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Development of predictive in vitro surrogate methods for traditional approaches assessing bioavailability and pharmacokinetics of lead compounds must be made to both keep pace with highthroughput (HT) lead identification and to mitigate the high costs associated with progression of compounds with poor chances of developmental success. Indeed opportunities for improvement still exist in the lead optimization phase versus the lead identification phase, where HT methodologies have been nearly optimized. Review of examples, limitations, and development of high-throughput microtiterplate-based assays for evaluating metabolic liabilities, such as in vitro radiometric and fluorometric assays for inhibition of cytochrome P450 (CYP) activity, determination of stability of a compound in liver microsomes, or cloned CYPs coupled to reconstituting systems are described. Parallel approaches to improve speed, resolution, sample preparation, as well as data analysis using LC/MS and LC/MS/MS approaches and technologies to assess compound integrity and biotransformation by automation and multiplexing are also discussed. Realization of the benefits in automation of cell-based models for determining drug permeability to predict drug absorption are still hampered by bottlenecks in analytical analysis of compounds. The implementation and limitations of surrogate physiochemical methods for passive adsorption such as immobilized artificial membranes (IAM) and parallel artificial membrane permeation assays (PAMPA), and compound solubility by laser nephelometry are reviewed as well. Additionally, data from a high-throughput 96-well equilibrium dialysis device, showing good correlation to classical methods, is presented. Finally, the impact of improvements in these downstream bottlenecks in lead optimization and preclinical drug discovery are discussed in this review.

The determination of aqueous solubility in a high-throughput screening environment is invaluable in the selection of the most promising potential drug candidates. We describe a fast method based on laser nephelometry that can determine the solubility of potential drug candidates (usually from combinatorial chemistry) supplied as dimethyl sulfoxide (DMSO) solutions in 96-well plates. In the sample, the percent DMSO is kept constant allowing direct comparison of results. The nephelometric method has been shown to produce results equivalent to those produced by an HPLC method and to be largely unaffected by colored solutions.

International audience; By contrast with photometry (i.e., the measurement of light transmitted through a particle suspension), nephelometry is a direct method of measuring light scattered by particles in suspension. Since the scattered light intensity is directly proportional to the suspended particle concentration, nephelometry is a promising method for recording microbial growth and especially for studying filamentous fungi, which cannot be efficiently investigated through spectrophotometric assays. We describe herein for the first time a filamentous fungi-tailored procedure based on microscale liquid cultivation and automated nephelometric recording of growth, followed by extraction of relevant variables (lag time and growth rate) from the obtained growth curves. This microplate reader technique is applicable for the evaluation of antifungal activity and for large-scale phenotypic profiling.

The authors have tested two formulas by means of which the filtrated and the intraocularly (intrathecally) synthesized fraction of particular protein can be distinguished. The laser nephelometric method was applied to determine the serum (IgG, IgA, IgM, C3, albumin), aqueous humor (IgA, IgM, albumin) and CSF (IgG, IgM, C3, albumin) protein values in 42 subjects to test two models in physiological conditions. It was confirmed that the best results were achieved using the formula after Stambuk. Computer simulation performed to compare the formulas after Stambuk and Reiber & Felgenhauer showed a strong correlation (r greater than 0.98) between the calculated filtrated fractions of both models in a wide range of different barrier permeabilities. Laser nephelometry proved to be a useful method to determine low IgA and IgM values in aqueous humor.

Summary: Concentrations of immunoglobuli ns were determined by laser nephelometry and, for comparison, by single radial immunodiffusion assay and a sandwich enzyme-linked immunosorbent assay (ELISA) in plasma samples of normal dogs. Correlation between methods was high, although the mean IgM concentrations determined by laser nephelometry were significantly higher than those determined by the single radial immunodirrusion assay or ELISA. In contrast, the mean IgA concentrations were significantly lower than those determined by ELISA. Laser nephelometry was the most reproducible method. The between-assay coefficient of variation was 4.12% for IgG, 6.98% for IgM and 6.35% for IgA. Laser nephelometry and ELISA showed similar accuracies, and both were more accurate than single radial immunodirrusion. Finally, laser nephelometry was much more sensitive than single radial immunodifrusion, but less sensitive than the ELISA method. In conclusion, results of the three methods were in general comparable. The higher range of linearity, precision and accuracy of laser nephelometry, and the availability of automated nephelometers make this the method of choice for quantifying canine plasma immunoglobulins.

The effects of detergents on antigens and allergens of birch and timothy grass pollen extracts, codfish, hen egg-white, cat dander and house dust mite were investigated by crossed immunoelectrophoresis (CIE), crossed radio-immunoelectrophoresis (CRIE), radio-allergosorbent test (RAST) inhibition and quantitative precipitation test using laser nephelometry. Nine household cleaning solutions and five chemical detergents were tested. Higher concentrations than recommended for regular cleaning purposes were used as no influence on the antigenic and allergenic activities could be detected using the usual concentrations. Soft soap, guanidine hydrochloride and sodium lauryl sulphate induced most modifications of the antigens and allergens tested. None of the detergents totally destroyed the antigenic and allergenic activities of the selected material, even when used in concentrations up to 10 times that recommended. The materials used for carpet cleaning would not be able to impose any denaturing effects on allergens left on carpets after cleaning.

The quantitative determination of glycoproteins and lectins by laser nephelometry is based on the fact that these two compounds form a complex comparable to the antigen-antibody precipitate. Due to the high sensitivity and reliability of this technique less than 25 pmol of glycoprotein or 4.5 pmol of lectin can be determined, thus providing a suitable method (a) to investigate the specificity of different lectins for certain carbohydrate residues, (b) to study the mode of interaction, and (c) to explore mammalian tissue for still-unknown lectin-like substances.

A nephelometric procedure for quantitative measurement of glycohemoglobin (Hb A1) was developed, evaluated, and compared with the semi-quantitative mini-column chromatographic procedure. Hb A1 was purified from human red cell hemolystate by Bio-Rex 70 ion-exchange liquid chromatography and was used for standards and immunization. The antisera raised in rabbits showed high cross-reactivities with normal human hemoglobin (Hb A). The latter was separated by mixing 25 μl of patients' hemolystate with 2 ml ion-exchange resin suspension for 15 minutes. One hundred microiters of the supernatant was incubated with 900 μl antiserum (dilution 1:50) for 45 minutes at room temperature. Samples were then read on a laser nephelometer. The sensitivity of the assay was found to be 0.1 mg Hb A1. The intraassay relative standard deviation (RSD) was 5.6% and the interassay RSD was 6.5%.

An adaptation of the nephelometric assay for serum immunoglobulins has been developed for detection and quantitation of extracellular immunoglobulins in cultures of lymphoblastoid cell lines. This assay employs the standard equipment for laser nephelometry and commercial reagents for immunoglobulin quantitation. By adjusting dilutions of controls and sample volumes of culture supernatants, amounts of IgG and IgM below 1 μg/ml can be detected in culture supernatants. At concentrations between 1 and 4 μg/ml, day-to-day and within-run variations for IgM assays were 16 and 11% respectively. The possibility of measuring immunoglobulins secreted by cell lines by conventional laser nephelometry opens several areas of application in the study of the functional activity of B cells and of cell-cell interactions.

A simple technique using high performance liquid gel permeation chromatography for determination of IgG in cerebrolspinal fluid (CSF) is described. Accurate determination of IgG was achieved by precolumn derivation with fluorescein isothiocyanate-conjugated anti-human IgG Fab, a substance that readily yields a fluorescent immune complex. Immune complexes separated as a well-delineated peak in the column void volume, and were measured by the fluorescence of the column eluates (Ex = 490 nm, Em = 520 nm. The results were reproducible with a deviation of less than 2% and a good linear relationship over the range of 2.0–24.5 mg/dl. The IgG concentration assayed by the column method correlated well with values obtained by laser nephelometry.

After quantitating immunoglobulins G, A, and M and complement C3c and C4 in serum by using a laser nephelometer coupled with a data processor, I compared these results with values obtained by an early-readout radial immunodiffusion method. Day-to-day precision was better for nephelometry than for radial immunodiffusion for all proteins analyzed. The average coefficient of variation was 6.0% for nephelometry and 9.9% for radial immunodiffusion. Comparison of these methods gave ranked correlation coefficients of 0.945, 0.981, 0.932, 0.803, and 0.792 for IgG, IgA, IgM, C3c, and C4, respectively. Nephelometry gave significantly higher values than radial immunodiffusion for IgG, IgA, IgM, and C3c, and significantly lower values for C4 (p less than 0.001). Part of this bias was found to be due to the equation programmed in the data processor for calculating the standard curves. Within 95% limits, nephelometry gave higher normal ranges than radial immunodiffusion for IgG, IgA, and IgM. Other possible factors that can produce this bias are discussed.

A laser nephelometric procedure to quantitate fibrinogen related antigens (FRA), that joined the best characteristics of the immunological available techniques, is proposed. It is sensitive, rapid, simple, reproducible, antiserum saving and accurate since it correlates very well with the tanned red cell hemagglutination inhibition immunoassay (TRCHII) at the most commonly found clinical levels (r = 0.98, p less than 0.0005), but with the advantage of a more objective quantitation and thus, reliable for any concentration of FRA. Prepared samples of different known levels of fibrinogen, assayed with the laser method and the TRCHII, showed a higher correlation with the laser procedure (r = 0.97, p less than 0.0005) than the obtained with the TRCHII (r = 0.85, p less than 0.001). The technic is useful in emergency situations, routine work and research.

SummaryLaser nephelometry is a technique which allows the evaluation of the concentration of several serum proteins and clotting factors. By means of this technique it is also possible to study the kinetics of the reaction between antigen and antibody. We studied the kinetics of the reaction between prothrombin and an antiprothrombin antiserum using several prothrombins namely: Prothrombin Padua, prothrombin Molise, which are two congenital dysprothrombinemias, cirrhotic, coumarin or normal prothrombins. Different behaviors in the kinetics of the reactions were shown even when the concentration of prothrombins was about the same in all plasma tested. These differences were analyzed by means of a computer (Apple II 48 RAM) programmed to solve four unknown equations (Rodbard’s equation). From the data so obtained one can see that when voltages at the beginning and at the end of the reaction are in all cases about the same, a clear difference in the time required to reach half the maximum value of the voltage can still be demonstrated. This parameter, which is expressed in minutes, is longer in coumarin and prothrombin Molise than in controls. On the contrary it is shorter in prothrombin Padua and has about the same value of controls in the cirrhotic patient. Moreover the time at which the maximum rate is obtained is longer in coumarin and prothrombin Molise than in controls and shorter in liver cirrhosis and prothrombin Padua. In conclusion data obtained show that coumarin prothrombin behaves in a different way from cirrhotic prothrombin and also that there is a different behaviour between the two congenital dysprothrombinemias.

The Hyland laser nephelometer PDQ system for the assay of apolipoprotein B (apo-B) in human serum is described. Within and between-batch precision, accuracy and reliability are discussed. This instrument represents an important development in the immunochemical assay of apo-B, and the speed, precision, and convenience of the methodology make such a system attractive. Quantitation of apo-B was assessed in normal and hyperlipaemic subjects. Comparisons were made with two other specific and sensitive immunological methods for quantifying apo-B: enzymeimmunoassay (EIA) and rocket immunoelectrophoresis (RIE). Results obtained by the three methods correlated very well.

Immunoglobulins G, A, and M were measured by radial immunodiffusion and by laser nephelometry in sera from 33 patients and in a preparation of purified colostral IgA. In general the results agreed for the two methods, with some exceptions attributed to the physicochemical characteristics of the immunoglobulins assayed: low-molecular-weight (monomeric) IgM gives falsely high values and IgA proteins (consisting predominantly of polymeric molecules) give falsely low values when assayed by radial immunodiffusion. Results from laser nephelometry apparently are not affected by the molecular-size characteristics of the proteins assayed.

Measurement of immunoglobulins (Ig) and of complement component C3 in human serum by automated and manual nephelometric techniques is tedious, and the effective linear range is too narrow. We describe a laser nephelometer/reagent system for measuring serum proteins by the use of forward light scatter (which enhances the ratio of reaction/blank), electronic blank subtraction, laser light (632 nm), and electronic signal selection. We report data establishing the range of linearity for immunoglobulins IgG, IgA, IgM, and complement C3 with this system, and correlations with results by a radial immunodiffusion. We also compared an electroimmunodiffusion system for quantitation of albumin and IgG in cerebrospinal fluid to our technique. The nephelometric system described provides a rapid, accurate, precise, and objective way to measure immunoglobulins and C3.

This rapid, reliable equilibrium turbidimetric immunoassay for serum C-reactive protein involves a potent monospecific antibody. Polyethylene glycol-6000 to accelerate and enhance the immunoprecipitation reaction, and Tween-20 surfactant to lower and stabilize the sample blank values. Grossly lipemic, icteric, or hemolyzed sera can be assayed. Values up to about 220 mg/L, for which the standard curve is linear, can be measured without sample dilution. Results by the proposed method and by radial immunodiffusion (r 0.989) or laser nephelometry (r = 0.957) correlated well. Analytical recovery averaged 101.3%. Within-, between-, and day-to-day CVs ranged from 0.9% to 3.5%, 0.8% to 5.5%, and 1.9% to 4.8%, respectively. The method is demonstrably superior to radial immunodiffusion or nephelometry. Any spectrophotometer that can measure turbidimetrically at 340 nm can be used.

Four methods (radioimmunoassay, electroimmunoassay, laser nephelometry and kinetic immunoturbidimetry) have been evaluated for the measurement of proteins with pregnancy-specific β 1 glycoprotein immunoreactivity. The influence of two maternal plasma proteins with different electrophoretic mobilities (α 2 and β 1 ) but with similar antigenic determinants has been assessed in each assay system. The radioimmunoassay method, which utilises a homogeneous preparation of pregnancy-specific β 1 glycoprotein for the preparation of iodinated tracer, and the kinetic immunoturbidimetric assay which measures antigen-antibody complex formation over the first minute of the reaction, were both considerably more specific for the β 1 component of pregnancy specific β 1 glycoprotein immunoreactivity than the laser nephelometric or electroimmunoassay methods.

Factor VIIIRAg was measured by laser nephelometry using a new diluent containing a citrate-carbonate buffer, pluronic polyol P94 and triton X100. No pre-treatment of the samples was required, the incubation time was 0.5 to 1.5 hours depending on the antiserum used, and the tests could be read during the following 4 hours (plasma) or 1.5 hours (intermediate purity F VIII). The CV for the assay was less than 10% for plasma or dilutions of plasma in which the minimum detectable amount of factor VIIIRAg was about 0.04 u/ml. A significant correlation was obtained between the nephelometric method and the Laurell rocket method using plasma, but agreement between the methods when therapeutic products were assayed was poor.

Four immunochemical methods for measuring C-reactive protein (CRP) in plasma were compared: radial immunodiffusion (RID), electroimmunoassay (EIA), immunoturbidimetry (IT), and laser nephelometry (LN). Close agreement was found between RID and EIA (EIA = 0.96 RID + 7.7 mg/L, r = 0.977, n = 100), IT and EIA (EIA = 1.11 IT - 15.8 mg/L, r = 0.951, n = 100), IT and RID (RID = 1.10 IT - 11.5 mg/L r = 0.959, n = 94). In initial studies in 1983, LN showed good agreement with RID (LN = 0.98 RID + 11.9 mg/L, r = 0.973, n = 60). Since then, normal CRP values measured by LN have tended to increase and, at the time of the present study (1986), LN agreed poorly with other methods. The reason for this change is obscure. Having excluded the possibility of enzymatic degradation of CRP, we conclude that instrumentation problems are involved, and therefore we no longer use this method.

We evaluated nephelometers from Behring, Hyland, and Beckman for IgG, IgA, and IgM quantitation in sera from patients with monoclonal gammopathies. The intra-batch precision of each instrument for each immunoglobulin class and for different concentrations of the same immunoglobulin was compared to the one obtained with the radial immunodiffusion method. No nephelometer showed a clearly better precision. The correlation with cellulose acetate electrophoresis was good for each of the three nephelometers. The mean value by the radial immunodiffusion method was higher than corresponding determinations by nephelometry.

Use of the newly developed laser nephclometer provides a sensitive immunochemical assay for Iysozyme in dog sera by detection of antigen-antibody complexes formed upon cross-reaction with sheep-anti-human Iysozyme. Results of lysoplate and nephelometric assays correlate well.

Laser nephelometry is a sensitive technique for measuring the degree of light scatter caused by the interaction of rheumatoid factors with heat aggregated human IgG. In measuring the rheumatoid factor (RF) level in rheumatoid serum and synovial fluid, it was found that nephelometry measured the combined amounts of IgM-RF and IgG-RF. However, on an equivalent weight basis, IgM-RF produced at least 17 times more scatter than IgG-RF. Low molecular weight IgM-RF had minimal light scattering activity in this assay. The total serum RF level, measured by nephelometry and expressed in terms of microgram/ml of IgM-RF equivalents, correlated strongly with IgM-RF measured by solid phase radio-immunoassay (r = 0.97, n = 30, P less than 0.001) and an equation was derived to equate the two variables. It is concluded that the nephelometric RF test is a simple and rapid method to measure the total polyclonal RF level in various biological fluids, although monoclonal or an occasional polyclonal preparation of rheumatoid factor may give atypical results.

In twenty-five ovine body fluids (serum, lung fluid and cerebro-spinal fluid), the concentrations of IgG1, IgG2, IgM and IgA were determined by laser nephelometry and radial immunodiffusion (RID). When nephelometric assays are carried out, antisera free from any turbidity are essential. Methods ensuring that goat and rabbit antisera will satisfy this requirement are described in the present paper. When sheep immunoglobulins were measured by laser nephelometry, adequate and reproducible results were obtained, comparable with those obtained by RID. Advantages of the nephelometric method include the speed of assay and its sensitivity, allowing precise determination of the very low concentrations of immunoglobulins in CSF.

The Laser-nephelometric technique is characterized by its easy handling, accuracy, good reproducibility and especially by the short time in which reliable results are available. A very good correlation was found between the results obtained by the electroimmunoassay and those obtained by Laser-nephelometry. In both techniques alpha2-macroglobulin is the largest serum protein that can be determined in normal unconcentrated cerebrospinal fluid (CSF). This is of diagnostic significance, since the serum/CSF ratios of both albumin and alpha2-macroglobulin are used as a parameter for the blood-CSF barrier condition.

Using a commercial source of peroxidase-labelled anti-C3d antibody (Dakopatts), an enzyme-linked immunosorbent assay (ELISA) has been developed to quantify the complement fragment C3d. The technique enables the detection of C3d in plasma, urine and cerebrospinal fluid (CSF). The C3d-ELISA therefore provides a very sensitive technique for the evaluation of complement activation in biological fluids. In both plasma and urine the technique is able to discriminate between samples from normal controls and patients with rheumatoid arthritis in whom complement activation is known to occur. A good correlation was found between results obtained by ELISA and those by laser nephelometry (r = 0.91, P less than 0.0001). Microtitre plates pre-coated with anti-C3d antibody and subsequently stored at -70 degrees C retained the ability to perform in this assay. The sensitivity, short assay time and use of commercial reagents and pre-coated plates give this technique numerous potential applications in the evaluation of complement activation.

Quantitative determination of proteins by immunological methods began with the quantitative precipitin technique of Heidelberger and Kendall in 1935’.

Transferrin determination was performed in 100 native human sera by RID, laser nephelometry and kinetic turbidimetry; the serum iron-saturation ranged between 0.03 and 1.00. No statistical difference could be observed between 3 immunochemical methods.

Antithrombin-III (AT-III) concentration in normal healthy volunteers and in an out-patient population was determined by a radial immunodiffusion (RID) and by a laser nephelometric technique, developed in our laboratory. In 43 normal individuals tested, the mean AT-III concentration by laser nephelometry was 24.1 ± 3.3 mg/dL or 108 ± 13% normal human plasma pool (NHP). The coefficient of variation (CV) of the assay was in the order of 7–10% and there was a good correlation (r=0.81) between RID and laser nephelometric method. Some developmental aspects of the laser nephelometric procedure and its advantages over the RID method are discussed.

A total of 1,002 urine specimens were evaluated by laser nephelometry. This technique was compared with both colony counts, done with a calibrated loop, and serial dilutions. For urine specimens containing between 10(4) and 10(5) bacteria per ml, laser nephelometry detected 75.4% of those detected by colony count and 65.6% of those detected by serial dilution. For specimens where the concentration of bacteria was greater than 10(5) per ml, laser nephelometry detected 95.8 and 92.4% of those detected by colony count and serial dilution, respectively. The mean detection time for bacteriuria varied from 1.57 h for more than 10(5) bacteria per ml to 4.47 h for more than 10(4) bacteria per ml. To determine the number of bacteria according to the voltage growth curve, the passage time at 3 V was used as an index. The mean passage time at 3 V decreased from 5.18 h for fewer than 10(4) bacteria per ml to 1.42 h for more than 10(6) bacteria per ml. The mean passage time at 3 V differed significantly for different concentrations of bacteria. Thus, this index allowed us to predict the number of bacteria in the urine specimens. Laser nephelometry has been used for many years for the immunological determination of proteins; it can now also be considered a tool for rapid screening in bacteriology.

It was noted while clarifying diluted sera for protein estimations by laser nephelometry that certain types of microfilters absorbed immunoglobulin G (IgG). A range of 10 microfilters (0.45 μ pore size) were evaluated for their ability to filter diluted sera (1:100 in saline) without removing IgG or albumin from the solutions. It was found that none of the filters removed albumin. However, most filters containing cellulose nitrate removed between 9–49% of the IgG from diluted sera. The details of this study are presented. The results from this study suggest that microfilters should be selected not only on their ability to remove particles of a nominated size but also on the ability of the membrane to be non-absorbent towards the material being filtered.