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The two equations were, respectively, y 35.936 + 9.271x - 0.413x2 - 0.021x3 (11.7) y 48.300 + 7.865x - 1.377x2 + 0.288x3 - 0.020X4 - 0.003x5 (11.8) For example, in the case of nicotinic acid, doses were assumed to be 1.0 and 2.0 for the standard and 0.8 and 1.6 for the unknown. Putting these values in Equation (11.7), responses are calculated. Then, putting the calculated responses into the standard formula for calculating potency estimate for a five-point assay, the unknown potency was...

The different sorts of microbiological assay

The main use of microbiological assay is in the determination of the potency of growth-inhibiting substances especially antibiotics and of growth-promoting substances amino acids and vitamins of the B group. There are three main assay procedures 1. The agar diffusion assay, which is applicable to both growth-inhibiting substances and growth-promoting substances 2. The flask or tube assay for growth-promoting substances 3. The tube assay for growth-inhibiting substances Although methods 2 and 3...

What Do We Want of an Assay How Do We Attain Our Goal

The determination of sample potency may be required for a variety of reasons. It is important for the analyst to consider the purpose of the analysis so that he or she may then select a procedure that will give results of the required quality by a method that is cost effective. A large variety of experimental designs is available to the analyst these designs and their capabilities will be described elsewhere in this book. Suffice it to say here that they range from simple tests of low precision...

Linearization of sigmoid curves

The need to transform sigmoid response lines into a more convenient form for arithmetical manipulation arose in connection with microbiological assays. Procedures for linearization were proposed independently by Hemmingsen (1933) and Gaddum (1933). They used the normal distribution curve in its cumulative form to manipulate quantal responses into a more manageable form. The two forms of the normal distribution curve are shown in Figures 4.5a and b. A quantal response is an all-or-none effect,...

Critical factors in the assay of growthpromoting substances

These are the critical factors and less-critical factors Dose levels. These should be high enough to permit adequate growth of the organism so that turbidity can be measured accurately. They should not be so high as to necessitate prolonged incubation to attain linearity of the dose-response line. Variation in size of inoculum in individual tubes. This is not very serious. Provided that incubation proceeds long enough, small initial differences should not influence final cell concentration....

Calculation of potency estimate and confidence limits

In the testing of a pharmaceutical substance or dosage form for quality control purposes, the records are generally liable to scrutiny by a national regulatory authority, in which case a statistical evaluation of each assay leading to potency estimate and confidence limits will be obligatory. In that case, the only practicable way is to use a computer to do the calculation. In other circumstances, such as a research or development program, it may not be necessary to do a statistical evaluation....

General Practical Aspects of Microbiological Assays

Microbiological assay (MBA) is defined here as the estimation of potency of a growth-promoting (GPS) or growth-inhibiting substance (GIS) by comparing its quantitative effect on the growth of a specific microorganism with that of a reference standard of defined potency. It is common belief that being a biological assay, MBA is subject to biological error and is, therefore, inherently less reliable than chemical or physicochemical methods. However, according to Coomber et al. (1982), it is...

Slope Ratio Assays Some Designs and Their Evaluation

It is generally in the case of turbidimetric assays of vitamins and amino acids that the slope ratio assay is applicable. There are official assay procedures described in the USP, United States Pharmacopoeia, but not in the EP, European Pharmacopoeia. Despite their absence from monographs, the EP, European Pharmacopoeia does give guidance on the evaluation of slope ratio assays. The ideal concepts on which slope ratio assays are based are 2. Responses that are directly proportional to dose...

The Agar Diffusion Assay Its Quantitative Basis

Penicillin was discovered by Alexander Fleming in 1929. It was in 1939 that a program of research began with a view to possibly manufacture penicillin or other substances having antibacterial activity. This project was at the Sir William Dunn School of Pathology in Oxford, England and was directed by Howard Florey and Ernst Chain. Penicillin proved to be the most promising candidate substance. Norman George Heatley, a biochemist, was assigned the task of developing conditions of growth of the...

Q

FIGURE 11.5 Illustration of the variation of bias due to curvature with changing true potency ratio of unknown to reference standard in a streptomycin assay using Bacillus subtilis in a 3 + 3 assay with 2 1 dose ratio. -0.4 -0.2 0.0 0.2 0.4 Logarithm of Known Potency Ratio

Other sources of error

Apart from the general sources of error described in Chapter 6, there are some sources of error peculiar to tube assays for growth-promoting substances 1. Cleanliness of glassware is of paramount importance. Traces of adsorbed vitamin, antibiotic, or detergent may affect growth in individual tubes, leading to erratic responses. 2. Because of the very low concentration of active substance in test solutions in the case of some assays, adsorption onto glassware during preparation of the test...

Index

Absorbance, 40, 41 Absorptiometry, See Staphylococcus aureus, 38-39 Agar diffusion assays, 9-21 applicability, 7 aseptic techniques, 73, 74 description, 3-4 dose-response line, 3 experimental designs for, 183-187 historical introduction, 9-10 inoculum preparation, 65 large plate using four-dose level standard curve, 186-187 potency estimate calculation, 18-20 preliminary evaluation, 90 reference standards for, 79-80 response measurement, 75-76 substituting values, 95 two-dose level using petri...

T4

Although ideally, the tubes should be placed in the rack in accordance with a randomized pattern, this may cause operational problems that are time consuming and accident prone. Problems of positional differences are perhaps better resolved by the use of a very well-stirred water bath. Kavanagh (1972) describes a high-precision water bath used in the Autoturb system. It is claimed that temperatures in different positions in the bath do not differ by more than 0.02 C. choosing a design for...

National and regional reference materials

The effort involved in establishing a standard subsidiary to the international standard is very substantial. It entails the selection of suitable material, its packing in a form that will ensure its stability over many years, then a collaborative assay by several laboratories, followed by the statistical evaluation of the raw data and allocation of a potency in accordance with the findings of the statistical evaluation. Many countries have established national reference standards. However, the...