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Background: COVID-19 convalescent plasma (CCP) ideally contains high titers of (neutralizing) anti-SARS-CoV-2 antibodies. Several scalable immunoassays for CCP selection have been developed. We designed an enzyme-linked immunosorbent assay (ELISA) that measures neutralizing antibodies (of all isotypes) in plasma by determining the level of competition between CCP and a mouse neutralizing antibody for binding to the receptor binding domain (RBD) of SARS-CoV-2., Methods: Plasma was collected from 72 convalescent individuals and inhibition of viral infection was determined by plaque reduction neutralization (PRNT50). The level of neutralizing antibodies was measured in the novel competition ELISA and in a commercially available ELISA that measures inhibition of recombinant ACE2 binding to immobilized RBD. These results were compared with a high throughput chemiluminescent microparticle immunoassay (CMIA)., Results: The results from both ELISAs were correlating, in particular for high titer CCP (PRNT50 ≥ 1:160) (Spearman r = .73, p < .001). Moderate correlation was found between the competition ELISA and CMIA (r = .57 for high titer and r = .62 for low titer CCP, p < .001). Receiver operator characteristic analysis showed that the competition ELISA selected CCP with a sensitivity and specificity of 61% and 100%, respectively. However, discrimination between low and high titer CCP had a lower resolution (sensitivity: 34% and specificity: 89%)., Conclusion: The competition ELISA screens for neutralizing antibodies in CCP by competition for just a single epitope. It exerts a sensitivity of 61% with no false identifications. These ELISA designs can be used for epitope mapping or for selection of CCP., (© 2021 The Authors. Transfusion published by Wiley Periodicals LLC on behalf of AABB.)

A sensitive staining method for alkali-labile phosphoproteins has been developed. As little as 0.2 nmol bound P/mm2 can be detected. The procedure is based on alkaline hydrolysis, phosphate capture, and formation of an insoluble rhodamine B-phosphomolybdate complex. A further modification for the qualitative detection of alkaline phosphatase activity on polyacrylamide gels is proposed. During incubation, the released Pi is precipitated as lead phosphate and subsequently stained with rhodamine B.

The cholinesterase (acylcholine acylkhydrolase, EC 3.1.1.8) of chicken egg yolk was partly purified and characterized. It was compared to homologous enzymes of liver and blood plasma of laying hens. During gel filtration, yolk and liver cholinesterase were resolved into two fractions. Blood plasma cholinesterase showed one form only, identical with yolk and liver cholinesterase 1 *** (EC 3.1.1.8). This form has an Mr of 440 000 and may be a tetramer of a cholinesterase form present in yolk and liver (Mr 104 000). Substrate specificity, pH optima, Km values, the influence of effectors (ammonium derivatives, choline, eserine, fluoride), gel filtration, gel electrophoresis, isoelectric focusing and affinity chromatography, all point to a very close similarity, if not identity, of the corresponding forms.

Hen's egg white and vitelline membrane nucleoside triphosphatases were purified resulting in active soluble subunits with MR 260,000 +/- 10,000. PH optima are divalent cation dependent and situated at pH 6.2 and 8.0 with ATP and at pH 6.15 with ADP as substrate. Ca2+ and Mg2+ are activators. Km and Ki values for Pi and PPi were determined. The enzymes are specific neither for ATP nor for ADP alone. No separation between nucleoside triphosphatase and nucleoside diphosphatase could be achieved. Differences found in their action can be due to differences in organization and properties of the (intermediary) enzyme-substrate complexes. A close relationship exists with homologous enzymes found in oviductal secretory cells and in oviductal secretions.

1. The non-specific hen's egg yolk alkaline phosphatase is a metalloprotein (Zn2+?) composed of two identical inactive subunits. 2. A second metal site preferably binds Mg2+ (15-fold activation). Me(II)(H2O)H+, a charged arginine, and tyrosine in the active site are involved in positioning and binding of the substrate and metal ion. 3. Substrate inhibition differs with pH. This may be related to the presence of two active sites in the enzyme, one in each subunit. 4. Uncompetitive inhibition with l-phenylalanine and analogues suggests a phosphorylated intermediate. 5. Inhibition is weakly competitive with Pi, strong non-competitive with PPi as compared to Mg2+- free PPi, and partially competitive with arsenate. 6. The purified enzyme is stabilized and activated by amines and proteins.

1. Hen's egg yolk alkaline phosphatase can transfer the donor substrate phosphoryl to a hydroxyl containing acceptor. 2. This acceptor contains preferably an alpha-amino or alpha-imino group. 3. The optimal pH of the transfer reaction is at 9.45 with Tris and at 9.60 with diethanolamine as acceptor. 4. Transfer products were isolated and characterized. 5. The ratio of products is independent of the donor substrate. ROH and R'OP production increase linearly with the acceptor concentration, while Pi release and Km remain constant. 6. This points to a mechanism involving two phosphorylated intermediates. 7. The acceptor bypasses the isomerization step of the phosphoryl enzyme, which is necessary before hydrolysis can take place.

A sensitive technique for inorganic phosphate determination was developed. It is based on the formation of an insoluble rhodamine B-phosphomolybdate complex. After it is washed with 1 N HCl the precipitate is dissolved in acetone and rhodamine B is measured spectrophotometrically at 555 nm. In 1 N HCl, the complex is composed of three molecules rhodamine B and one molecule phosphomolybdate. Due to the high molar absorbance of rhodamine B in acetone and to the threefold amplification of dye concentration compared to Pi concentration in the precipitated complex, a molar absorption coefficient of 330,000 +/- 5000 M-1 cm-1 (SD) is obtained. This allows the determination of quantities as low as 1.5 nmol Pi with good precision, while quantities as low as 0.5 nmol Pi are detectable. The effect of anions and buffers was studied. Some possible applications of the method are illustrated, as, e.g., enzyme activity measurement at very low substrate concentration and determination of small quantities of Pi and total phosphate in (biological) samples.