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R F Ritchie

Publications and source records attributed to R F Ritchie.

At least 37 records · Page 2Linked to original sources
PubMed1994

ELISA of IgG antibody to oxidized low-density lipoprotein: effects of blocking buffer and method of data expression.

We describe an ELISA for serum IgG antibodies against malondialdehyde-modified low-density lipoprotein (mLDL). Optimal antigen concentration, serum dilution, and dilution of enzyme-conjugated second antibody were 25 mg/L, 1:250, and 1:5000, respectively, when 5 g/L human serum albumin was used for blocking. When data were expressed as mLDL/LDL (the ratio of IgG binding to mLDL vs LDL), within-run and between-run CVs were 7.0% and 8.9%, respectively. Antibody concentrations expressed as mLDL/LDL or as mLDL-LDL (the difference between IgG binding to mLDL and to LDL) were higher in women with systemic lupus erythematosus (n = 20) than in controls (n = 20) (P < 0.001). With bovine serum albumin or Superblock blocking buffers, only the mLDL-LDL data were significant. Thus, the choice of blocking agent and the method of data expression should be carefully considered when assaying IgG antibodies against mLDL.

Adolescent↗
PubMed1994

New international reference preparation for proteins in human serum (RPPHS).

Quality-control surveys in recent years, in various parts of the world, have shown poor between-laboratory agreement for measurements of plasma proteins. Despite the existence of international reference materials distributed by the World Health Organization, standards produced by diagnostics manufacturers and professional organizations differ significantly in their ascribed values. The reasons for this are complex but include poor availability of the primary materials, confusion about their use, and the fact that their turbidity on reconstitution precludes their use in modern optical immunoassays. This unfortunate situation led to an important initiative to produce sufficient quantities of a widely available, optically clear secondary reference material for plasma proteins that could be used worldwide by manufacturers, professional organizations, and laboratories. Here we present an overview on how the laboratory community, including manufacturers, clinical laboratories, professional societies, and regulators, has reached what we consider is a successful conclusion to a difficult problem.

Blood Proteins↗
PubMed1993

The relationship between serum levels of lipoprotein(a) and proteins associated with the acute phase response.

The association of serum lipoprotein(a) (Lp(a)) with inflammation was investigated in a primarily rheumatologic study group (n = 570; 202 males and 368 females) by studying the relationship between serum levels of Lp(a) and a panel of acute phase proteins (C-reactive protein (CRP), alpha 1-antitrypsin (AAT), alpha 1-acid glycoprotein (AGP), haptoglobin (HPT), complement components 3 and 4 (C3, C4), prealbumin (PAL), albumin (ALB) and transferrin (TRF)). Lp(a) data were adjusted for age and sex, but not clinical condition as no significant differences in Lp(a) levels were observed, using analysis of variance, among the 15 diagnostic categories in the study group. Univariate analyses revealed significant positive associations between Lp(a) levels and levels of C4, AGP, C3 and HPT. Multivariate analysis revealed that C4 and AGP (in descending order of significance) were significant independent predictors of Lp(a) concentration, together accounting for 2.9% of the variability in Lp(a) concentration in the present study group. The data indicate that confounding effects of an acute phase response should be considered in epidemiologic studies, if a high prevalence of inflammation is suspected.

Acute-Phase Proteins↗
PubMed1993

Background staining in immunoblot assays. Reduction of signal caused by cross-reactivity with blocking agents.

Certain serum samples produce high background in Western and direct immunoblot assays that detect human serum IgG against specific antigens. We determined that this was due to a reaction between endogenous IgG and the membrane blocking agent (we refer to this as blocking-specific background). Using milk as blocking agent, we screened 107 sera by Western immunoblot or checkerboard immunoblot assays, and found that 6.5% of sera had background intensities sufficient to interfere with the interpretation of final results. Blocking-specific background was also observed using bovine serum albumin and other animal protein-based blocking agents. As the primary antibody in these immunoblot assays was human IgG, we investigated human serum albumin as a blocking agent; this approach eliminated the problem of blocking-specific background.

Cross Reactions↗
PubMed1993

Application of checkerboard immunoblotting (CBIB) to the detection of anti-viral IgG in human serum.

In the present study, we have begun to investigate the possibility of using checkerboard immunoblotting (CBIB) as a semi-quantitative screening tool for detecting human serum IgG against specific viral antigens. The viral antigens studied were Epstein-Barr, herpes simplex I and II, cytomegalovirus, varicella zoster, rubella, rubeola, and mumps. Western immunoblotting experiments using these partially purified preparations demonstrated that there were apparently no interactions between IgG from non-immune sera and the respective viral antigen preparations. The CBIB assay was evaluated using sera of known positive or negative immune status for the viral antigens. There was excellent agreement between the results of CBIB and the results of alternative methods for evaluating immune status: all discrepancies (1/18 sera for mumps, 3/18 sera for rubeola, and 1/28 sera for rubella) involved sera with borderline results, either by CBIB or by the alternative method. Therefore, although further work is required to define the method in terms of sensitivity and clinical specificity, and to refine positive/negative cutpoint criteria for certain antigen components, our preliminary experience suggests that CBIB has considerable potential in the efficient and inexpensive screening of sera for the presence of IgG against a panel of viral antigens, so as to identify subjects at risk for infection.

Antibodies, Viral↗
PubMed1993

The molecular weights of twelve apolipoprotein(a) variants, determined using haptoglobin 2-2 polymer molecular weight standards.

Apolipoprotein(a) [apo(a)] variants were characterized in 398 sera by immunoblotting: (a) by molecular weight, using a haptoglobin 2-2 polymeric series as standards, and (b) by nomenclature, using serum pools containing previously characterized apo(a) variants as standards. The haptoglobin 2-2 standard curve (172-859 kDa) alleviates the necessity of obtaining molecular weights by extrapolation. Among the 398 sera, 40.2% had double apo(a) bands (54 phenotypes), 58.0% had a single apo(a) band and 1.8% were null (no bands observed). An inverse, though non-monotonic, relationship was observed between apolipoprotein(a) molecular weight and serum lipoprotein(a) [Lp(a)] concentration. Due to the large size of apo(a) and the relatively small increment between variants (15-16 kDa), molecular weight could not be used alone to characterize variants. Even with a CV of 3-4%, there was an overlap between variant molecular weight estimates. However, in combination with the identification of variants by comparison with standards, the haptoglobin 2-2 standard curve could be used to obtain mean molecular weight estimates for each variant. 12 distinct variants were identified among the sera, with apparent mean molecular weights of 314, 388, 410, 433, 454, 466, 503, 519, 528, 543, 553 and 572 kDa, respectively. These molecular weight estimates are consistent with the theoretical molecular weight range for apo(a) variants, calculated from sequence and carbohydrate analysis, of 238-643 kDa.

Apolipoproteins A↗
PubMed1992

A flexible, efficient, checkerboard immunoblot system for the detection and semiquantitation of specific antinuclear antibodies.

In this paper an uncomplicated method for the simultaneous detection and semiquantitation of 11 of the 12 commonly studied antinuclear antibodies (ANA) in a single run is described. This new application of checkerboard immunoblotting (CBIB) is based upon available technology and employs purified antigens which can be either purchased or produced in-house. CBIB requires no electronic instrument, can be formatted to meet the needs of the user, is rapidly performed, and has acceptable labor and materials costs. Data on the use of the method to examine available reference antisera is presented. CBIB has also proven practical for the clinical study of 18 sera, at two dilutions per membrane, for each set of specific antinuclear antibodies, also at two or more dilutions.

Antibodies, Antinuclear↗
PubMed1991

An improved method for determining high molecular weights by protein immunoblotting: application to apolipoprotein(a) phenotyping.

The determination of very high molecular weights (greater than 500 kd) by protein immunoblotting is limited by a lack of commercially available molecular weight standards in that range. We have therefore investigated the use of the human haptoglobin 2-2 phenotype polymeric series as a high molecular weight standard curve. Using pre-electrophoresed 3-12% SDS polyacrylamide gradient gels, non-dissociated haptoglobin 2-2 serum was separated, transferred and probed with anti-human haptoglobin. We were able to visualize up to 13 bands, with a theoretical molecular weight range of 171.9-859.5 kd, in non-dissociated haptoglobin 2-2 serum. Molecular weights up to 584 kd were confirmed by comparison with available standards. The method was then applied to the determination of apolipoprotein(a) [apo(a)] phenotype molecular weights. Precision of the method was excellent, with intra-run CVs of less than 2.9% and inter-run CVs of less than 3.2% for apo(a) molecular weight. This method of molecular weight calibration is applicable to the characterization of any high molecular weight protein that can be successfully electrophoresed, transferred and visualized.

Apolipoproteins A↗
PubMed1991

International Federation of Clinical Chemistry standardization project for measurements of apolipoproteins A-I and B.

To minimize differences in apolipoprotein measurements among laboratories and methods, a standardization program involving common suitable reference material is needed. The Committee on Apolipoproteins of the International Federation of Clinical Chemistry initiated a collaborative study for the standardization of test systems for measuring apolipoproteins (apo) A-I and B, with 25 company laboratories and three research laboratories involved in apolipoprotein analysis to: (a) evaluate calibration differences among the test systems; (b) evaluate whether comparability of the data can be achieved with the use of frozen serum pools to recalibrate the different systems; and (c) evaluate and select suitable candidate reference material. We used 26 test systems for apo A-I and 28 for apo B. Relatively modest differences were found in calibration for apo A-I, but very wide differences were observed for apo B methods. After uniform calibration, the overall among-laboratory CV for apo B decreased from 19% to 6%. Three lyophilized serum preparations for apo A-I and three liquid-stabilized serum preparations for apo B were selected for further evaluation as candidate international reference materials.

Apolipoprotein A-I↗
PubMed1983

Elevated serum acute phase protein levels as predictors of disseminated breast cancer.

Serum levels of four acute phase proteins, alpha 1-acid glycoprotein (AAGP), alpha 1-antitrypsin (AAT), haptoglobin (Hpt), and C3, were measured prior to biopsy in 38 women subsequently shown to have Stage I and II breast cancer and prior to treatment in 16 women with Stage IV disease. Sixty-one women with benign and 28 women with no breast disease served as controls. Mean serum levels of all four proteins were significantly elevated in women with stage IV disease as compared to women with Stage I or II disease or controls. Normal versus elevated levels for each protein were defined and AAGP was found to be the single most sensitive predictor of disseminated disease among the four. AAGP was elevated in 81.3% of Stage IV, 25% of Stage II, 14.3% of Stage I, and 12.4% of controls. Women with multiple proteins elevated were most likely to have advanced stage disease. Composite analysis of all four proteins using number of proteins abnormal or logistic regression analysis gave results similar to AAGP, both showing increasing numbers of proteins abnormal with increasing stage of breast cancer. These results indicate that measurement of serum acute phase proteins may be useful in initial staging of breast cancer patients and in following patients for indications of disseminated disease.

Breast Neoplasms↗
PubMed1983

Disparity in antiserum avidity as measured by nephelometry, radial immunodiffusion, and column-binding strength.

When antiserum potency is being assessed, avidity is often inferred from affinity measurements or from measurements obtained from completely unrelated immunoassay techniques. The present experiments describe an instance where antibodies that have very high column-binding strength, obtained by elution from immobilized antigen with dissociating agents, have low avidity in nephelometry and radial immunodiffusion. This low avidity appears not to be due to molecular damage since the eluted antibodies do not contain molecular aggregates and retain their column-binding strength when reapplied to the immobilized antigen. We theorize that in nephelometry and radial immunodiffusion, lattice formation may require a heterogeneous population of antibodies and that the low avidity fractions may have too limited a specificity. We support this theory by combining two different populations of low avidity antibodies and finding that the combination produces a nephelometric response greater than that of either fraction alone.

Antibody Affinity↗
PubMed1982

Performance on immunoglobulin IgG, IgA, and IgM tests in CAP survey specimens.

The performance of participants in nine consecutive College of American Pathologists (CAP) Survey specimens during 1979-1981 are listed for IgA, IgG, and IgM. The majority of participants used either radial immunodiffusion (RID) (51.4%) or nephelometric kits (33.6 per cent) manufactured by major firms and reported results in mass units (93.5 per cent). When tabulated by kit, prominent differences in performance were noticed. Furthermore, definite trends in methods employed were evident during the two-year period.

Humans↗
PubMed1981

Serum protein changes in women with early breast cancer.

Lowered serum concentrations of albumin, IgG, IgM, and transferrin have been identified preoperatively in a population of otherwise healthy white women over age 40 with early stage breast cancer. Definition of low values for each of the four serum proteins has been arrived at via comparison with age-matched controls, consisting of disease-free women and women with benign breast lesions. Thus defined, low values for the individual serum proteins have been found to occur in malignant/control study subjects at the following frequencies: albumin 68%/4.7% (P less than 0.0001); IgG 56%/21% (P less than 0.02); IgM 59%/19% (P less than 0.001), and transferrin 50%/4.7% (P less than 0.0001). Among the relevant historical and pathologic data evaluated in addition to the presence or absence of malignancy, only age has been found significant in influencing serum protein concentrations, and this has been taken into account in analyzing results. Forty-four percent of study subjects subsequently found to have breast cancer have low concentrations of at least three of the four discriminant proteins simultaneously in the pre-operative sample. None of the controls have these findings. Twenty-nine percent of women with a malignant breast lesion and 9% of controls have simultaneously low concentrations of two of the four discriminant proteins. Using these measurements of serum proteins it thus becomes possible to assign risk of malignancy when a woman is found to have a breast mass.

Adult↗
PubMed1981

Method for testing antiserum titer and avidity in nephelometric systems.

Antiserum performance in a nephelometric system can be characterized by parameters derived from measuring reaction rates. The characterization process is derived from a series of dose-response curves (elicited nephelometric response vs antigen concentration) generated from various dilutions of the antiserum being tested. Antiserum titer can then be calculated by plotting the antigen concentration found at one-half the maximum nephelometric response (Hmax) of each dose-response curve (C50) vs the corresponding antiserum dilution. Antiserum avidity can be calculated by plotting Hmax against its corresponding antiserum concentration. After general expressions are determined for C50 and Hmax vs antiserum concentration, a single dose-response curve suffices for characterizing antisera with respect to titer and avidity. Direct evidence is provided for the validity of C50 and Hmax as measures of titer and avidity by correlating these parameters with antiserum binding strength and with the number of antibodies eluted from immobilized antigen. This method can be applied to evaluate and compare different antiserum lots having the same specificity, to identify reagent inadequacies by comparing antisera of different specificity, and to predict the optimal antiserum dilution to use in performing an assay.

Antigen-Antibody Complex↗