Differential assay of salivary and pancreatic alpha-amylase in serum and urine, with use of monoclonal antibody to human salivary amylase immobilized on bacterial cell wall

We previously reported (Clin Chim Acta 1986;159:89) that bacterial cell wall chemically coated with a monoclonal antibody specific to human salivary (S) amylase (EC 3.2.1.1) could be successfully used to separate S and pancreatic (P) amylase in solution. We have now applied this method to serum and urine samples and found that the activities of S and P amylases so measured correlated well with those measured by the isoamylase inhibitor method. The present method is simple and reliable for routine clinical tests.     

胰型淀粉酶、脂肪酶在急性胰腺炎中的临床应用

目的为了评价急性胰腺炎中胰型淀粉酶（P—AMY）的临床应用价值,对总淀粉酶（α-AMY）、P—AMY、脂肪酶（lipase,LPS）进行方法学评价。方法选择急性胰腺炎患者126例、其他疾病患者对照组105例、健康对照组95例,用日立7600全自动生化分析仪测定α-AMY、P—AMY、LPS。本实验室临界值定为α-AMY216 U／L、P—AMY 115U／L、LPS 200U／L。结果α-AMY、P-AMY、LPS灵敏度分别为73．1％、88．5％、92．3％,特异性分别为70．5％、81．9％、82．9％,ROC工作曲线下α-AMY、P-AMY、LPS灵敏度分别为65．4％、88．5％、88．5％,特异性分别为86．7％、80．9％、89．5％,α-AMY和P—AMY之间差异有统计学意义（P=0．045）,P-AMY和LPS之间差异无统计学意义（P=0．613）。结论P-AMY检测比α-AMY更敏感、更特异性地反映急性胰腺炎。LPS也同样在急性胰腺炎中具有高灵敏度和特异性,临床上应同时检测P-AMY和LPS作为诊断急性胰腺炎的指标,具有更重要意义。     

Characterization of amylase linked immunoglobulin G to distinguish human salivary and pancreatic isoamylases

An immunoglobulin G of the kappa type linked to salivary amylase was identified in the serum of a patient with colon cancer and persistent hyperamylasemia. The binding site on the immunoglobulin in the complex is located in F(ab')2 portion. The purified IgG recombined only with purified human salivary amylase, and could be used to separate human isoamylases.     

Pancreatic amylase measured in serum by use of a monoclonal antibody immunochemically immobilized to a solid phase

We studied a method for measuring the pancreatic isoenzyme of amylase (EC 3.2.1.1) by use of a mouse monoclonal antibody against human salivary-type amylase (Clin Chem 1985;31:1283) coupled indirectly to particles of polyvinylidene fluoride via polyclonal goat anti-mouse immunoglobulin. These particles, in 200 microL of a suspension, could remove salivary amylase (activity 2200 U/L) from an equal volume of serum in 5 min. Measurement of amylase activity in the supernatant fluids from treated sera thus provided an assay of pancreatic amylase. Precision studies at three activity concentrations yielded within-run CVs of 1.6% to 1.7% (n = 25) and total CVs of 2.2% to 5.1% (20 days). Salivary amylase added to each of 10 sera was completely (99.8%, SD 1.6%) removed. The new method (y) showed the following regression statistics when compared with an electrophoretic method (x): slope = 0.989 (SD 0.019), intercept = -0.220% (SD 1.48%), SEE 4.0%, n = 51. Similar respective regression values were found for urine samples: slope = 0.934 (SD 0.053), intercept = 2.3 U/L (SD 3.2), SEE 8.4 U/L, n = 26. The following respective values were found when the new method (y) was compared with the previously described immunoprecipitation assay (x): slope = 1.02 (SD 0.02), intercept = 2.2% (SD 1.4%), SEE 3.3%, n = 23 sera. Reference intervals for pancreatic amylase activity in serum were established for three different substrates: maltotetraose, maltopentaose, and p-nitrophenylheptaoside.     

Rapid quantitative, specific measurement of pancreatic amylase in serum with use of a monoclonal antibody

In this rapid quantitative assay for pancreatic alpha-amylase (EC 3.2.1.1) in serum, we precipitate salivary amylases by 10-min incubation with monoclonal anti-salivary amylase antibody immobilized on particles of polyvinylidene fluoride. We then centrifuge the serum mixture and measure the pancreatic amylase activity remaining in the supernate by a kinetic method. The assay requires 50 microL of serum and the standard curve is linear to at least 1300 U of pancreatic amylase per liter of serum. CVs were 1.3% within-run, 6-8% day-to-day. Apparent analytical recovery of pancreatic amylase activity added to serum was 101% +/- 2%. Addition of purified salivary amylase, 356 U/L, to sera gave a value for apparent pancreatic amylase of less than 4 U/L, or 1% of the added salivary amylase activity. This assay correlated well with an electrophoretic method (slope, 0.97-0.99; intercept, 0.5 to -4 U/L; correlation coefficient, 0.946-0.990; and standard error of the estimate 3-5 U/L). Estimated normal reference intervals with maltotetraose as substrate were: total amylase, 39-118 U/L; pancreatic amylase, 11-50 U/L; and salivary amylase, 18-79 U/L.     

Suitability of control materials in the differential inhibition assay for human pancreatic and salivary amylase

We investigated the behavior of 26 quality-control sera with the inhibitor method for differential amylase (EC 3.2.1.1) assay. We also studied the sensitivity to the wheat-derived inhibitor of pancreatic amylases from 10 different animals in comparison with human pancreatic and salivary amylase. The results indicate that only control materials containing human amylases can be measured accurately. The animal amylases (bovine, equine, porcine) used in many quality control sera are relatively insensitive to the inhibitor as compared with human pancreatic and salivary amylase.     

Evaluation of a new assay for pancreatic amylase: performance characteristics and estimation of reference intervals

We have evaluated a new assay for the specific determination of pancreatic (P) isoamylase, the principle being based on a synergistic inhibitory effect of two monoclonal antibodies directed towards salivary (S) amylase. After 3 min incubation, activities were determined with maltoheptaoside-PNP as substrate on a Hitachi 705 analyzer at 25, 30 and 37 degrees C, respectively. Coefficients of variation ranged from 0.6 to 5.4% for within-run and 2.1 to 9.9% for day-to-day precision. Linearity held up to 1200 U/L (25 degrees C) and 2300 U/L (37 degrees C). Comparison of the new method with the wheat germ inhibitor technique showed an excellent correlation, with coefficients ranging from 0.990 to 1.00. Using purified P- and S-amylase we observed no inhibiting cross-reactivity of the antibodies with the P-isoenzyme, but an incomplete blockage of S-amylase: residual activity was approximately 2% at 25 and 30 degrees C, and 2.5% at 37 degrees C. The distribution pattern of P-, S- and total amylase activity in serum of healthy subjects was only slightly skewed to the right. We found neither an influence of sex nor of age on the reference ranges. In random urine samples, distribution of activities was strongly skewed. However, if the activity was related to the urinary creatinine concentration, an approximately normal distribution was obtained, allowing, as in serum, the establishment of upper and lower reference values.     

Quantitative immunoenzymatic assay of human lutropin, with use of a bi-specific monoclonal antibody

In this immunoenzymatic assay for human lutropin (hLH) we used bi-specific antibodies (BiAbs) obtained from the fusion of two hybridomas producing antibodies to beta-D-galactosidase and hLh. The BiAb complexed with the enzyme beta-D-galactosidase was used as tracer in a double-determinant assay. We compared the assay involving the BiAb (Bi-EIA) with an immunoenzymatic assay (EIA) in which the same capture antibody was used but the tracer was an enzyme-conjugated hLH-specific monoclonal antibody produced by the same parental cell line used to produce the BiAb. The coefficient of correlation (r) between the two assays was 0.979 but the Bi-EIA was more sensitive (detection limits: 0.8 int. units/L for the Bi-EIA, 2.0 int. units/L for the EIA) and more specific (less than 0.04% vs less than 1.2% cross-reactions with human choriogonadotropin). Mean intra- and interassay CVs for the Bi-EIA were 2.9% and 5.9%, respectively. Correlation (r) with an immunoradiometric assay (IRMA, Serono kit) was 0.960, with radioimmunoassay (RIA, Biodata kit) 0.909, and with an enzyme-linked immunosorbent assay (ELISA kit, Specialty Medical Industries Inc.) 0.888, (n = 25). Evidently, bi-specific antibodies can be used successfully in immunoenzymatic assays, and with potentially greater sensitivity and specificity than assay with a traditional antibody-enzyme conjugate.     

Isolation and characterization of a pancreatic elastase from plasma of patients with acute pancreatitis

1. An elastase-like enzyme in plasma of patients with acute pancreatitis was purified by DEAE-cellulose column chromatography and polyacrylamide-gel disc electrophoresis. 2. In this way 0.24 mg of purified enzyme with a specific activity of 3.94 succinyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide units/mg of protein was obtained from 10 ml of plasma. 3. The purified material was homogeneous as ascertained by sodium dodecyl sulphate/polyacrylamide-gel disc electrophoresis and had an apparent molecular weight of 24 000 as measured by gel filtration on Sephadex G-100. 4. This enzyme hydrolysed denatured casein and Congo Red-elastin as well as succinyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide. Its amidolytic activity was inhibited by soya bean trypsin inhibitor, but not by aprotinin. 5. Although the enzyme was immunologically similar to elastase 2, its kinetic properties and substrate specificity were apparently different. 6. We propose that an elastase-like enzyme, probably different from elastase 1 or elastase 2, is liberated from the pancreas into blood during acute pancreatitis and becomes combined with alpha 2-macroglobulin.     

Time course of changes in serum activity of the P3 isoform of pancreatic amylase isoenzyme in patients with acute pancreatitis

We studied the temporal sequence of serum P3 isoamylase changes in patients with acute pancreatitis, in comparison with serum pancreatic lipase (LPS) activity. Twenty-four hospitalized patients with acute pancreatitis, the onset not more than 12 h previously, were admitted to the study. Serum samples were collected daily for 6 days after onset for enzyme determinations. Measurements in the 48-h period after the onset of acute pancreatitis showed clinical sensitivities of total amylase, P3 isoform and LPS of 100%. During the following period, LPS and P3 isoform activities decreased with time with parallel changes, still showing diagnostic sensitivity significantly higher than total amylase. P3 isoform and LPS appear to be interchangeable markers of pathological release of pancreatic enzymes into the bloodstream during acute pancreatitis. However, LPS determination is more convenient because of its simplicity and shorter turnaround time.     

血清淀粉酶和脂肪酶联合测定在急性胰腺炎诊断中的意义

目的 探讨血清淀粉酶（AMY）和脂肪酶（LIP）测定在急性胰腺炎诊断中的价值。方法 对54例急性胰腺炎患者、58例胆结石患者、47例肾结石患者和50例健康对照组人员进行血清淀粉酶（AMY）和脂肪酶（LIP）测定。结果 54例急性胰腺炎中血清淀粉酶和脂肪酶升高的敏感性分别为87．0％和92．6％；联合测定AMY和LIP单项指标阳性率为98．1％；105例非急性胰腺炎患者中，AMY阳性的有12例，LIP阳性的有10例，AMY和LIP的特异性分别为88．6％和90．5％，AMY和LIP同时阴性的特异性为100％。结论 联合测定血清AMY和LIP可提高急性胰腺炎诊断的敏感性和特异性，对急性胰腺炎具有重要的诊断意义。     

Radioimmunoassay for human pancreatic amylase: comparison of human serum amylase by measurement of enzymatic activity and by radioimmunoassay.

A radioimmunoassay (RIA) for human pancreatic amylase has been developed for the determination of human serum amylase content. The assay was shown to be sensitive (7 ng/ml), reproducible and specific, but human pancreatic amylase and salivary amylase could not be distinguished by the antiserum used. In normal subjects, the mean concentration of amylase determined by the RIA was found to be 122.1 ng/ml (range: 55--250 ng/ml). A good correlation was observed between the concentration of amylase and its enzymatic activity in normal subjects. In some instances with high amylase activity, however, the rise in enzymatic activity was not accompanied by increasing amount of amylase content.     

Pleural effusion volume in patients with acute pancreatitis: a retrospective study from three acute pancreatitis centers

ObjectiveTo assess the value of pleural effusion volume (PEV) quantified on chest computed tomography (CT) in patients with early stage acute pancreatitis (AP).MethodsData of PEV, and C-reactive protein (CRP) levels as well as Ranson, bedside index of severity in acute pancreatitis (BISAP), Marshall, acute physiology and chronic health evaluation II (APACHE II), CT severity index (CTSI), and extra-pancreatic inflammation on computed tomography (EPIC) scores in patients with AP were collected. Duration of hospitalization, severity of AP, infection, procedure, intensive care unit (ICU) admission, organ failure, or death were included as the outcome parameters.ResultsIn 465 patients, the mean PEV was 98.8 ± 113.2 mL. PEV showed strong and significant correlations with the CRP levels, duration of hospitalization as well as the Ranson, BISAP, Marshall, APACHE II, CTSI, and EPIC scores (p < .05). PEV demonstrated significant accuracy in predicting severity, infection, procedure, ICU admission, organ failure, and death (p < .05).ConclusionPEV quantified on chest CT positively associated with the duration of hospitalization, CRP levels, Ranson, BISAP, Marshall, APACHE II, CTSI, and EPIC scores. It can be a reliable radiologic biomarker in predicting severity and clinical outcomes of AP.

KEY MESSAGES

• Pleural effusion is a common chest finding in patients with acute pancreatitis.
• Pleural effusion volume quantified on chest CT examination positively associated with the duration of hospitalization, CRP level, as well as Ranson, BISAP, Marshall, APACHE II, CTSI, and EPIC scoring systems.
• Pleural effusion volume can be a reliable radiologic biomarker in the prediction of severity and clinical outcomes of acute pancreatitis.

     

Diagnostic utility of a new monoclonal antibody pancreatic isoamylase assay in chronic pancreatic diseases

In order to evaluate the efficacy of a monoclonal pancreatic (P) isoamylase assay in the diagnosis of chronic pancreatic disease and to compare the behavior of this test with that of amylase and elastase 1, these three enzymes were measured in the sera of 39 healthy controls, 28 patients with pancreatic cancer, 50 with chronic pancreatitis and 60 with extra-pancreatic diseases. In patients with chronic relapsing pancreatitis, increased P-isoamylase and elastase 1 values were found in similar percentages (about 70%), whereas the percentage for elevated amylase values was lower (52%). Elastase 1 was increased in 52% of patients with pancreatic cancer, while the other two enzymes were only occasionally elevated. The levels for all three enzymes were abnormal in some patients with extra-pancreatic diseases. It may be concluded that this assay for P-isoamylase determination is sufficiently sensitive and reliable in detecting pancreatic inflammation, even though some limitations concerning its specificity should be born in mind.     

Analysis of a high-throughput HLA antibody screening assay for use with platelet donors

BACKGROUND: Passive infusion of HLA antibodies has been implicated in transfusion reactions. A rapid, inexpensive method of screening blood donors for HLA antibodies might reduce the incidence of reactions. A high-throughput microbead-flow analyzer HLA antibody detection technique was compared with an enzyme-linked immunosorbent assay (ELISA) method. STUDY DESIGN AND METHODS: Ninety-six apheresis platelet (PLT) donors were tested for antibodies to Class I and II HLA antigens with mixed-antigen microbead-flow analyzer and ELISAs. For both assays, samples reactive in the mixed-antigen assay were tested with a panel-reactive antibody (PRA) assay. Samples reactive in both the mixed-antigen and the PRA assays were considered positive. RESULTS: In the mixed-antigen microbead assay, 46 (48%) samples were reactive to Class I antigens and 20 (21%) to Class II. Further testing in the microbead PRA assay revealed that 34 (35%) had antibodies to Class I antigens, 18 (19%) to Class II, and 42 (44%) to either Class I or Class II. Class I antibodies were present in 56 percent of females and 36 percent of males. In the mixed-antigen ELISA, 4 samples were reactive with Class I antigens, 4 with Class II antigens, and 5 with Class I or Class II. All 5 reactive samples were also reactive in the ELISA PRA assay and were from females. CONCLUSION: The microbead assay was more sensitive than the ELISA and detected antibodies in a large proportion of donors. Samples reactive in the mixed-antigen microbead assay should be confirmed by a second assay before concluding that antibodies are present.