Serum batch test

Rapid and accurate assessment of viable cell number is an important requirement in many experimental situations involving animal cell culture, e.g. measurement of growth factor activity, serum batch testing and toxicity assays. 

Colorimetric assays that can be miniaturized into 96-well plates and measured using an ELISA reader allow many samples to be analysed rapidly, and also reduce medium and plastics costs (Cook Mitchell, 1989). The acid phosphatase assay, terminated by NaOH addition (AP NaOH assay), has been found to be particularly useful for both serum batch testing and toxicity assessment. The usefulness and limitations of the following colorimetric assays are described. 

After an appropriate growth period (e.g. 4-6 days, depending on cell line, for serum batch testing), determine the endpoint (as below). For toxicity tests, incubate cells in 100 xl of medium for 24 h prior to addition of test substance (at twice the final concentration in 100 xl of medium). Incubation with toxic substance prior to endpoint measurement is usually for 6 days. For measurement of Trypan blue exclusion, trypsinize cells with 0.5% trypsin (Gibco) and... 

Fetal calf serum (FCS) for bioreactor medium (see Note 6). Batch test before purchase (see Note 7). Inactivate the complement by heating to 56°C for 30 mm and store in 100-mL aliquots at -20°C... 

Because bovine serum (foetal, newborn, adult) remains fundamental to most tissue culture, it is still a necessity to batch test a lot prior to purchase. A reliable batch of serum is a major investment for any tissue culture laboratory. Assays based on the intended use should be in place (often there are more exacting requirements for hybridoma culture) and comparisons made between the material available from alternative suppliers. If a requirement for a particular project is the shipping of materials from one country to another, then the origin of the serum is also a consideration (e.g. US Department of Agriculture restriction on importation) (Stacey et al, 1998). 

DIA is produced by transient expression in COS cells using the method described in ref. 21. Serial dilutions of the medium are tested on ES cells plated in 24-well plates. A 100-fold higher dilution than the minimal dilution required to keep ES cells undifferentiated is typically used. Serum batches are tested for their ability to sustain the growth, differentiation, and viability of ES cells grown at clonal density in the presence and absence of DIA. 

More variability may perhaps occur in UHMWPE wear testing results due to inconsistent volumes (per station) of diluted serum lubricant used, its cleanliness, consistency of serum replacement, and regular replenishment with deionized water and the onset of deterioration, protein deposits, and calcification. Attention to those areas may be more rewarding in achieving more consistent results. For this reason, additives are typically used to attempt to overcome such problems or delay their influence until a serum batch is due to be replaced (e.g., after nearly 1 week of, say, 0.5 millions walking cycles at IHz simulator frequency). In the author s laboratory and many others, 2 g/1 of sodium azide is usually added to the lubricant solution to slow bacterial growth, and 20mM (7.45 g/1) of ethylenediami-netetraacetic acid (EDTA) is added to reduce calcification and protein precipitation. 

A control sample is a sample for which the concentrations of the test analyte is known and which is treated in an identical manner to the test samples. It should ideally be of a similar overall composition to the test samples in order to show similar physical and analytical features. For instance, if serum samples are being analysed for their glucose content, the control sample should also be serum with a known concentration of glucose. A control sample will be one of many aliquots of a larger sample, stored under suitable conditions and for which the between batch mean and standard deviation of many replicates have been determined. It may be prepared within the laboratory or purchased from an external supplier. Although values are often stated for commercially available control samples, it is essential that the mean and standard deviation are determined from replicate analyses within each particular laboratory. 

Extraneous contamination can be a problem and the use of human serum is discouraged since early batches of yellow fever vaccine became contaminated with hepatitis B virus. For similar reasons normal human diploid fibroblast strains are the preferred cells as they can be shown to be free, not only of contaminating virulent viruses but also of transforming viruses (Furesz et al 1988). Such cells have replaced primary cells but they must be regularly tested for... 

For the determination of the antiplasmodial activity of the compounds, the multi-resistant Dd2 strain of Plasmodium falciparum was used and [8-3H]-hypoxanthine incorporated into the parasitic nucleic acids measured. The plasmodia were incubated with 0.3% parasitemia and an erythrocyte hematocrit of 2.5% in the presence of different concentrations of selected experimental agents in a final volume of 200 xl. The medium employed was RPMI 1640 containing 10% of heat-treated human serum and 3 mg/1 of gentamycin. In the incubations, the concentrations of the compounds varied from 0.3 to 100 xM. After 48 hours, each batch was treated with 50 xl [8-3H]hypoxanthine and incubated additional 18 hours. Testing results are provided in Table 1. 

The classical column procedures for the preparation of lysozyme are difficult to use with high viscosity solutions such as serum. A batch method was developed based on affinity chromatography using deami-nated chitin [82]. The method was found to give a one-step purification of nearly theoretical amounts in tissue homogenates tested. These included tissue homogenates from humans, primates, avian egg white and plants. The use of a chitin-coated cellulose affinity column for purification of lysozyme has also been described [83]. 

The authors performed RAST inhibition to measure the relative potencies of the different venom extracts using the patient s serum as a source of IgE anti-venom. Although they initially suspected the new source of Polistes wasp venom, the relative potency tests showed greater variation in the honeybee venom. The IgG concentrations were consistent with this finding. This report emphasizes the care that must be taken with the preparation of each injection, especially when using a new batch of antigen. 

The application of quality control procedures to ensure that satisfactory analytical performance of enzyme assays is maintained on a day-to-day basis is complicated by the tendency of enzyme preparations to undergo denaturation with loss of activity. This maltes it difficult to distinguish between poor analytical performance and denaturation as possible causes of a low result obtained for a control sample introduced into a batch of analyses. Assured stability within a defined usable time span is therefore the prime requirement for enzyme control materials, as it is for enzyme calibrators. However, specifications for the two types of materials can differ in other respects. Because the function of a calibrator is to provide a stated activity under defined assay conditions, it is not necessary for it to show sensitivity to changes in the assay system identical to those of the samples under test therefore within certain Umits, enzymes from various sources can be considered in the search for stability. However, it is the function of a control to reveal small variations in reaction conditions, so it must mimic the samples being analyzed. The preparation of enzymes from human sources is not by itself a guarantee of an effective control. For example, human placental ALP is very stable, but it differs significantly in kinetic properties from the liver and bone enzymes that contribute most of the ALP activity of human serum samples it is therefore not an ideal enzyme for use in control material for the determination of ALP. 

The older RIA kit procedures required about 1 hour incubation either at room temperature or at 37 °C. Overall, a batch of 20 patient specimens, 2 or 3 controls, and 6 calibrators (each in duplicate) required less than 3 hours to process. Sample volumes per test were small (about 25 iL). Interassay reproducibility, expressed as a coefficient of variation, was typically 7% at a T4 concentration of 3.8 Xg/dL (50 nmol/L) cross-reactivity of T3 was about 5% at the 50% displacement concenti ation. The detection limit of these assays (defined as mean minus 2 standard deviations [SD] of the zero cahbrator) was approximately 0.4 fig/dL (5 nmol/L). This limit of detection was not sufficient to measure T4 dhectly in dialysates or ultrafiltrates of serum. Highly sensitive T, RlAs that have a detection limit of 0.8 pg per tube or 0.16 ng/dL of dialysate were eventually developed. 

The conditions of cell culture are critical for chemical testing. Particular attention must be given to the medium used for the cultivation of cells. While the use of serum, usually foetal bovine serum (FBS), has long been customary to provide isolated cells with a pool of nutrients, attachment factors, and hormones, several issues are encouraging scientists to move towards serum-free hormonally defined medium [53]. In particular, the composition of FBS varies from batch to batch, rendering highly discriminative analyses such... 

Suppliers of materials and reagents should be expected to operate according to standards appropriate to their supply and use, and to provide relevant quality control documentation. The developer has the responsibility to confirm that critical materials detailed in the SOP(s) are suitable for their intended purposes. Furthermore, it is important to monitor different batches of materials with regard to changes or variations, which may affect their use. For certain reagents, such as serum, testing prior to use may be necessary. 

Elements in an immunoassay that could impact its robustness include incubation temperatures, light exposure (enzyme-linked immunosorbent assay, ELISA), and different lots of matrix (plasma, serum CSF). The ruggedness of the analytical method can be tested by implementing changes to the analysts, different instruments, batch size, and the day, time, or other environment factors otherwise should not greatly impact the consistency of the assay. 

If available, a potency assay developed for the batch release of the biological is a good starting point because it can often be adapted for use as a Nab bioassay. For example, a neutralization assay for GM-CSF can be a modification of the proliferation assay used for potency assessment for GM-CSF. This approach is advantageous, as optimization of cell line maintenance and culture conditions had already been performed. However, this assay needs to be tailored for use as a neutralization assay, as it may not be adequately sensitive for the purpose in its original format. Therefore, the performance of the potency assay in the presence of test sample (appropriate species serum) and response to the therapeutic to yield a sufficiently sensitive assay requires validation. Ideally, the cell line should yield a functional end point upon treatment with the therapeutic protein, the assay should be simple to perform, and the biological end point should be tolerant of the test sample matrix and perform adequately over a range of concentrations. 

The use of serum and other undefined and complex media additives for the growth of mammalian cells may result in problems of reproducibility in the process. To avoid batches with negative impact on cells, testing procedures must be incorpo-... 

The German "Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area, Working Group Analytical Chemistry" has developed a continuous nebulization FAAS method for copper in serum based on a 1 -r- 1 dilution (dilution depends on the applied burner system) with a determination limit of 0.1 mg/L At a mean copper concentration of 1.24 mg/L the within batch precision was 2.4% and the day-to-day precision 3.2% (Winter and Schaller, 1985). Accuracy was checked against certified reference materials. The method was applied on fortified samples from a serum pool of healthy persons and compared by a GF/ AS method developed and tested at the same time (Angerer et al., 1985). The FAAS method is still in successful routine use in a number of German laboratories because of its reliability, simplicity and speed (Schaller, 1993). 

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