Bromcresol Green Method Research Articles

Interference by an IgM paraprotein in the bromcresol green method for determination of serum albumin.

Journal Article Interference by an IgM paraprotein in the bromcresol green method for determination of serum albumin. Get access R G Reed R G Reed Search for other works by this author on: Oxford Academic Google Scholar Clinical Chemistry, Volume 33, Issue 6, 1 June 1987, Pages 1075–1076, https://doi.org/10.1093/clinchem/33.6.1075a Published: 01 June 1987

Underestimation of monoclonal proteins by serum protein electrophoresis on cellulose acetate.

Our study of 95 serum samples from 37 patients with monoclonal gammopathy revealed distorted irregular monoclonal (M) protein bands after serum protein electrophoresis (SPE) on cellulose acetate membrane. In 71 (75%) of the 95 sera, the M-protein was underestimated and the albumin concentration overestimated. Dilution of the serum sample before SPE eliminated the abnormality of the M-protein bands. By SPE, the mean albumin concentration in these 71 undiluted sera was 45.8 (SD 7.4) g/L vs 37.9 (SD 5.8) g/L for the diluted sera; moreover, this was true of individual samples: measured albumin concentration in each diluted serum sample was always less than in the undiluted serum. As measured by the bromcresol green dye-binding method, the albumin concentration was 32.8 (SD 5.9) g/L. Similarly, the M-protein concentration in SPE was 49.5 (SD 12.3) g/L for the undiluted sera vs 61.8 (SD 15.1) g/L for the diluted sera, and the M-protein concentration in each diluted serum sample always exceeded that in the undiluted serum. Underestimation of M-protein limits the usefulness of M-protein measurement in evaluating the patient's response to therapy and for early detection of disease progression. SPE strips should be carefully inspected visually, and sera with M-protein band abnormalities should be diluted and re-assayed if SPE is to quantify concentrations of M-protein and albumin accurately.

A sensitive spectrophotometric method for the determination of human serum albumin with chrome-azurol S aluminium chelate.

A simple and highly sensitive method for the determination of serum albumin based on the formation of a ternary complex from chrome-azurol S aluminium chelate was developed. A nonionic detergent, polyethylene glycol-p-nonylphenyl ether n=10, acted as a stabilizer of the ternary complex in the present system. The formation of the ternary complex caused a large increase in absorbance at 630 nm, and the calibration curve was linear between 10 and 200 μg of albumin in the final colored solution (8.0 ml). The sensitivity of the present method was about eight times that of a bromcresol green control method. Heparin and bilirubin did not interfere with the determination.

A sensitive spectrofluorimetric method for the determination of human serum albumin with chrome-azurol S.

An extremetly sensitive spectrofluorimetric method for assay of human serum albumin (HSA) was developed, based on binding of chrome-azurol S(CAS) by HSA. The CAS-HSA complex shows a fluorescence at 616nm, whereas CAS alone exhibits only a weak fluorescence at this wavelength. A nonionic detergent, polyethylene glycol-p-nonylphenyl ether n=10, acts as a stabilizer of the CAS-HSA complex. The calibration curve was linear in the range from 5 to 80 μg of HSA in the final solution. Relative standard deviation was 1.3% (n=10). The method requires less than one-thirtieth of the amount of HSA required by the control bromcresol green method. HSA values in control sera and spinal fluid assayed by the new method were in agreement with those obtained by the control bromcresol green method.

Serum protein reference values in foals during the first year of life: comparison of chemical and electrophoretic methods.

Normal reference values for serum proteins of foals from birth to 1 year of age have been established. Chemical and electrophoretic/refractometric methods for total protein, albumin, total globulin and Albumin/Globulin (A/G ratio) have also been compared. The biuret total protein method and Bromcresol Green (BCG) method on the Technicon SMA 12/60 autoanalyzer were used and compared with total protein determined via refractometry and albumin determined by Cellulose Acetate (CA) electrophoresis/densitometry. Globulin and A/G ratios were calculated from the chemical method data and compared with that obtained electrophoretically. Total protein, albumin, total globulins and A/G ratios all were in agreement at all sample times studied. Data on the subfractionation of serum globulins via CA electrophoresis is also presented. Wide variations in alpha and beta globulin levels were noted among the foal sera early in life. As a result, two distinct populations of foals with respect to both globulin content and A/G ratio were identified. One of these populations (Group A) appeared to have obtained passive immunity more slowly than the other (Group B) animals. Comparison of these data with clinical cases of foals in which failure of passive transfer was a part suggests that the A/G ratio may be useful in assessing adequate colostral antibody levels in the newborn foal.

Multilayer-film bromcresol green method for albumin measurement compared with three others.

Multilayer-film bromcresol green method for albumin measurement compared with three others.

Fluorimetric Assay for Serum Albumin Based on Its Enzymatic Activity

ABSTRACT Serum albumins are known to catalyze the hydrolysis of aryl esters. Both human and bovine albumins are active against Naphthol AS acetate, resulting in a fluorescence excited at 320 nm and monitored at 500 nm. HSA was more active than BSA. At pH 8.0 the reaction is activated by cetyltrimethylammonium bromide. Other esterases in serum require either calcium or a higher pH for activity. The assay is conducted with albumin diluted to about 10−7 M or less, thus dissociating many potentially interfering ligands. Palmitic acid did not interfere. The interference by bilirubin is minimized by using highly dilute albumin. The present method gives results with serum which correlate well with the widely used Bromcresol Green method. Limit of detection for HSA is 14 picomoles.

Decreased bilirubin binding by albumin in late pregnancy

It is known that the albumin concentration of maternal plasma is reduced at term and that the binding of certain drugs and bilirubin’ in plasma is likewise reduced. This decreased plasma binding of bilirubin may be due to decreased albumin concentrations alone or a decrease in the ability of albumin to bind bilirubin. It is possible that the same factor(s) which causes decreased binding ability in cord blood may decrease the binding ability in maternal blood. We undertook this study to determine if the quantitative binding of bilirubin to albumin (binding fraction) was changed in pregnant women at term. We collected heparinized venous plasma from 19 normal pregnant subjects at term (38+ gestational weeks) and 19 normal, nonpregnant control subjects. Informed consent was obtained from all subjects in this study. Apparent total bilirubin binding capacity was measured by the peroxidase method with pooled human bilirubin-enriched plasma used as a control. Albumin was determined by the bromcresol green method. All assays have been described in detail elsewhere.’ The binding fraction was expressed as the total bilirubin binding capacity/albumin molar ratio. Statistical analysis of the data included calculations of Pearson’s product-moment correlation coefficients and multiple regression analysis. Correlation coefficients were tested for significance with the use of one-tailed t tests. The total bilirubin binding capacity in the pregnant subjects was 20.9? 2.7 (mean? SD) compared to 30.5 2 3.4 in our nonpregnant sample. The albumin concentration was 3.6 * 0.26 compared to 4.6 -C 0.29 and the binding fraction was 0.66 ‘0.11 versus 0.78 * 0.10 for the nonpregnant sample. Fig. 1 shows the relationship of total bilirubin binding capacity to binding fraction in the two groups, along with the respective linear regression equations. We found that the relationship between total bilirubin binding capacities and binding fraction was stronger in nonpregnant women (r = 0.87, p < 0.001) than in pregnant women (r = 0.76, p < O.OOl), probably because there was less spread in the total bilirubin binding capacity in the former group. The binding fraction of the pregnant group was about 15% lower than the binding fraction of the nonpregnant group, whereas

A comparison of dye binding methods for albumin determination: the effects of abnormal sera, reaction times, acute phase reactants and albumin standards

Four bromcresol green methods were evaluated, three of which were used with a reaction time of 15 seconds. An immunonephelometric assay and a bromcresol purple method were used as comparison methods. The bromcresol purple method agreed well with the more specific immunonephelometric procedure. Forty-seven human sera, thirty with low albumin concentrations and seventeen with high albumin concentrations were used in each assessment. The best bromcresol green methods were those using low dye and surfactant (Brij-35) concentrations and the shortest possible reaction times. Except in one bromcresol green method with a long reaction time of 10 minutes, there was no significant difference in values overall between sera having a low albumin concentration with either raised or normal acute phase reactants. Comparable results were obtained using standards of human and bovine albumin preparations in bromcresol green methods but not in the bromcresol purple method.

Continuous-flow serum albumin determination by reaction with picrate ions, with use of a flow-through picrate ion electrode.

An automated potentiometric method for serum albumin determination by use of the picrate/albumin reaction is described. A continuous-flow system and a specially designed flow-through picrate ion electrode were used in making the measurements. Various factors affecting the reaction, such as pH, picrate ion concentration, and reaction time, were studied. Peak height in millivolts and albumin concentration were linearly related in the range 10-70 g/L. Both within-run and day-to-day, the CV for the method was about 2%. Analytical recovery of albumin added to serum samples ranged from 97.0 to 110.3%, averaging 102.2%. Results compare favorably with those by the established bromcresol green method. The proposed method is suitable for routine use and for screening tests.

Eine verbesserte Methode zur selektiven Albuminbestimmung mit Bromkresolgrün auf dem SMAR 12/60

The estimation of serum albumin obtained with SMAR 12/60 by the bromcresol green method are in good agreement with values determined by electrophoresis only in the normal range. Interestingly marked discrepancies occur in the clinically relevant cases of hypalbuminosis. The shortening of the reaction time between bromcresol green and serum results in a better agreement between the values obtained with SMAR 12/60 and the values determined by electrophoresis over the whole range.

Optimizing determination of plasma albumin by the bromcresol green dye-binding method.

Some modifications of the conditions of the reaction between plasma and bromcresol green have led to an improved method for determination of plasma albumin with the Vickers M300 multichannel analyzer. Dye concentration and reaction time are the factors principally influencing method specificity, but variable protein composition of human plasma also affects it, so that optimal reaction conditions vary from specimen to specimen. Thus a compromise must be reached such that the best conditions for determining plasma albumin over a range of different protein concentrations are achieved. In the proposed method for the Vickers M300 a reaction time of 12 s (the minimum possible) is used. Comparison with "rocket" immunoelectrophoresis gave the following linear regression: y = 10 + 0.79 x (n = 91; r = 0.96).

3] Indirect quenching fluoroimmunoassay

This chapter discusses the development and use of indirect quenching fluoroimmunoassay (FIA). The development of the first radioimmunoassay (RIA) for insulin has proved to be an important milestone. It is found that such assays are extremely sensitive, highly specific, practical, and applicable to a wide range of proteins, glycoproteins, and haptens. The key reactant in a RIA is the antibody and the isotopically labeled antigen is employed only as a tracer. Nonisotopic immunoassays can be classified into those that require a separation step and those that do not. In indirect quenching FIA antibodies against fluorescein are added after the completion of the first antigen-antibody reaction and bind to and quench the fluorescein groups of the free fraction of the fluorescein-labeled antigen. The nonseparation FIA established for human albumin is sufficiently sensitive for all clinical purposes and the amount of sample needed is small. Results on serum, cerebrospinal fluid, and urine samples are correlated closely with those obtained by the two existing methods. Results suggest that the bromcresol green method overestimated low hSA values.

Comparison of manual methods of determination of albumin in human cerebrospinal fluid by the bromcresol green and immuno-precipitation methods

Simultaneous determinations of albumin concentrations in human cerebrospinal fluid were made by three different manual methods: bromcresol green dye-binding technique of Doumas et al., a modification of the latter with color measurement shortly after the addition of dye as proposed by Ingwersen and Raabo and finally by an immuno-precipitation technique. Results obtained by the immuno-precipitation method correlated significantly with the dye-binding method of Doumas et al., (r = 0.93) and the procedure of Ingmersen and Raabo (r = 0.94). Data from the two dye-binding techniques correlated significantly with one another also (r = 0.98). However, the immuno-precipitation technique gives albumin values lower than the dye-binding procedures. The latter presumably estimates other proteins which can also bind bromcresol green. The data corroborates the conclusions of earlier investigators that immuno-precipitation method is more specific for albumin assays.

Urinary albumin determination by the immediate bromcresol green method

It has been demonstrated recently that the reaction of serum samples with bromcresol green (BCG) reagent proceeds in two steps. Albumin is responsible for the immediate reaction while other serum proteins produce the slow reaction. In this paper the immediate BCG reaction has been used for the determination of urinary albumin concentration in patients with proteinuria by a slightly modified method with a primary pH adjustment of the urine and the use of a urine blank. Comparison of the immediate BCG method (γ) with Laurell “rocket” technique (χ) gave the following equation: γ = 17.2 +1.006 χ ( n = 98; r = 0.99) mg/1. The coefficient of variation (within-day), C.V. (%), ranged between 0.9 and 2.7% depending on the albumin concentration. It is thus possible to carry out rapid, accurate and precise albumin determinations in urine samples using this simple method.

The influence of citrate and phosphate on the mancini single radial immunodiffusion technique and suggested improvements for the determination of urinary albumin

When adapting the immediate bromcresol green method for urinary albumin determination a correlation study with the Mancini single radial immunodiffuon (SRID) method was performed. This study showed large disparities between the two methods, SRID giving the higher results. The unsuitability of the currently used SRID methods is demonstrated and improvements to the method are suggested. Known amounts of albumin were added to urine samples as well as to a “synthetic urine”, both giving falsely elevated results with the SRID method. On investigating the different components of the “synthetic urine”, it was found that the disparities were due to the influence of citrate and phosphate. On addition of citric acid or phosphate to the dilution buffer and/or the gel buffer, the results of the SRID method agreed with those of other methods and with the expected values. The findings presented in this paper can probably be extended to other immunological methods too since it seems to be the antigen-antibody reaction which is affected.

Continuous-flow analysis for serum albumin, by use of the immediate reaction with bromcresol green.

Albumin reacts with bromcresol green reagent almost immediately, other serum proteins more slowly. This phenomenon can be exploited to improve the specificity of this routine method for determining serum albumin. I describe a 30-s reaction as adapted to continuous-flow analyzers, with no interference from other serum proteins. The method shows linearity to 72 g/liter. Results of this method correlate very well with those by the Laurell "rocket" and manual (30-s) bromcresol green methods. This procedure gives a considerable improvement in specificity for continuous-flow analyzers as compared with current bromcresol green methods.

Improved and more specific bromcresol green methods for the manual and automatic determination of serum albumin

The bromcresol green (BCG) method for determination of serum albumin has been modified and the specificity improved. A manual and a continuous flow method is described. Both methods are suitable for routine use and the results agree well with those obtained by an immunonephelometric method.

Improved continuous-flow (SMAC) determination of serum albumin.

The albumin values determined by the bromcresol green methods do not compare well with values by more specific methods for albumin determination. The discrepancies have been related to, among other things, acute-phase reactants and are especially pronounced in the lower albumin range. These disadvantages are also inherent in a routine continuous-flow method for albumin (SMAC). The bromcresol green method has been improved considerably by shortening the reaction time before the absorbance is measured, as is described here. The modified method yields values that better agree with those by more specific methods and an influence of acute-phase reactants is no longer observed.

New automated dye-binding method for serum albumin determination with bromcresol purple.

We describe a new automated dye-binding method for serum albumin determination with bromcresol purple (BCP) that has several advantages over an existing bromcresol green (BCG) method. The continuous-flow method is sensitive, linear, and precise, with negligible sample interaction at an analytical rate of 60 samples per hour. Unlike BCG, BCP did not react with an albumin-free serum globulin preparation or pure human transferrin solutions. Reaction with serum was instantaneous; in contrast, BCG exhibits a slow nonspecific reaction with some specimens. The specificity of BCP was demonstrated by good agreement with results of "rocket" immunoelectrophoresis (EIA) where y(BCP) = 0.95X(EIA) + 1.72. The BCG method overestimated serum albumin concentration where y(BCG) = 1.01X(EIA) + 6.77. Precipitation, which affects the BCG method, was not observed with BCP. Blank corrections were negligible, salicylate did not interfere, and bilirubin affected the method only if present in very high concentration. The method offers a solution to the poor accuracy of existing BCG methods while retaining many of their desirable features.