Laser nephelometry and radial immunodiffusion compared for immunoglobulin quantification in pathological sera

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.     

Comparison of radial immunodiffusion and laser nephelometry for quantitating some serum proteins

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.     

Comparison of nephelometry and immunofluorescence for immunoglobulin quantitation in pathologic sera

Nephelometers from Beckman, Hyland and Technicon, and the immunofluorescent system from International Diagnostic Technology were evaluated for IgG, IgM, and IgA quantitation in pathologic sera. Within-run, between-run, and between-day imprecision of each instrument varied for each immunoglobulin and for different levels of the same immunoglobulin. Thus, no instrument showed clearly superior precision. However, for elevated immunoglobulin levels, Technicon had poorer between-day precision than the other instruments (p < 0.05). Comparison of monoclonal immunoglobulin quantitation with monoclonal protein quantitation by serum protein electrophoresis in 13 patients showed best correlation for the Beckman instrument (r = 0.942). Quantitation of IgG, IgM, and IgA in 50 specimens required approximately 330, 390, 480, and 480 min to complete with Hyland, International Diagnostic Technology, Beckman, and Technicon, respectively. Our evaluation suggests that the proportion of abnormal specimens in the workload and the availability of reagents for desired assays should be considered in determining the suitability of one of these instruments for a particular laboratory.     

Rate nephelometry and radial immunodiffusion compared for measuring serum prealbumin

We compared a rate-nephelometric method and a radial immunodiffusion (RID) assay for measurement of prealbumin (transthyretin) in 55 samples of serum from healthy children. The mean prealbumin concentration as measured by the Beckman Auto ICS nephelometer was 188 mg/L (range 128-350); the mean by RID was 221 mg/L (range 125-419). This difference was statistically significant by Student's t-test (p less than 0.05), but the correlation coefficient (r) was 0.95. To determine a reference interval for prealbumin in children by the Auto ICS method, we assayed samples from 93 healthy children between the ages of one day and 18 years (55 boys, 38 girls). The mean was 191 mg/L, the reference interval (mean +/- 2 SD) 109-273 mg/L. There was no significant difference in prealbumin concentrations between girls and boys (Student's t-test, p greater than 0.05). Evidently the Beckman Auto ICS method measures prealbumin in serum rapidly and accurately.     

Comparison of specific protein assays in biological fluids by radial immunodiffusion and laser nephelometer.

Laser immuno-nephelometry is presently being used for measuring concentrations of specific proteins in biological fluids. Our findings have not substantiated the highly correlative results with radial immunodiffusion that have been reported. The reason for our poor correlation was found in the lack of uniformity in expressing concentrations of the different specific proteins in the calibrating standards. One value of the laser nephelometer was the greater precision obtained when comparing results to those measured by radial immuno-diffusion. Also, the laser was found to be more sensitive for measuring low level concentrations.     

Accuracy of quantitative immunoglobulin assays by simple radial immunodiffusion

Investigations of the accuracy of quantitative immunoglublin assays (IgG, IgA, IgM) by simple radial immunodiffusion are reported. The normal values (n = 79) were in good agreement with those reported by other authors. The method is characterized by satisfactory precision and high sensitivity. Tests for accuracy resulted in almost complete agreement between expected and measured immunoglobulin concentrations. The specificity of the antibodies incorporated in the agar gel was determined by immunoelectrophoretic analyses. Additional tests on the influence of deep-freeze storage of the specimen sera on the immunoglobulin content showed that with increasing time of storage only the IgM concentrations decreased. Moreover, it was found that differences in filling volume, diffusion time and temperature of incubation considerably altered the diameter of the precipitate. The influence of these parameters on the quality of the analyses can be avoided by standardizing the test conditions.     

Pregnancy-specific beta 1-glycoprotein (SP1) determined by means of electroimmunoassay, radial immunodiffusion and nephelometry.

A comparison has been made between rocket-immunoelectrophoresis (RIE), radial immunodiffusion (RID) and automated immunonephelometry (AIP) in the assay of pregnancy-specific beta 1-glycoprotein (SP1) in serum from pregnant women. Using RIE an interaction was demonstrated between the various SP1-reactive molecular populations causing a bias of up to 10%. An interaction corresponding to this phenomenon cannot be demonstrated when using RID and AIP. When correlating the serum-SP1 concentration of samples containing various ratios of SP1-reactive molecules by means of RIE, RID and AIP, it was demonstrated that there was no correlation between the results achieved using one method compared to the results achieved by either of the other methods. The results achieved using one method can therefore exclusively be judged from reference values determined using the same method. The analysis time is essentially shorter with AIP than with RIE and RID.     

Multiple precipitation of sera containing monoclonal IgG proteins in radial immunodiffusion

Twenty-nine sera containing monoclonal IgG proteins have been examined by radial immunodiffusion using commercially available plates. Double precipitation by IgG myeloma sera was demonstrated to be due to the deficiency of specific antibody determinants to the Fd fragment of the monoclonal proteins.Quantitation of polyclonal- and monoclonal IgG in myeloma sera, based on ring size measurements of the two precipitation rings, leads to erroneous values. The quotient of the ring areas, however, may be useful in the evaluation of chemotherapeutic treatment.     

Comparison of serum IgE determination by radioimmunoassay and by single radial immunodiffusion

Two commercially available methods of total serum IgE determination have been evaluated, viz: the radioimmunosorbent test (RIST) and the assay by single radial immunodiffusion (RID). RIST was found a suitable and rapid method for the wide range of IgE concentrations to be expected in the sera of an allergic population. The RID method has a lower limit of detection of about 1000 I.U./ml; over this value, both techniques provided statistically correlated results. However, for technological reasons RID was considered less suitable for routine application.     

Immunochemical determination of human lysozyme by laser nephelometry.

The methods currently available for quantitative determination of lysozyme are based on the capacity of the enzyme to lyse Micrococcus lysodeikticus. Previous attempts to develop immunochemical assays have been reported, but the sensitivity was inferior to that of the most widely used lysoplate method. We describe the development of a sensitive immunochemical method for lysozyme assay in which the recently developed laser nephelometer is used. The sensitivity so achieved is at least comparable to that of the lysoplate method, and in some circumstances better. Results obtained by the lysoplate assay and the nephelometric assay correlate well. Among the advantages of our method are easier handling of larger numbers of samples, quicker results, and the possibility of assaying enzymatically inactive forms of the enzyme.     

Immunochemical determination of human apolipoprotein B by laser nephelometry.

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.     

Measurement of serum glycosaminoglycans by laser nephelometry

A simple, sensitive micromethod for the assay of serum hyaluronic acid and chondroitin sulphates is presented. The method is based on the binding of the quaternary ammonium salt, cetylpyridinium chloride (CPC) to serum polyanions, and quantitation of the complexes by laser nephelometry. Measurement of the CPC complexes in serum before and after digestion with specific enzymes enables quantitation of hyaluronic acid and chondroitin sulphates in less than 100 μl serum. Using this technique, hyaluronic acid is detectable in a small number of normal human sera at concentrations up to 4 mg/l, and chondroitin sulphates are consistently present at concentrations ranging from 2 to 25 mg/l.     

Multiple precipitation in IgA radial immunodiffusion by non-identical antigens

By radial immunodiffusion of several sera in commercially available IgA plates two precipitation rings were obtained. Double precipitation was demonstrated to be due to the presence of pregnancy-associated α₂-glycoprotein in sera of some adult females.Calculation of the concentration based on ring size measurement of the two precipitation rings in these instances does not lead to erroneous values if the diffusion is allowed to reach completion.     

非浓缩尿蛋白电泳法和免疫散射比浊法测定尿免疫球蛋白G的比较

目的比较非浓缩尿蛋白电泳法和免疫散射比浊法测定尿免疫球蛋白G的临床应用及价值。方法分别采用十二烷基磺酸钠-琼脂凝胶电泳技术(SDS-AGE)与免疫散射比浊法分别测定2010年7月至2011年7月兴化市人民医院62例干化学法尿蛋白(+～++++)的住院患者非浓缩尿液标本中免疫球蛋白G的百分含量,并按尿总蛋白定量及尿IgG电泳百分比计算尿IgG含量,与免疫散射比浊法定量做相关分析。结果 62例非浓缩蛋白电泳法尿IgG结果阳性率53%,免疫散射比浊法阳性率64%,电泳法和免疫散射比浊法测定尿中IgG阳性率差异无统计学意义(P>0.05),两种方法在检测非浓缩尿IgG上完全符合率达89%,相关系数为0.95,呈正相关(P<0.01)。结论非浓缩蛋白电泳法与免疫散射比浊法检测尿IgG结果差异无统计学意义,有显著相关性,且结果准确,灵敏度高。     

Determination of thyroxine-binding globulin in human serum by single radial immunodiffusion and radioimmunoassay.

Two immunochemical methods for determination of thyroxine-binding globulin in human serum were developed, in which the purified globulin and monospecific antiserum to it are used. One method, based on radial immunodiffusion, has good precision and values for analytical recovery. Reference values obtained for men were 9.8-17.8 mg/liter and for women 11.3-20.5 mg/liter. The sex-related difference was significant. The other method is based on radioimmunoassay, with use of an iodinated acylating agent for the labeling of thyroxine-binding globulin. The relative merits of the two methods are discussed.