Rapid measurement of serum pancreatic amylase.

A simple, rapid assay for the pancreatic isoenzyme of human serum amylase was developed. The assay utilized an immunoabsorbent prepared by coating latex beads with a monoclonal antibody specific for pancreatic amylase. Treatment of patient serum with immunoabsorbent removed pancreatic amylase, and measurement of residual amylase activity with standard total amylase methodology allowed estimation of the pancreatic amylase content. Extraction efficiency of pancreatic amylase was consistent at amylase concentrations up to 1,000 U/L (y = 0.97 x +16.7 U/L; r = 0.9995). The assay was standardized with purified pancreatic amylase added to neonatal serum (low endogenous activity). A comparison of patient specimen results with the results of a standard technique (cellulose acetate electrophoresis) yielded an excellent correlation (immunoabsorption result = 0.96 electrophoresis result + 1.2 U/L; r = 0.987). Salivary amylase did not interfere with the assay until levels exceeded 1,000 U/L. Daily analysis of a frozen serum pool yielded a coefficient of variation of 9.2% at mean pancreatic amylase value of 54 U/L (+/- 5 U/L). A normal range study found a strong influence of age, with pancreatic amylase levels increasing dramatically in the first 3 years of life, to stabilize at a range of 0-66 U/L thereafter.

Eliminating interference from heterophilic antibodies in a two-site immunoassay for creatine kinase MB by using F(ab')2 conjugate and polyclonal mouse IgG.

Heterophilic antibodies interfere in two-site immunoassays. Our purpose was to screen for the samples containing heterophilic antibodies that react with mouse immunoglobulins and to use them to determine how to eliminate interference in various two-site immunoassays being developed in our laboratory. Of approximately 2600 samples screened, 81 had heterophilic antibodies. When creatine kinase MB (CKMB) concentration was measured with intact antibody conjugate in these 81 samples, 18 (22%) samples had apparent CKMB values significantly greater than values measured with Hybritech's "Tandem -E CKMB immunoenzymetric" assay and Corning agarose-gel electrophoresis. Adding up to 133 mg/L of polymerized IgG or up to 1666 mg/L polyclonal mouse IgG in the assay did not eliminate the interference in all the samples. However, adding F(ab')2 conjugate plus 40 mg/L of polymerized IgG or 83 mg/L of polyclonal mouse IgG eliminated the interference in all the samples. This approach was also effective in eliminating the interference in 15 samples containing 4.7-165.2 mg/L of human antimouse antibody (HAMA). Combined use of F(ab')2 conjugate and polyclonal mouse IgG is recommended to eliminate interference from heterophilic antibodies that react with murine immunoglobulins or HAMA in two-site murine-antibody-based assays.

Immunoaffinity purification of creatine kinase-MB from human, dog, and rabbit heart with use of a monoclonal antibody specific for CK-MB.

We describe a simple, rapid immunoaffinity procedure for purifying the MB isoenzyme of creatine kinase. Immunoaffinity gel is prepared by linking a monoclonal antibody ("Conan-MB"), specific for this isoenzyme, to Sepharose 4B. Heart tissue is homogenized and fractionated with 40-70% saturated ammonium sulfate before it is applied to the immunoaffinity gel. CK-MB activity, retained on the gel, is then eluted with a high-pH diethylamine buffer (0.1 mol/L, pH 10.5). The purified CK-MB isoenzyme is stabilized by collection directly into tubes containing glycerol (to prevent dissociation of the enzyme subunits) and pH-neutralizing buffer. This procedure compares favorably in yield, specific activity, and technical ease with a multi-column purification method previously used in our laboratory. We have used the immunoaffinity procedure to purify to homogeneity CK-MB from human, dog, and rabbit heart, with yields of 50.0%, 53.1%, and 49.3% and specific activities of 540, 477, and 377 kU/g, respectively. The preparations are pure as judged by protein staining with silver nitrate after electrophoresis on sodium dodecyl sulfate/polyacrylamide gel.

Quantification of lactate dehydrogenase-1 in serum with use of an M-subunit-specific monoclonal antibody.

We have developed a rapid, one-step assay for measuring lactate dehydrogenase-1 (LD-1) activity in serum after extraction of LD-2, LD-3, LD-4, and LD-5 isoenzymes by an immobilized M-subunit-specific monoclonal antibody (D.8.1). In the assay, 100 microL of serum is mixed with 50 microL of a suspension of 0.8-micron-diameter latex particles coated with 30 micrograms of the monoclonal antibody D.8.1, then incubated at room temperature for 5 min. The latex particles, to which LD-2 through LD-5 are bound, are pelleted by centrifugation for 2 min at 12,000 X g, and the LD-1 activity is measured kinetically in the supernatant fluid. We optimized the assay for antibody immobilization, antibody concentration, and time and temperature of incubation. Serum bilirubin concentrations up to 0.33 g/L (0.56 mmol/L) did not interfere in the assay. Hemolysis interfered solely through LD-1 released from erythrocytes. The within-assay CV for low-concentration quality-control material (LD-1 33 U/L) was 3.5% (n = 9) and for high-concentration material (LD-1 185 U/L) was 1.9% (n = 8); the between-assay CVs for the two materials were 6.1% (n = 9) and 2.5% (n = 10), respectively. The LD-1 activity measured in 98 samples by our assay compared well with a two-step polyclonal antibody-based assay (Isomune-LD, Roche Diagnostic Systems; r = 0.998) and with an electrophoretic method (Paragon, Beckman Instruments; r = 0.956).

Extremely high values of prostate-specific antigen in patients with adenocarcinoma of the prostate; demonstration of the "hook effect".

We reviewed 721 consecutive samples submitted for measurement of prostate-specific antigen (PSA) over five months. We identified three patients with extremely high PSA concentrations: 650, 1840, and 3280 micrograms/L (their acid phosphatase activities were 3.2, 1337, and 2.8 U/L, respectively), and present case reports for the latter two. Serial dilutions of samples obtained from the patient with the highest PSA concentration indicated that the one-step Tandem-PSA assay gave falsely low values for high concentrations of PSA, an observation consistent with the phenomenon of the "hook effect." This effect was not observed when the sample was reanalyzed for PSA by a two-step procedure.

Semi-automated direct colorimetric measurement of creatine kinase isoenzyme MB activity after extraction from serum by use of a CK-MB-specific monoclonal antibody.

This semi-automated colorimetric assay for the MB isoenzyme of creatine kinase (EC 2.7.3.2) is based on a monoclonal antibody ("Conan-MB") specific for this isoenzyme and is a modification of a previously published method (Vaidya et al., Clin Chem 1986;32:657-63). A 0.64-cm bead coated with 2 to 3 micrograms of antibody is incubated with 100 microL of serum and 10 microL of 0.2 mol/L beta-mercaptoethanol for 1 h at room temperature, to extract CK-MB. The beads are washed with de-ionized water and incubated with CK substrate for 45 min at 37 degrees C. A solution containing trans-1,2-diaminocyclohexane-N,N,N', N'-tetraacetic acid, p-iodonitrotetrazolium violet, and diaphorase is added and the resulting colored product is measured at 492 nm. The standard curve is linear to 200 U of CK-MB per liter, and analytical recovery is 97-113%. Total assay CV for low (9.7 U/L) and high (50.7 U/L) quality-control materials was 14.1% (n = 1878) and 11.6% (n = 1842), respectively. CK-MB activity correlated well (r = 0.978, n = 226) with CK-MB measured by a two-site mass immunoassay, and 99.4% of samples with CK-MB greater than or equal to 12 U/L (n = 347) were verified by electrophoresis on agarose.

Quantitation of serum lactate dehydrogenase-5 with monoclonal antibodies.

Spleen cells from BALB/cJ mice which had been immunized with human lactate dehydrogenase-1 (LDH-1) were fused with SP2/0-Ag14. Two hybridomas were produced which recognized the antigen. Competitive RIA revealed that one antibody ('Smit-LDH') recognized the H subunit of LDH while the other ('Hem-LDH') recognized both H and M subunits of LDH. With the use of these antibodies we developed an assay for LDH-5 activity in which serum is incubated for 30 min at room temperature with the two antibodies ('Smit-LDH' and 'Hem-LDH' in the ratio 64:1.3, micrograms/ml) immobilized on latex beads to extract LDH-1 through LDH-4. After centrifugation, the LDH activity of the supernatant is measured and represents LDH-5 activity. Latex beads coated with bovine serum albumin were used as control. The LDH-5 activity as determined by our assay correlated well (r = 0.98) with the values obtained by an electrophoresis method. There was no interference due to LDH-1 through LDH-3 up to 3,000 U/l and LDH-4 up to 350 U/l. Serum samples with total LDH activity above 1,000 U/l were appropriately diluted in order to avoid interference by LDH-4. Use of these monoclonal antibodies allows precise, rapid and direct measurement of LDH-5 activity in serum.

Direct measurement of creatine kinase-MB activity in serum after extraction with a monoclonal antibody specific to the MB isoenzyme.

Fusion of splenocytes from A/J mice immunized by creatine kinase (EC 2.7.3.2)-MB isoenzyme (CK-MB) with SP2/0-Ag14 myeloma cell line generated hybridomas producing monoclonal antibodies specific to CK-MB. One of these monoclonal antibodies ("Conan-MB") was used to develop a direct assay for CK-MB activity. In the assay, serum is incubated for 30 min at room temperature with "Conan-MB" immobilized on latex beads. The beads are then washed, and CK-MB activity bound to the antibody is measured after incubation with CK enzyme reagent for 30 min at 37 degrees C. Results with the assay correlated well (r = 0.997) with those for CK-MB concentration as measured by a two-site immunoassay. Neither CK-MM, CK-BB, mitochondrial CK, nor a hemolysate of erythrocytes interfered. Use of this unique monoclonal antibody allows rapid, precise, and direct determination of CK-MB activity.

Inadequacy of traditional ELISA for screening hybridoma supernatants for murine monoclonal antibodies.

Hybridomas producing anti-creatine kinase (CK) and anti-lactate dehydrogenase (LDH) antibodies were screened by enzyme linked immunosorbant assay (ELISA) with antigen coated onto plastic wells. Out of seven antibodies positive for each isoenzyme of CK, four antibodies failed to bind to the radiolabeled antigen in solution phase radioimmunoassay (RIA) or native antigen in competitive ELISA. Moreover, out of nine antibodies shown reactive with LDH-1 and seven antibodies binding to LDH-5 by ELISA, not a single antibody bound to either radiolabeled or native antigen in solution. Our results along with other recent studies strongly suggest that antigen conformation on solid phase may frequently be different from in solution and that screening by ELISA in which antigen is attached to solid phase is often inappropriate for determining the presence of useful monoclonal antibodies.

7S RNA, containing 5S ribosomal RNA and the termination stem, is a specific substrate for the two RNA processing enzymes RNase III and RNase E.

The 7S RNA, a precursor of 5S rRNA that contains 5S rRNA and the termination stem and loop, is a substrate for RNase E and is also a substrate for RNase III. The cleavage by RNase III is in the stem, 11 nucleotides downstream from the 3' end of the mature 5S rRNA and 8 nucleotides downstream from the RNase E cleavage site. Near the cleaved nucleotides there are three base pairs that appear in the same relative positions in most known RNase III cleavage sites. The large product of the RNase III cleavage reaction, which is a 5S rRNA that contains 11 extra nucleotides at the 3' end, is a substrate for RNase E. This suggests that the information for the 3'-end cleavage by RNase E resides mainly in the 5S rRNA itself. Using rnc rne strains, carrying the plasmid that leads to the accumulation of 7S RNA, we showed that the 7S RNA does not result from an RNase III cleavage but is apparently a proper transcription termination product.

Alkaloid production by Claviceps sp. SD-58; involvement of phosphatase isozymes.

Simultaneous reduction in alkaloid yield and level of phosphatases by high concentrations of phosphate was observed in Claviceps sp. SD-58. Tryptophan-induced culture showed an increase in alkaloid yield and the level of phosphatases. Phosphate caused repression of both acid phosphatase (isoenzyme I) and alkaline phosphatase (isoenzymes III and V).

A new method for isolation of saprophytic cultures of Claviceps fusiformis from sclerotia.

The proposed method includes primary and secondary treatments. Of the various disinfectants tested, 1-propanol and 1-butanol as a primary treatment and phenol, resorcinol, HgC12 or NaCl as a secondary treatment completely eliminated the contamination hazard. 1-Butanol and phenol were not useful disinfectants since they inhibited the growth of C. fusiformis along with the other contaminants. Primary and secondary treatment of sclerotia with 1-propanol and resorcinol, respectively, produced the maximum of stable cultures. Out of 120 cultures tested, 5 cultures demonstrated an appreciable yield of alkaloids under submerged cultural conditions.