Marilyn F. Collins

Analytical evaluation of particle-enhanced immunonephelometric assays for C-reactive protein, serum amyloid A and mannose-binding protein in human serum.

Against a background of growing interest in more sensitive assays for quantifying various acute phase proteins, we evaluated the performance of recently developed tests for C-reactive protein (CRP), serum amyloid A (SAA) and mannose-binding protein (MBP) on the Behring nephelometer II (BN II). Sample results outside the calibration ranges of 3·5 to 220 mg/L for CRP, 3·3 to 215 mg/L for SAA and 0·09 to 5·6 mg/L for MBP were automatically re-measured at another dilution. The lower limits of detection were 0·01, 0·7 and 0·01 mg/L for CRP, SAA and MBP, respectively. The coefficients of variation (CV) for intra- (n ⩾ 20) and inter- (n ⩾ 15) assay precision were < 5·2% and < 8·5%, respectively, for the three proteins at concentrations representing low, normal and high. Linearity for each method was within 5% of the expected values throughout the calibration range. We observed no significant interference from bilirubin (up to 300 mg/L) or haemoglobin (up to 10 g/L) for the three tests. Method comparison studies performed for CRP and SAA yielded the following results: y (CRP on BN II) = 0·75x (ELISA, Hemagen) −0·25 mg/L (r = 0·981, Sy/x = 2·1 mg/L; y (SAA on BN II) = 1·44x (ELISA, Hemagen) −9·9 mg/L (r = 0·972, Sy/x = 6·9 mg/L), where ELISA is enzyme-linked immunosorbent assay. Reference intervals established in 261 adult blood donors (aged 36·2 ± 9·0 years) were found to be log-normal with 2·5th, 50th and 97·5th centiles of < 0·17, 100 and 10·1 mg/L for CRP, < 0·84, 2·10 and 9·70 mg/L for SAA; and 0·30, 1·28 and 4·10 mg/L for MBP. We observed no relationship with CRP concentration and age; however, SAA levels increased with age while MBP levels decreased. The BN II provides a simple, rapid and sensitive system for measuring CRP, SAA and MBP in human serum.

Maternal obesity and markers of inflammation in pregnancy.

OBJECTIVES To evaluate whether obesity is associated with changes in pro-inflammatory and immunomodulatory cytokines in pregnancy. METHODS We performed a cross-sectional study using maternal serum from the early second trimester to examine biomarkers associated with inflammation in relation to maternal body mass index (n=80 total). RESULTS Leptin and high sensitivity C-reactive protein were significantly different between groups and increased with increasing body mass index. MCP-1 was significantly increased in the morbidly obese mothers. Interleukin-2 exhibited a U-shaped relationship with body mass index; transforming growth factor-beta1 demonstrated a nonsignificant negative trend with body mass index; and the levels of hepatocyte growth factor and tumor necrosis factor-alpha did not differ appreciably between groups. CONCLUSIONS Maternal obesity in pregnancy is associated with changes in cytokines, protein hormones and acute phase proteins in the second trimester, with an increase in MCP-1 in the morbid obesity category, and an increase in Leptin and hsCRP with increasing BMI category.

Quantitation of immunoglobulin adsorption to plastics

Adsorption of 125I-labelled rabbit IgG to plastics (cellulose nitrate, polyallomer, polystyrene, polyvinyl) was primarily dependent on initial antibody concentration and, to a lesser extent, on time allowed for adsorption. The highest concentration tested (100 mug/ml) gave the highest quantities adsorbed after 18 h at room temperature. This concentration, however, gave the lowest percent adsorption (6.5 to 12.0%) of the initial amount. IgG concentration of 10 mug/ml resulted in 25.0 to 65.1% adsorption over the same time period; at 1 mug/ml, 47.0 to 96.6% of the initial amount was adsorbed. All of the plastics tested adsorbed IgG to approximately the same degree, with the exception of cellulose nitrate. This plastic adsorbed 32 to 49% less than the others, under maximal adsorption conditions.

Evaluation of the recombinant VlsE-based liaison chemiluminescence immunoassay for detection of Borrelia burgdorferi and diagnosis of Lyme disease.

ABSTRACT Recent efforts to improve the serologic diagnosis of Lyme disease have included the use of a synthetic peptide (C6) that reproduces the sequence of invariable region 6 of VlsE, the variable surface antigen of Borrelia burgdorferi. In the present study, the diagnostic performance of DiaSorins recombinant VlsE-based chemiluminescence immunoassay in 1,947 human serum samples was evaluated. Sensitivity was determined using two serum panels from the CDC. For panel I, we observed sensitivities of 68.4% and 75.6% for subjects with early, localized (n = 19) or disseminated (n = 41) disease, respectively. For panel II, we observed sensitivities of 61.5% and 100% for subjects with early (n = 26) or late-stage (n = 11) disease, respectively. We observed a specificity of 99.5% for healthy donors (n = 600) living either in regions of the United States where the disease is endemic or in regions where it is not endemic. Overall, specificity among 207 potentially cross-reactive sera from subjects who had other spirochetal infections, nonspirochetal infections including bacterial and viral infections, or autoimmune or neurologic disease; who were positive for rheumatoid factor or anti-mouse antibodies; or who had been previously vaccinated for Lyme disease was 93.7%. In a direct comparison of 1,038 prospectively collected samples for Lyme disease testing we observed a relative sensitivity of 70%, a relative specificity of 99.1%, and an overall agreement of 97.1% between the DiaSorin recombinant VlsE chemiluminescence immunoassay and the Immunetics peptide-based C6 enzyme-linked immunosorbent assay.

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.

Development of immunoturbidimetric assays for fourteen human serum proteins on the Hitachi 912.

Abstract Many laboratories rely on dedicated nephelometers or turbidimeters and commercial reagent kits for the evaluation of serum proteins. However, with growing emphasis on cost containment, laboratories are forced to seek additional operational efficiencies by capitalizing on the use of existing analyzers whenever possible. In the present paper we describe the development of immunoturbidimetric assays for routine analysis of 14 human serum proteins (α1-antitrypsin, α2-macroglobulin, albumin, apolipoproteins AI and B, complement components 3 and 4, haptoglobin, immunoglobulins A, G, and M, orosomucoid, prealbumin, and transferrin) on the Hitachi 912™, a general chemistry analyzer. With this system, we obtained excellent precision at levels corresponding to low, normal, and high physiologic concentrations of each protein (within-run imprecision CVs ≤3.4%, total imprecision CVs ≤4.1%). Linearity for each method was within 5% of the expected value throughout the calibration range, and method comparisons with either the Roche turbidimetric or Dade Behring nephelometric assays were in good agreement (r >0.97). We observed no significant interference from bilirubin (up to 718 μmol/l), hemoglobin (up to 8 g/l), triglyceride (up to 14.7 mmol/l) or rheumatoid factor (up to 4140 IU/ml). Calibration for the 14 protein assays was stable for at least 7 days and onboard refrigerated reagents were stable for at least 3 months. The instruments automated sample re-run feature minimized sample handling and helped to conserve specimens. In conclusion, the newly developed assays on the Hitachi 912™ offer high throughput (>250 tests per hour) without the associated cost of a dedicated instrument for protein assays.

Commutability of the CRM 470 C-reactive protein value in the Dade Behring N High Sensitivity CRP assay.

Abstract Certified Reference Material 470 (CRM 470) demonstrates commutability with both the manufacturers calibrator and with dilutions of serum pools in the Dade Behring N High Sensitivity assay for C-reactive protein (CRP). Both regression and back calibration show similar nonlinearity for all materials, largely due to the method of calibration curve fitting used in this assay. Significant differences in values among the currently available commercial assays can be largely overcome by using appropriate calibration curve fitting and the recommended value transfer protocol, which includes a minimum of two assay runs on each of at least 3 separate days, with weight correction of all reconstitutions and dilutions. An initial weight-corrected dilution should be made each day because of the relatively high level of CRP in CRM 470. In our opinion, the degree of nonlinearity, imprecision, and differences in values in currently available assays renders the use of fixed clinical decision cut-points questionable for high-sensitivity CRP. An alternative approach is suggested.

Serologic associations of anti-cytoplasmic antibodies identified during anti-nuclear antibody testing

Abstract Background: There are currently no guidelines concerning additional laboratory testing for specific autoantibodies among anti-nuclear antibody-negative sera with an anti-cytoplasmic staining pattern identified by indirect immunofluorescence assay. Moreover, few data are available that address this laboratory situation. Methods: We performed specific autoantibody assays in 200 sera with an anti-nuclear antibody titer ≤1:32 and a cytoplasmic titer (undefined staining pattern) of ≥1:64, identified sequentially in the course of routine anti-nuclear antibody testing. Results: A total of 85 sera (42.5%) were positive in one (n=57) or more (n=28) of the specific autoantibody tests performed. Autoantibodies identified were antimitochondrial (15%), antimicrosomal (13%), anti-neutrophil cytoplasmic (10%), anti-smooth muscle (6%), anti-parietal cell (4%), and extractable nuclear antigen (8.5%, including histones, SSA, SSB, Sm, Jo-1 or Scl-70). A positive result in one or more of these assays was more frequent at anti-cytoplasmic titers ≥1:1024 (77.8%) than at titers of 1:64–1:128 (7%) (χ2=25.3, p<0.001). Conclusions: The present data demonstrate that undefined anti-cytoplasmic staining in anti-nuclear antibody-negative sera is associated with, although not necessarily caused by, a high frequency and wide range of specific autoantibodies. Further work is needed before specific recommendations can be made concerning follow-up in subjects with this laboratory finding. Clin Chem Lab Med 2006;44:1283–6.