Determination of blood glucose using 4-amino phenazone as oxygen acceptor

Author(s): Trinder P


Microtechniques are now used extensively in diagnostic as well as immunological studies for viruses, protozoa, In microtitrations constant volumes of fluids are picked up and delivered by specially constructed and calibrated metallic microdiluters. According to the Microtiter Instruction Manual of the Cooke Engineering Company (1965), their high titanium, stainless steel, new-pattern microdiluters with a precisely slotted tip are not supposed to rust, but in the hot and humid climate of Calcutta they do rust, though they continue to deliver fluids properly when tested on the special 'go-no-go' delivery tester supplied by the manufacturer. Many of the rust particles become detached from the microdiluters during their agitation in the wells of the dilution plates and produce, both with human group 0 and goose erythrocytes, an atypical settling pattern at the bottom of disposable plastic V plates. There is a small central button of erythrocytes, showing as well as the peripheral deposit, small black particles rimmed by a clear space. The peripheral concentric erythrocyte deposit closely simulated the typical haemagglutination pattern. Microscopically many black rust particles of various sizes and shapes were observed among discrete erythro-cytes, and we have called this atypical pattern pseudo-haemagglutination. The rusted particles did not alter the typical agglutina-tion pattern or the titre of some of the haemagglutinin-positive enteroviruses and arboviruses. They, however, produced pseudo-haemagglutination with the haemag-glutinin-negative enteroviruses as well as the uninoculated tissue culture maintenance medium. It appears that the rusted microdiluters, if used for diluting the antisera or the haemagglutinin-positive antigens, are not likely to modify the positive haemagglutination patterns. But one has to be cautious in using the rusted microdiluters for screening antigens as the detached rust may of itself produce a false-positive haemagglutination pattern. Although the dl adrenaline method has worked very well in this laboratory (Trinder, 1969), the manual version is a little insensitive and the colour development time is rather long. I have now worked out a method of using 4-amino phenazone which is three times as sensitive as the dl adrenaline method, uses only two relatively stable solutions , and requires only a 10-minute colour development. The two solutions required are: 1 PROTEIN PRECIPITANT Prepared exactly as for the dl adrenaline method but containing 01 % w/v phenol. Keeps indefinitely. 2 COLOUR REAGENT Fermcozyme 653 AM, 5 ml, and 5 ml of 0-1 % peroxidase (R.Z. 0-6) are added to 300 ml of a solution containing 1% w/v Na5HP04, 0-1%

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