2-Fold dilutions

In the third test, the activity was titrated using serial 2-fold dilutions. All four levels were fully active, and this time there was little sign of toxicity. A few cultures had been confirmed active previously, but none had exhibited a separation of toxicity from activity, let alone activity over at least an 8-fold range. After an Inauspicious start, the activity of culture OS-3153 had been firmly established. Success had been achieved in the quest for a fermentation product with anthelmintic activity. In fact, as events would soon prove, the newly found product had an even broader activity than had been anticipated. 

The calculations are done in whichever transformation is necessary to allow parallel straight lines to be fitted. The estimation of potency from the vertical distance between the line requires also an index of slope for the portion of the curve being employed, which is given by the difference between the high and low responses of the two lines, divided by the log distance between the dilutions tested. This distance is the log of the dilution factor when serial dilutions are employed, e.g., log 2 for a series of 2-fold dilutions, log 4 for a 4-fold series. The estimate of potency is obtained from the ratio of the difference between patient and control readings to the slope of the lines (see below). 

In a one-stage factor VIII assay employing serial 2-fold dilutions the following clotting times were obtained. 

If 2-fold dilutions are used, the quantities Xp, x , [ip], and may be obtained from Table 3. The products [xy] are calculated as Sxy — (SxSy/n), where Sxy indicates the sum of the products of each log plasma dilution with its corresponding clotting time in working units, and SxSy/n is the correction for the mean and is the product of the sum of the log dilutions (from Table 3) and the sum of the clotting times n is the number of plasma dilutions tested. 

Quantities Requibed in the Calculation of Potency Ratios fbom Clotting-Factob Assays Based on Single Readings ON Dipfebing Numbebs op 2-Fold Dilutions of Patient and Control Plasmas ... 

Figure 1. Immunoblot analysis of various acyl-CoA dehydrogenases in rat heart mitochondria. Heart mitochondria were isolated from ctMttrol and VPA-treated rats. Serial 2-fold dilutions of mitochondrial suspmsion (2.0, 1.0, 0.5, 0.25 and 0.125 tig protein) were subjected to immunoblot analysis.

Figure 3. Slot-blot analysis of inRNAs for SCAD, IVD and p-attin from VPA-treated rat liver and heart, RNA was prepared from the livers and hearts of 10 control and 10 VPA-treated rats, respectively. Serial 2-fold dilution of total RNA (4,2.1 pg) were hybridized to the individual cDNA probes.

Figure 4. Tissue specific changes in the amount of mRNAs for various acyl-CoA dehydrogenases caused by VPA administration. Adult Wistai rats were divided into two groups of ten rats (Control and VPA-treated). Total RNA was prepared from liver, psoas muscle, heart, kidney and small intestine. Serial 2-fold dilutions of total RNA (4,2,1 ng) were blotted onto a nylon membrane and hybridized with the radiolabeled cDNA probes for various acyl-CoA dehydrogenases. The results on the amount of each mRNA were quantitatively analyzed using a densitometer, and expressed as a shift from the control level in percentage. Each value represents the mean of three experiments.

The odor threshold values were determined in aqueous solutions by a published method (12) using polyethylene bottles. The concentration was reduced by 2-fold dilutions, and the samples and a blank (water) were presented to the subjects. The threshold concentration was the lowest concentration at which more than 50% of the subjects could discriminate the odor between the sample being tested and the blank. The odor thresholds determined were (I) 40 ppb and (U) 3.5 ppb, as shown in Table III. The volatiles from the fruit obtained by steam distillation included I and n in concentrations of approximately 21 ppm and 840 ppm, respectively. Since each amount exceeded the threshold concentration by a large extent, it is clear that both of them significantly contributed to the smell of the fruit. 

The agglutinating activity was assayed by using horse or mouse erythrocytes in microtiter plates. In some experiments, human erythrocytes was also used. 25 pi of 2.5 % (v/v) suspension of erythrocytes in 6.4 mM phosphate buffer saline, pH 7.2 (PBS) was added to 50 pi of serial 2-fold dilutions of the lectin fractions in PBS. The plates were incubated at room temperature for 1 hr. The results were expressed as the minimum concentration of lectin fractions (pg/ml). The agglutination inhibition was expressed as the minimum concentration of each sugar required for inhibition of the lectin fractions. 

---