCC following initial therapy. In these patients, elevations of serum PIVKA-II levels determined by ECLIA system preceded the HCC relapse detected by imaging diagnostic procedures. In summary, this assay system is suitable for detecting small increases in PIVKA-II concentrations. Determination of PIVKA-II by this assay system is found to be useful for the early detection of HCC.

[Clinical usefulness of lectin-reactive fraction of alpha-fetoprotein in hepatocellular carcinoma].

Alpha-fetoprotein (AFP) is well known as a tumor marker of hepatocellular carcinoma (HCC). AFP shows micro heterogeneity because of a structural variance in its sugar chain, and the sugar chain obtained from HCC patients has a high affinity to lectins such as Lens culinaris agglutinin. The lens culinaris agglutinin A-reactive fraction of alpha-fetoprotein (AFP-L3) was detected by a sensitive method using lectin-affinity electrophoresis coupled with antibody-affinity blotting. Of 62 HCC patients examined before initial therapy, 23 patients (37.1%) had an AFP-L3 level greater than 15%. In contrast, none of the patients with liver cirrhosis or chronic hepatitis tested were positive for AFP-L3. Seven (26.9%) of 26 patients with small HCC less than 2 cm in diameter measured on ultrasonography or computed tomography showed elevated AFP-L3 levels. Because there was no correlation observed between AFP-L3 and PIVKA-II, the combined measurement of these two complementary markers appeared to be useful in the diagnosis of HCC. When the results of both assays were combined, 34 (54.8%) of the 62 patients with untreated HCC showed elevated levels of one or both markers. The use of both markers together with imaging modalities during regular follow-up of chronic liver disease patients who are at high risk for HCC might be of great benefit.

[Simultaneous presence of macroamylase and macrolipase in a patient].

Here we report a case demonstrating simultaneous macroamylasemia and macrolipasemia. The patient showed persistently elevated serum amylase and lipase activities. On thin-layer gelfiltration, normal serum amylase migrated as a round spot behind albumin. However, serum amylase from this patient migrated slightly ahead of albumin. This finding indicated that serum amylase from this patient had a molecular weight greater than that of normal amylase. Lipase of normal serum was eluted from a Sephacryl S-300 column after the 4S protein. Lipase in the serum from this patient was eluted from the column slightly behind the 19S protein. This finding indicated that serum lipase from this patient also had a molecular weight greater than that of normal lipase. The conversion of macroamylase and macrolipase was observed when the normal serum was mixed with the patient's serum. To determine the isotypes of immunoglobulin (Ig) that was bound to amylase and lipase in the patient's serum, we investigated the effect of adding various anti-human Ig sera to the patient's serum. Amylase activity was precipitated only with anti-IgA lambda and lipase activity was precipitated both with anti-IgA lambda and anti-IgA kappa (anti-IgA kappa > anti-IgA lambda). Taken together, these data indicate that the material that enhanced amylase and lipase activities in the patient's serum was monoclonal IgA; IgA lambda for amylase and both IgA lambda and IgA kappa for lipase.

[Demonstration of PIVKA-II and AFP production by gastric cancer by indirect immunoenzymatic staining of paraffin sections].

The patient had elevated plasma PIVKA-II and serum AFP levels. However, no tumor was detected with an ultrasonography, angiography, abdominal computed tomography (CT), and magnetic resonance imaging (MR). Gastric endoscopic examination disclosed gastric cancer and then subtotal gastrectomy was done. Shortly after the surgery, both plasma PIVKA-II and serum AFP levels returned to each normal level. The extirpated tumor revealed histologically immature and mature cancer cells. It was pathologically diagnosed as IIc early cancer. The localization of PIVKA-II and AFP in gastric cancer cells was demonstrated by the immunostaining method using monoclonal antibody. Taken together, these results indicate that cancer cells may produce PIVKA-II or AFP in this patient.

[Early diagnosis of acute myocardial infarction by an immunoinhibition method for analysis of creatine kinase isoforms].

Conventional isoenzyme and enzyme values in serum usually are normal during the first few hours of acute myocardial infarction (AMI). Thus definitive diagnosis may be delayed. Measurement of serum creatine kinase (CK) isoform has begun to attract attention. In this study, we measured CK isoform with an immunoinhibition method in the first available samples from patients with AMI and from healthy subjects. In the 394 healthy subjects, the mean ratio of MM3 to MM1 of CK isoform was 0.494 +/- 0.1495 (SD). The upper limit of the reference values for this ratio was considered to be 0.793 (mean + 2 SD). In 40 of 48 patients, this ratio in the first available samples from patients with AMI was greater than 0.793. In 15 of 20 patients whose total CK activity was less than 260 IU/l, this ratio was greater than 0.793, while CK-MB activity measured with the immunoinhibition method was well within the reference range in all of these patients. Our results show that in the first available samples from patients with AMI, measurement of the ratio of MM3 to MM1 of CK isoform has the highest diagnostic efficiency. Thus, measurement of CK isoform with the immunoinhibition method can be applied for early diagnosis of AMI.

[Studies on a serum factor inhibiting lactate dehydrogenase (LDH) activity in a patient's serum].

We found extremely low activity of serum lactate dehydrogenase (LDH, EC. 1.1.1.27) in a 70-year-old female patient. The decrease of LDH activity was observed when the normal serum was incubated with the patient's serum. Inhibition rate of LDH activity by the patient's serum was higher at 4 degrees C than at 37 degrees C. The patient's serum inhibited both subunits of LDH, and inhibited more strongly the M-subunit than the H-subunit LDH isoenzymes. IgG (lambda type) in the patient's serum was found to be responsible for the inhibition of LDH activity. The mechanism why IgG inhibiting LDH activity developed in the patient's serum remain undetermined.