Nephelometry has become a very reliable and convenient method for immunoassays. Recent innovations in polymer chemistry have led to the design of highly sensitive and reproducible immunoassays for the Behring Nephelometer Analyzer. The principle of particle‐enhanced immunoassays has led to the development of new polymer particles. Key advances include the preparation and use of particles with polystyrene cores covered by thin shells with chemically reactive groups. The cores are prepared by emulsion polymerization, giving particles with a well‐defined size. The particle size has been optimized to fulfill the requirements for nephelometric analysis. The shells consist of a polystyrene/polymethacrylate/polymethacrylamidoacet‐aldehyde dipentylacetal copolymer, which allows a covalent binding of the immunochemicals of interest. Using these particles, a method for the determination of C‐reactive protein has been worked out.
Vertebrate and invertebrate L-lactate dehydrogenases (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) are effectively bound to oxamate-diaminohexyl-Sepharose, whereas several D-lactate dehydrogenases (D)lactate:NAD+ oxidoreductase, EC 1.1.1.28) do not bind to the same Sepharose. One explanation for our findings is that the enzymes' substrate is oriented in a reversed manner in the active center of the D-and L-lactate dehydrogenases.
MATERIALS AND METHODSSepharose 4B was purchased from Pharmacia, 1,6-diaminohexane was from Calbiochem, and all other reagents were from Sigma. LDHs from horseshoe crab skeletal muscle (8), snail and abalone foot (7), barnacle (6), and chicken heart (9) were purified in this lab by published procedures. Pig heart and rabbit muscle LDHs were purchased from Sigma.Lobster tail muscle LDH (isoenzyme 5) was prepared by initial homogenization and fractionation (35-55% saturated ammonium sulfate) as described by Eichner and Kaplan (10). The 55% ammonium sulfate precipitate was dialyzed extensively against 0.05 M potassium phosphate buffer (pH 6.8) containing 0.15 M KCI (irrigation buffer) at 40C, with several changes of the dialyzing buffer. The affinity chromatography column containing oxamate-diaminohexyl-Sepharose gel was preequilibrated with irrigation buffer containing 0.2 mM NADH. After addition of NADH (0.2 mM), the enzyme solution was applied to the column. The column was washed with irrigation buffer containing the NADH until the absorbance of the eluate at 280 nm was zero compared to fresh buffer. The elution of the purified enzyme from the column was then acThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
5832complished with a 0.05 M potassium phosphate buffer (pH 6.8) containing no NADH and 10 mM pyruvate. To separate the LDH isoenzyme 5 (LDH 5) from the contaminating hybrid LDH 4, isoelectric focusing was carried out in a 110-ml preparative isoelectric focusing apparatus (48 hr, 600 V, 3°C) with a 1% ampholine (pH 5-7; LKB, Sweden) gradient as described by Haglund (11).Lobster leg LDH (isoenzyme 1) was prepared by grinding whole legs with a commercial meat grinder, followed by homogenization in an appropriate amount of extraction buffer as described for lobster tail LDH. After the first centrifugation, the muscle tissue could be easily separated from the exoskeletal debris for a second extraction. The ammonium sulfate fractionation was the same as for lobster tail LDH. The viscosity and the protein content of the sample was further reduced by a heat step (incubation for 5 min at 45°C after dialysis against the affinity chromatography irrigation buffer containing 10 mM 2-mercaptoethanol and 1 mM EDTA) and subsequent centrifugation (40,000 X g). Oxamate-Sepharose affinity chromatography was performed as described for lobster tail LDH 5. After elution of the purified enzyme, the sample was concentrated and the pyruva...
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