Endotoxin concentration

Where the test is conducted as a limit test, the specimen is determined to be positive or negative to the test judged against the endotoxin concentration specified in the individual monograph. Where the test is conducted as an assay of the concentration of endotoxin, with calculation of confidence limits of the result obtained, the specimen is judged to comply with the requirements if the result does not exceed (1) the concentration limit specified in the individual monograph and (2) the specified confidence limits for the assay. In either case the determination of the reaction endpoint is made with parallel dilutions of redefined endotoxin units. 

Prevalence of byssinosis correlates better with airborne endotoxin concentration than with total dust (65). Also, gramnegative bacteria levels in the mill correlate well with disease (66). It has been hypothesized that endotoxins elicit symptoms of byssinosis by activation of both the classical and the alternative pathway of complement with subsequent release of anaphylatoxins, which lead to airway narrowing, and chemotaxins, which cause the influx of PMNs followed by release of lysosomal enzymes and, ultimately, tissue damage. In experiments with guinea pigs using bract, cotton, and gin mill trash extracts, there is a strong correlation between number of PMNs recruited to airways and level of endotoxin (67). 

On completion of intermediate purification, most contaminants are removed. For example, endotoxin concentrations are reduced by more than two orders of magnitude, while the specific activity of recombinant protein, a measure of purity, is increased several-fold. The purified material emerging from this stage of downstream processing is composed of recombinant... 

The purpose is to establish a standard test method for endotoxin concentration monitoring of in-process or finished product by LAL gel formation method. 

The determination of the endotoxin with limulus amebocyte lysate (LAL) is based on gel formation of a mixture consisting of a solution of endotoxin of Gram-negative bacteria with a solution of lysate. The extent and speed of the reaction depend on the endotoxin concentration, pLL, and temperature. The reaction requires the presence of certain cations, a proclotting enzymes system, and clottable protein, which are produced by lysate. 

Label standards with endotoxin concentration in EU/ml, initials, and preparation date. 

For semiquantitative gelation method, the geometric mean of the endotoxin concentration = antilog (Xe//), where Xe is the sum of the log endpoint concentrations of the dilution series used, and/ is the number of replicate test tubes. 

To determine the endotoxin concentration of solution A, calculate the endpoint concentration for each replicate series of dilution factor by X. The endotoxin concentration in the sample is the geometric mean of the endotoxin concentration of the replicates. 

The preparation being examined passes the test if the endotoxin concentration of each of all tested samples is less than the endotoxin limit concentration specified in the specification and the test meet the following three conditions ... 

If the endotoxin concentration of one of the two solutions is lower and the other one is higher than this limit, repeat the test. 

The polymyxins are cationic, basic, and amphipathic poljtpeptides that interact with lipopolysaccharides in the outer membrane of bacilli. They potently neutralize endotoxin, reduce blood endotoxin concentrations in patients with septic shock during direct hemoperfusion over immobilized poljmyxin B fibers, and are bactericidal for many Gram-negative rods, even in resting bacteria. Alteration of the cell wall is also thought to be the mechanism of damage to renal epithelia and to the nervous system. 

Figure 7. Standard curve of gel purified 60 kd protein endotoxin of Btk was generated using a double antibody sandwich ELISA. The arrows indicate dilutions of two Btk formulations absorbance values were used to determine the endotoxin concentrations of the formulations, based on the standard curve. The formulation dilutions gave curves that were virtually superimposable on the standard curve. Such similarity in shape and slope indicate that the antibody is likely binding to a specific determinant common to the purified Btk and the Btk in the formulation. In general immunoassays for biopolymers are much easier to develop than assays for small molecules. However, only recently has an interest in trace analysis of such materials begun to develop in the environmental field. Thus, sample cleanup and handling is not as sophisticated as with small molecules.

Filter Area 3.9 cm2 Endotoxin Concentration Before Filtration pg/ml Endotoxin Concentration in Filtrate pg/ml ... 

With turbidimetric end-point assays, turbidity is measured after a fixed incubation period. By inclusion of standards alongside dilutions of the material under test, a standard curve can be created and the endotoxin concentration can be read off this curve for the material under test. Equipment using microtiter plates is commercially available to minimize the amounts of LAL reagent necessary and to maximize the number of test sets per set of standards. The period of incubation over which turbidimetric end-point assays is conducted is critical. All samples will be equally turbid with overlong incubation if incubation times are too short none of the samples will be measurably turbid. It is not possible to stop the reaction to take the readings. 

Magneto elastic sensors have also been used for the detection of endotoxin, which can cause sepsis in immunocompromised patients. The system was based on the use of magnetoelastic sensors to monitor gel formation (clot formation), which changes the viscosity of the limulus amoebocyte lysate (LAL) assay in response to endotoxin. By correlating different endotoxin concentration and the LAL assay clot rate, it was found that the maximum clot rate was directly proportional to the endotoxin concentration. Endotoxin concentrations as low as 0.0105 EU/mL have been detected in approximately 20 min (Ong et al. 2006). 

Many diseases in many industries have been linked to inhalation of endotoxin. Some are shown in Table 7.3. A chronic respiratory disease of textile workers called byssinosis has recently been linked to exposure to endotoxin in dirty cotton. Castallen et al. " and Kennedy et al. have shown linear relationships between airborne endotoxin concentration and decrease in lung function. However, there was no relationship between decrease in lung function and the dust levels in the cotton works. The organism most commonly found in cotton and linked for these symptoms is Enterobacter agglomerans. 

For proteases (detergent enzymes) voluntary exposure limits of 0.4 (xg/ m have been set." By comparison, the limits on an 8-hour personal exposure to total inhalable dusts is 10 mg/m, i.e. 25 000 times higher than protease and 100 000 higher than the endotoxin concentration." ... 

Allermann, L. Poulsen, O.M. 2000. Inflammatory potential of dust from waste handling facilities measured as IL-8 Secretion from lung epithelial cells in vitro. Annals of Occupational Hygiene 44(4) 259-269. Berndt, A., Derksen, F.J. Robinson, N.E. 2010. Endotoxin concentrations within the breathing zone of horses are higher in stables than on pasture. The Veterinary Journal 183 54-57. 

In a subsequent study on fiber grown in the same three areas, Olenchock et al. (1985) found that the California-grown cottons generated the lowest endotoxin contamination of the air they also reported that decreases in acute pulmonary function during exposure paralleled endotoxin concentrations. 

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