Development of a radial immunodiffusion technique employing monoclonal antibodies for apolipoprotein B determination in human plasma

Twenty monoclonal antibodies obtained from two different fusions of SP2/0-Ag 14 cell line (non-secretor hybridoma) with human plasma low density lipoproteins have been selected. We found that a mixture formed by the 20 monoclonal antibodies was able to form a single precipitin line with human plasma low density lipoproteins by a double gel diffusion technique. Further studies revealed that only two monoclonal antibodies were needed to precipitate low density lipoproteins in gel. However, a minimum of four particular monoclonal antibodies was required to obtain an optimal precipitin ring and a linear standard curve within 24 h using a radial immunodiffusion technique. We have then compared the radial immunodiffusion performed with a monoclonal antibody mixture to those employing conventional goat and rabbit antibodies in terms of plasma apolipoprotein B determinations. The apolipoprotein B values determined by monoclonal antibodies significantly correlate with the values obtained by the technique using goat (r = 0.95; p less than 0.001) and rabbit (r = 0.95; p less than 0.001) antibodies. Our data indicate that a mixture of monoclonal antibodies can mimic conventional antibodies in terms of immunoprecipitation and apolipoprotein B determination.     

Rate immunonephelometry and radial immunodiffusion compared for apolipoproteins AI and B assay

We measured apolipoproteins (apo) AI and B in fresh plasma samples and serum control pools by using two rate immunonephelometric assay (INA) methods (Beckman "Array" and Behring) and radial immunodiffusion (RID). Both INA methods on average gave apoAI values similar to those by RID in fresh plasma samples. The coefficients of correlation for the two INA-RID method pairs were as follows: Beckman INA vs RID, n = 94, r = 0.73; and Behring INA vs RID, n = 112, r = 0.66. The bias between the INA and RID methods was reflected reasonably well by either lyophilized or frozen control pools. For apoB, the Beckman INA measurements in fresh plasma averaged 40% lower than RID values, due almost entirely to the values assigned to calibration sera, but results by the two methods were highly correlated (n = 94, r = 0.93). The Behring INA values for fresh plasma averaged only 13% lower than RID values, but the two methods were less well correlated (n = 112, r = 0.74). Frozen control pools were most suitable for apoB analysis.     

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.     

Serum A1 and B apolipoprotein determination: comparison of an immunoturbidimetric method with a monoclonal-antibody-based radial immunodiffusion assay.

Epidemiological and clinical evidence have indicated that apolipoprotein A1 and B determination can better define the lipoprotein pattern in normal subjects and in subjects with coronary heart disease. In this paper, a recent immunoturbidimetric method for routine apolipoprotein A1 and B measurement (using the Turbitimer system and commercially available antisera) has been evaluated. The precision and the accuracy of the method have been previously considered. Within-run and between-run coefficients of variation (ranging from 1.67% to 5.04%) for both assays indicate good precision of the method. Accuracy was evaluated on 2 consecutive days (n = 10 each run) using a standard serum for apolipoprotein A1 and B. The bias obtained was 3.79% for apolipoprotein A1 and 2.30% for B. Apolipoproteins A1 and B were then measured in 100 normal and hyperlipemic sera with the immunoturbidimetric assay and radial immunodiffusion (using the monoclonal antibodies). The data obtained were evaluated by linear regression analysis (Al, r = 0.893; B, r = 0.862). The good correlation between the two methods suggests that the immunoturbidimetric assay can be usefully performed for routine apolipoprotein A1 and B determination because of its lower cost, rapidity, and simplicity.     

Serum A1 and B apolipoprotein determination: comparison of an immunoturbidimetric method with a monoclonal-antibody-based radial immunodiffusion assay

Summary Epidemiological and clinical evidence have indicated that apolipoprotein A1 and B determination can better define the lipoprotein pattern in normal subjects and in subjects with coronary heart disease. In this paper, a recent immunoturbidimetric method for routine apolipoprotein A1 and B measurement (using the Turbitimer system and commercially available antisera) has been evaluated. The precision and the accuracy of the method have been previously considered. Within-run and between-run coefficients of variation (ranging from 1.67% to 5.04%) for both assays indicate good precision of the method. Accuracy was evaluated on 2 consecutive days (n=10 each run) using a standard serum for apolipoprotein A1 and B. The bias obtained was 3.79% for apolipoprotein A1 and 2.30% for B. Apolipoproteins A1 and B were then measured in 100 normal and hyperlipemic sera with the immunoturbidimetric assay and radial immunodiffusion (using the monoclonal antibodies). The data obtained were evaluated by linear regression analysis (A1,r=0.893; B,r=0.862). The good correlation between the two methods suggests that the immunoturbidimetric assay can be usefully performed for routine apolipoprotein A1 and B determination because of its lower cost, rapidity, and simplicity.     

The quantitative determination of apolipoprotein A-I (apo-lp-Gln I) in human serum by radial immunodiffusion assay (RID).

A radial immunodiffusion (RID) assay was developed to determine the concentration of the major apolipoprotein (apolipoprotein A-I, apo-lp-Gln I) of human high density lipoproteins (HDL): (1) In a random population sample of apparently healthy persons (104 women, 95 men) aged 40-49 years, serum levels of apo A-I were determined. For both women and men a value of 137 +/- 23 mg/dl was found. (2) Values of 139 +/- 21 mg/dl were found in a group of 31 women taking oral contraceptives, i.e. an insignificant increase in apo A-I level. (3) In two volunteers, apo A-I levels showed no significant variation in a 24-h period.     

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 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.     

Measurement of serum apolipoprotein B by radioimmunoassay

Abstract. A sensitive and specific double antibody radioimmunoassay for the major apolipoprotein (apo B) of human serum very low density lipoprotein (VLDL) and low density lipoprotein (LDL) is described. Using anti-LDL and anti-apo B antibodies the immunoreactivity of LDL and apo B were compared. Human LDL and its isolated apo B were not immunologically identical when each antiserum was used with its homologous label; a population of antibodies was selected which reacted with antigenic sites unique to the antigen itself as well as to those which were common to the closely related protein. When the heterologous label was used with either antiserum, a population of antibodies directed against antigenic sites shared by the LDL and apo B molecules was selected.
Apo B in sera samples can be measured using either anti-LDL or anti-apo B antibodies provided that intact LDL was used for preparation of the iodinated tracer and standard. Serum apo B levels in healthy normolipi-daemic males and females were 0.93 ±0.25 g/l (range 0.58–1.39) and 0.90 ± 0.15 g/l (range 0.58–1.12), respectively. The total cholesterol and apo B, and phos-pholipid and apo B concentrations for both males and females were significantly correlated (P<0.05). In another normolipidaemic population ( n = 52), total serum apo B values correlated positively with LDL cholesterol ( r= 0.92, P< 0.001).
Apo B was measured in sera from patients with abetalipoproteinaemia, familial hypercholesterolaemia and Tangiers disease. Apo B was not detected in the serum of subjects with abetalipoproteinaemia, while the apo B level in the familial hypercholesterolaemic subjects was significantly elevated (range 3.26–4.94 g/l) compared to normals (P<0.001). Serum apo B (0.80 g/l) of the subject with Tangier disease was within the normal range.     

A simple radial immunodiffusion method for assay of beta 2-microglobulin in serum

In this method for clinical measurement of beta 2-microglobulin, a 10 g/L agarose gel containing anti-beta 2-microglobulin is used, with subsequent staining with Coomassie Blue. Although the method is slow (requiring 30 h), it is inexpensive, reliable, and accurate over the range of 1 to 10 mg/L, and is useful in the determination of beta 2-microglobulin in serum and cerebrospinal fluid. A modified procedure in which staining with silver is used may be sufficiently sensitive for the clinical assay of urine.     

Radioimmunoassay for human plasma apolipoprotein B.

We have developed a simplified double antibody radioimmunoassay for human apolipoprotein beta. The purified antigen, a narrow density subclass of beta lipoprotein (d 1.030-1.050 g/ml; was isolated by a combination of gel filtration and ultracentrifugationmthis material gave a single immunoprecipitin arc on crossed immunoelectrophoresis into an antibody to whole human serum. The antigen was radiolabelled using iodine monochloride at pH 10. The iodinated antigen was indistinguishable immunochemically from native material and eluted as a single radioactive peak from a Sephadex G-200 column. The lower limit of sensitivity of the assay was 15 ng protein, and the working range, 15-200 ng. The mean apolipoprotein B level (+/-1 S.D.) in 128 healthy control subjects was 86.6 +/- 29.6 mg/100ml and is in agreement the values published by other workers using unmodified assays.     

von Willebrand factor antigen: a radial immunodiffusion method evaluated and compared with an ELISA method.

A new commercial kit method for the quantification of von Willebrand factor antigen (vWFAg) by radial immunodiffusion was compared to an established ELISA technique. Major discrepancies were found between the two methods. The radial immunodiffusion method had poorer intra- and inter-assay coefficients of variation than the ELISA method, although there was adequate inter-method agreement when 100 plasma samples from controls and patients were compared. However, there was a great difference in values obtained for vWFAg in the reference sample supplied with the commercial kit and that obtained directly from the National Institute for Biological Standards and Controls. There were also differences in levels of significance when vWFAg was measured by the two techniques in different clinical groups, and standard deviations were larger when the kit method was used. It is suggested that on scientific and economic grounds, the commercial radial immunodiffusion kit does not offer a competitive advantage over the ELISA method.     

Semi-automated enzyme-linked immunosorbent assay (ELISA) for the quantification of apolipoprotein B using monoclonal antibodies

A semi-automated competitive, double-antibody, solid-phase enzyme-linked immunosorbent assay for apolipoprotein B (Apo B) has been developed which utilizes microtiter plates with commercially available monoclonal antibodies and alkaline phosphatase-conjugated second antibody. The working range of the assay is 20–200 ng. The concentration of plasma Apo B was 0.88 ± 0.20 g/l (n = 40) for a random sample of normal adults. The correlation coefficient for this assay, compared to a radial immunodiffusion assay, was 0.95 (slope = 1.13, INTERCEPT = −15). The quantification of the samples was not influenced by freezing and thawing, storage at −20°C for up to 9 mth, or the lipoprotein particle on which the Apo B was present. The method is suitable for measurement of apolipoprotein B in either normal or pathological plasma, lipoprotein density classes, and is sensitive enough to quantify Apo B in cell biological and molecular biological investigations.     

Radial immunodiffusion assay of apolipoprotein B in blood dried on filter paper--a potential screening method for familial type II hypercholesterolaemia

In the search for an effective neonatal screening programme for familial type II hypercholesterolaemia, we have developed and validated a radial immunodiffusion assay for measuring, in dried blood spots, the concentration of apolipoprotein B. The method has advantages over previously described methods, being cheaper, more robust, and suitable for partial automation. In 34 adults there was a close linear relationship between capillary and venous blood spot apolipoprotein B concentrations (r = 0.94), between capillary blood spot and serum apolipoprotein B also measured by radial immunodiffusion (r = 0.90), and between capillary blood spot apolipoprotein B and LDL cholesterol measured by ultracentrifugation (r = 0.85). Apolipoprotein B levels in serum and LDL cholesterol also correlated closely (r = 0.95, n = 40). In 30 normal 3- to 5-day old babies, heel prick blood spot apolipoprotein B levels were 280 +/- 70 mg/l (mean +/- SD) and 790 mg/l in one presumed heterozygote for familial hypercholesterolaemia. We conclude that serum apolipoprotein B measured by radial immunodiffusion accurately reflects adult LDL cholesterol levels, and that the assay, using dried blood spots on filter paper should permit both neonatal and adult screening programmes to detect familial hypercholesterolaemia.     

Structural relationship of human apolipoprotein B48 to apolipoprotein B100.

Although the complete amino acid sequence of human apolipoprotein (apo) B100 is known (4536 amino acids), the structure of apo B48 has not been defined. The objective of our study was to define the structure of apo B48 and its relationship to apo B100. Antibodies were produced against 22 synthetic peptides corresponding to sequences in human apo B100. The levels of immunoreactivity of the antipeptides to apo B100 and apo B48 were used to define the structural relationship between these two species of apo B. Six antibodies from sequences in the amino-terminal half of apo B100, including antipeptide 2110-2129, bound to both apo B100 and apo B48. 15 other apo B-specific antipeptides from sequences carboxyl-terminal to residue 2152 bound to apo B100, but not to apo B48. Immunoblots of cyanogen bromide digests of apo B100 and apo B48 with antipeptides 2068-2091 and 2110-2129 detected a 16-KD fragment (residues 2016-2151) in the apo B100 digest and a fragment of identical size in the apo B48 digest. Because apo B48 appears to contain the apo B100 cyanogen bromide fragment 2016-2151 and because an antiserum specific for the peptide 2152-2168 does not bind to apo B48, we conclude that apo B48 represents the amino-terminal 47% of apo B100 and that the carboxyl terminus of apo B48 is in the vicinity of residue 2151 of apo B100.     

Gold-labeled nanoparticle-based immunoresonance scattering spectral assay for trace apolipoprotein AI and apolipoprotein B

BACKGROUND: Apolipoprotein AI (ApoAI) and ApoB are risk indicators of cardiovascular disease. We describe the use of immunoresonance scattering to measure the ApoAI and ApoB in serum. METHODS: We used a trisodium citrate method to prepare 9.0-nm gold nanoparticles labeled with goat anti-human ApoAI and ApoB antibodies. The immune reaction between gold-labeled antibodies and antigens took place in Na2HPO4-NaH2PO4 buffer solution (pH 6.4 for ApoAI and pH 6.0 for ApoB) in the presence of 75 g/L polyethylene glycol (PEG). We used a transmission electron microscope to observe the shape of the gold nanoparticles. Results were compared with those obtained by immunoturbidimetric methods. Twenty-five human serum samples were assayed by the immunoresonance scattering assay preset with the data indicated and by an immunoturbidimetric assay. RESULTS: The presence of PEG greatly enhanced the intensity of resonance-scattering peaks at 560 nm. The intensity (DeltaI) was proportional to concentration at 0.00833-0.3333 mg/L ApoAI and 0.00197-0.1972 mg/L ApoB. The detection limits were 2.04 and 0.96 microg/L for ApoAI and ApoB, respectively. The results for human serum samples were in agreement with those obtained with an immunoturbidimetric method. Linear regression analysis revealed a correlation coefficient, slope, and intercept of 0.915, 0.966, and 68.53 mg/L, respectively, for ApoAI and 0.919, 0.996, and 15.46 mg/L for ApoB. CONCLUSION: This method showed high sensitivity and good selectivity for quantitative determination of ApoAI and ApoB in human serum, with satisfactory results.     

Electroimmunoassay of a subunit protein in a macromolecular complex (apolipoprotein B in human plasma very low density lipoprotein); implications for other electroimmunoassay systems.

With an electroimmunoassay ("rocket") system for the apolipoprotein B component of the plasma very low density lipoprotein complex we obtained results which were similar to those obtained by a colorimetric tetramethylurea extraction method. Results were up to twice as high as those using radioimmunoassay. Low density lipoprotein containing apolipoprotein B as the only demonstrable protein component was used as the standard for these assays. This protein produced larger and higher rockets at pH 8.6 when the negative particle charge was increased by maleylation. The very low density lipoprotein complex has a higher negative charge at pH 8.6 than low density lipoprotein. These findings suggest that some apolipoprotein B in vary low density lipoprotein is not "recognised" by anti-apolipoprotein B antibodies, hence radioimmunoassay results are lower than those obtained with the tetramethylurea extraction method. The higher negative charge on very low density lipoprotein particles (compared with low density lipoprotein), as a factor tending to increase rocket area and height, is counterbalanced by reduced recognition by antiapolipoprotein B antibodies. The net result of these opposing tendencies is that the rocket electroimmunoassay of apolipoprotein B in very low density lipoprotein fortuitously gives valid results, under the specified assay conditions. We conclude that electroimmunoassays of complex proteins are not necessarily valid if protein subunits are used for standards. This has implications for the electroimmunoassay of other apolipoproteins.