Agar-Gel Precipitin-Inhibition Technique for C-Reactive Protein Determinations

An application of the agargel precipitin-inhibition technique of Ray and Kadull detects the C-reactive protein present in the acute-phase human sera. The sensitivity of this procedure is compared with the capillary tube-precipitin method of Selman and Halpern.     

Agar-Gel Precipitin-Inhibition Technique for C-Reactive Protein Determinations: II. Degree of Sensitivity and Reproducibility

The agar-gel precipitin-inhibition test (AGPI) for C-reactive protein determinations proved more sensitive than the capillary tube-precipitin procedure. Furthermore, titers of positive CRP sera were obtained by this AGPI technique. This technique was compared with other indices of infectivity, namely, the white blood cell count and the erythrocyte sedimentation rate. The ease and reproducibility of this recommended test procedure were demonstrated by technicians unfamiliar with this procedure.     

Agar-Gel Precipitin-Inhibition Technique for Histoplasmosis Antibody Determinations

The agar-gel precipitin-inhibition technique has been modified to detect antibodies of Histoplasma capsulatum in sera from human clinical cases and experimental animals infected with this organism. By use of this modified technique, the histoplasmosis can be detected more consistently and reliably than with the direct agar-gel diffusion test, and titers are comparable to those attained by the complement-fixation serological technique.     

Agar-Gel Precipitin-Inhibition Technique for Plague Antibody Determinations

An application of the agar-gel precipitin-inhibition technique we described previously can be used to detect plague antibodies in human and animal sera after a series of plague vaccine inoculations or after exposure to Pasteurella pestis. Determination of the minimal reacting concentrations of the plague antigen and antibody reagents, methods for combining reagents, and length of incubation periods are discussed.     

Agar-Gel Precipitin-Inhibition Test for Coccidioidomycosis: II. Serological Test Antigen Studies in Agar-Gel

In this study of saprophytic and parasitic growth-phase extracts of Coccidioides immitis, an antigen from the spherule culture supernatant fluid detected a specific antibody in heretofore serologically negative suspect coccidioidomycosis human sera when diffused in agar-gel. This antigen-antibody reaction occurred also in some of the serologically positive human coccidioidomycosis sera. This study indicates that this antigen-antibody reaction should be utilized as a possible routine serological test for complete serodiagnosis.     

Agar-Gel Precipitin Technique in Anthrax Antibody Determinations

A modification of the agar-gel precipitation inhibition technique of Thorne and Belton for detecting anthrax antibodies reduces inconsistency of visually determined end points on the same sera observed by different technicians. Determination of the minimal reacting concentrations of the anthrax antigen and antibody reagents, modifications of the visualization apparatus, methods for combining reagents, and length of incubation periods contribute to the ease of the end-point determinations and the uniformity of results. When compared with the previous technique, the modified procedure is less time-consuming while retaining satisfactory reproducibility, simplicity, specificity, and sensitivity.     

Agar-Gel Precipitin-Inhibition Test for Coccidioidomycosis: I. Preliminary Evaluation of the Complement-Fixation and Agar-Gel Precipitin Tests in the Serodiagnosis of Human Coccidioidomycosis

The agar-gel precipitin-inhibition serological test for coccidioidomycosis was a more sensitive indicator of Coccidioides immitis antibodies than the tube precipitin, the agar-gel immunodiffusion, in the complement-fixation tests in assaying monkey sera, whether these sera were from prechallenge-vaccinated or postchallenged animals. When applying this technique to the assay of human sera, an analogous finding generally persisted. However, some human sera were positive by the complement-fixation test and negative by the agar-gel precipitin-inhibition test. These sera were diffused in agar-gel against various coccidioidin complement-fixation, tube precipitin, and agar-gel precipitin-inhibition test antigens with essentially negative results.     

Maternal Serum C-Reactive Protein in Women with Preterm Prelabor Rupture of Membranes

Objective

This study evaluated maternal C-reactive protein (CRP) as a predictor of microbial invasion of the amniotic cavity (MIAC) and histological chorioamnionitis (HCA) in women with preterm prelabor rupture of the membranes (PPROM) before and after 32 weeks of gestation.

Methods

This study was a prospective observational cohort study of 386 women. Maternal serum CRP concentrations were evaluated, and amniotic fluid samples were obtained via transabdominal amniocentesis at the time of admission. Placentas underwent histopathological examination after delivery. MIAC was defined based on a positive PCR for Ureaplasma species, Mycoplasma hominis and Chlamydia trachomatis and/or positive 16S rRNA gene amplification. HCA was defined based on the Salafia classification.

Results

Maternal CRP was significantly higher in women with MIAC and HCA (median 9.0 mg/l) than in women with HCA alone (median 6.9 mg/l), MIAC alone (median 7.4 mg/l) and without MIAC or HCA (median 4.5 mg/l) (p<0.0001). CRP was a weak predictor of the occurrence of MIAC and HCA before and after 32 weeks of gestation. Only the 95^th percentile of CRP and PPROM before 32 weeks exhibited a false-positive rate of 1%, a positive predictive value of 90% and a positive likelihood ratio of 13.2 to predict MIAC and HCA. However, the low sensitivity of 15% limits the clinical utility of this detection.

Conclusion

CRP is a poor predictor of the occurrence of MIAC and HCA, even at early gestational ages.     

A Genetic Association Study of Serum Acute-Phase C-Reactive Protein Levels in Rheumatoid Arthritis: Implications for Clinical Interpretation

Background

The acute-phase increase in serum C-reactive protein (CRP) is used to diagnose and monitor infectious and inflammatory diseases. Little is known about the influence of genetics on acute-phase CRP, particularly in patients with chronic inflammation.

Methods and Findings

We studied two independent sets of patients with chronic inflammation due to rheumatoid arthritis (total 695 patients). A tagSNP approach captured common variation at the CRP locus and the relationship between genotype and serum CRP was explored by linear modelling. Erythrocyte sedimentation rate (ESR) was incorporated as an independent marker of inflammation to adjust for the varying levels of inflammatory disease activity between patients. Common genetic variants at the CRP locus were associated with acute-phase serum CRP (for the most associated haplotype: p = 0.002, p<0.0005, p<0.0005 in patient sets 1, 2, and the combined sets, respectively), translating into an approximately 3.5-fold change in expected serum CRP concentrations between carriers of two common CRP haplotypes. For example, when ESR = 50 mm/h the expected geometric mean CRP (95% confidence interval) concentration was 43.1 mg/l (32.1–50.0) for haplotype 1 and 14.2 mg/l (9.5–23.2) for haplotype 4.

Conclusions

Our findings raise questions about the interpretation of acute-phase serum CRP. In particular, failure to take into account the potential for genetic effects may result in the inappropriate reassurance or suboptimal treatment of patients simply because they carry low-CRP–associated genetic variants. CRP is increasingly being incorporated into clinical algorithms to compare disease activity between patients and to predict future clinical events: our findings impact on the use of these algorithms. For example, where access to effective, but expensive, biological therapies in rheumatoid arthritis is rationed on the basis of a DAS28-CRP clinical activity score, then two patients with identical underlying disease severity could be given, or denied, treatment on the basis of CRP genotype alone. The accuracy and utility of these algorithms might be improved by using a genetically adjusted CRP measurement. Please see later in the article for the Editors'' Summary     

The Prognostic Value of C-Reactive Protein Serum Levels in Patients with Uterine Leiomyosarcoma

Objective

C-reactive protein (CRP) has previously been shown to serve as a prognostic parameter in women with gynecologic malignancies. Due to the lack of valid prognostic markers for uterine leiomyosarcoma (ULMS) this study set out to investigate the value of pre-treatment CRP serum levels as prognostic parameter.

Methods

Data of women with ULMS were extracted from databases of three Austrian centres for gynaecologic oncology. Pre-treatment CRP serum levels were measured and correlated with clinico-pathological parameters. Univariate and multivariable survival analyses were performed.

Results

In total, 53 patients with ULMS were included into the analysis. Mean (SD) CRP serum level was 3.46 mg/dL (3.96). Solely, an association between pre-treatment CRP serum levels and tumor size (p = 0.04) but no other clinic-pathologic parameter such as tumor stage (p = 0.16), or histological grade (p = 0.07), was observed. Univariate and multivariable survival analyses revealed that CRP serum levels (HR 2.7 [1.1–7.2], p = 0.037) and tumor stage (HR 6.1 [1.9–19.5], p = 0.002) were the only independent prognostic factors for overall survival (OS) in patients with ULMS. Patients with high pre-treatment CRP serum levels showed impaired OS compared to women with low levels (5-year-OS rates: 22.6% and 52.3%, p = 0.007).

Conclusion

High pre-treatment CRP serum levels were independently associated with impaired prognosis in women with ULMS and might serve as a prognostic parameter in these patients.     

Gene-Specific DNA Methylation Association with Serum Levels of C-Reactive Protein in African Americans

A more thorough understanding of the differences in DNA methylation (DNAm) profiles in populations may hold promise for identifying molecular mechanisms through which genetic and environmental factors jointly contribute to human diseases. Inflammation is a key molecular mechanism underlying several chronic diseases including cardiovascular disease, and it affects DNAm profile on both global and locus-specific levels. To understand the impact of inflammation on the DNAm of the human genome, we investigated DNAm profiles of peripheral blood leukocytes from 966 African American participants in the Genetic Epidemiology Network of Arteriopathy (GENOA) study. By testing the association of DNAm sites on CpG islands of over 14,000 genes with C-reactive protein (CRP), an inflammatory biomarker of cardiovascular disease, we identified 257 DNAm sites in 240 genes significantly associated with serum levels of CRP adjusted for age, sex, body mass index and smoking status, and corrected for multiple testing. Of the significantly associated DNAm sites, 80.5% were hypomethylated with higher CRP levels. The most significant Gene Ontology terms enriched in the genes associated with the CRP levels were immune system process, immune response, defense response, response to stimulus, and response to stress, which are all linked to the functions of leukocytes. While the CRP-associated DNAm may be cell-type specific, understanding the DNAm association with CRP in peripheral blood leukocytes of multi-ethnic populations can assist in unveiling the molecular mechanism of how the process of inflammation affects the risks of developing common disease through epigenetic modifications.     

Association of Ideal Cardiovascular Metrics and Serum High-Sensitivity C-Reactive Protein in Hypertensive Population

Increased levels of the inflammatory biomarker high-sensitivity C-reactive protein (hs-CRP) are associated with increased risk of cardiovascular disease. However, ideal cardiovascular health indicates lower risk of cardiovascular disease. This study aimed to investigate the effect of ideal cardiovascular health behaviors and health factors on hs-CRP levels among a hypertensive population. From 2006 to 2007, a cross-sectional study was conducted to survey 41,476 hypertensive subjects among the employees of Kailuan Corporation. Data from unified questionnaires and blood biochemical examinations were collected. The effects of ideal cardiovascular health behaviors and health factors on hs-CRP levels were evaluated through multivariate logistic regression analysis. A negative correlation was observed between hs-CRP levels and the number of ideal cardiovascular health metrics. The mean hs-CRP levels of subjects with zero to one, two, three, and four to six ideal cardiovascular health metrics were 1.11, 0.96, 0.90, and 0.80 mg/L, respectively (P<0.01). Multivariate logistic regression analysis revealed that after adjustment for sex, age, triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and other risk factors, the risks for subjects with two, three, and four to six ideal health metrics with serum hs-CRP >3 mg/L were lower than those with zero to one ideal health metrics, with corresponding odd ratios of 0.86 (95%CI: 0.79–0.93, P<0.01), 0.76 (95%CI: 0.69–0.83, P<0.01), and 0.68 (95%CI: 0.64–0.75, P<0.01), respectively. This finding suggests that ideal cardiovascular health behaviors and health factors were related to decreased hs-CRP levels in a hypertensive population.

Clinical Trial Registration

Unique identifier: ChiCTR-TNC-11001489.     

The Impact of Baseline Serum C-Reactive Protein and C-Reactive Protein Kinetics on the Prognosis of Metastatic Nasopharyngeal Carcinoma Patients Treated with Palliative Chemotherapy

Background

The aim of this study was to determine whether baseline C-reactive protein (CRP) levels and CRP kinetics predict the overall survival in metastatic nasopharyngeal carcinoma (mNPC) patients.

Methods

A total of 116 mNPC patients from January 2006 to July 2011 were retrospectively reviewed. Serum CRP level was measured at baseline and thereafter at the start of each palliative chemotherapy cycle for all patients.

Results

Patients with higher values of baseline CRP (≥ 3.4 mg/L) had significantly worse survival than those with lower baseline CRP values (< 3.4 mg/L). Patients were divided into four groups according to baseline CRP and CRP kinetics: (1) patients whose CRP < 3.4 mg/L and never elevated during treatment; (2) patients whose CRP < 3.4 mg/L and elevated at least one time during treatment; (3) patients whose CRP ≥ 3.4 mg/L and normalized at least one time during treatment; and (4) patients whose CRP ≥ 3.4 mg/L and never normalized during treatment. The patients were further assigned to non-elevated, elevated, normalized, and non-normalized CRP groups. Overall survival rates were significantly different among the four groups, with three-year survival rates of 68%, 41%, 33%, and 0.03% for non-elevated, elevated, normalized, and non-normalized CRP groups respectively. When compared with the non-elevated group, hazard ratios of death were 1.69, 2.57, and 10.34 in the normalized, elevated, and non-normalized groups (P < 0.001).

Conclusions

Baseline CRP and CRP kinetics may be useful to predict the prognosis of metastatic NPC patients treated with palliative chemotherapy and facilitate individualized treatment. A prospective study to validate this prognostic model is still needed however.     

Standards for Immunohistochemical Imaging: A Protein Reference Device for Biomarker Quantitation

We are developing a reference device to be used in the validation of immunohistochemical imaging of biomarkers by microscopy. The prototype device consists of p53 protein immobilized at various concentrations on a glass slide. The device is designed as a reference control to be used with assays that incorporate commercially available anti-p53 antibodies. p53 protein was characterized by mass spectrometry and covalently immobilized through amide linkage to the (3-aminopropyl)trietoxysilane-modified glass surface. This procedure is reproducible and provides a chemically stable product in high yield. The surface-bound protein was shown to be immunoreactive by its specific interaction with anti-p53 antibody (Ab) and detection by absorbance and fluorescence spectroscopy. Also, comparison was made with microscopic images of Ab-stained tissue samples, known to stain positive for p53. Further development will be required to establish accurate surface protein concentrations in the range required for specific clinical applications. (J Histochem Cytochem 58:1005–1014, 2010)     

Obesity Associated Molecular Forms of C-Reactive Protein in Human

Objective

To describe novel C-reactive protein (CRP) molecular forms (mf) in human plasma.

Design and Methods

Five novel CRP-mfs, disctinct from the previously described native (nCRP) and modified (mCRP) C-reactive proteins, were separated from human plasma by polyacrylamide gel electrophoresis and immunodetected by western blot in subjects with or without increased BMI, cardiovascular disease (CVD), and diabetes (n = 1800).

Results

Three of the five CRP-mfs were present in all samples. One, CRPmf-4, was present in a subgroup of subjects and its presence was associated with elevated body mass index (BMI). CRP-mf-5 was present in about 2% of the subjects and was not associated with any other parameters. The presence or distribution of the 5 CRP-mfs were not Ca²⁺-dependent. Crossed immuno-localization experiments indicated that none of the CRP-mfs were complexed with any of the lipoprotein classes or with signature proteins of the complement-factor. Moreover, the distribution of CRP-mfs were not significantly correlated with plasma CRP levels. CRP-mf-4 was significantly associated with increased BMI, but not with other parameters of the metabolic syndrome (HDL-C and triglyceride levels, and diabetes).

Conclusions

We have identified five new CRP-mfs out of which CRP-mf-4 was significantly associated with obesity. We have shown that oligomerization of CRP was not calcium dependent. We hypothesize that adipose tissue produces a factor which influences the formation of CRP mf-4. CRP-mfs might be used as an obesity-associated inflammatory marker.     

A Comparison of Protein Quantitation Assays for Biopharmaceutical Applications

Dye-based protein determination assays are widely used to estimate protein concentration, however various reports suggest that the response is dependent on the composition and sequence of the protein, limiting confidence in the resulting concentration estimates. In this study a diverse set of model proteins representing various sizes of protein and covalent modifications, some typical of biopharmaceuticals have been used to assess the utility of dye-based protein concentration assays. The protein concentration assays (Bicinchoninic acid (BCA), Bradford, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA), DC, Fluorescamine and Quant-i) were compared to the 'gold standard' assay, quantitative amino acid analysis (AAA). The assays that displayed the lowest variability between proteins, BCA and DC, also generated improved estimates when BSA was used as a standard, when compared to AAA derived concentrations. Assays read out by absorbance tended to display enhanced robustness and repeatability, whereas the fluorescence based assays had wider quantitation ranges and lower limits of detection. Protein modification, in the form of glycosylation and PEGylation, and the addition of excipients, were found to affect the estimation of protein concentration for some of the assays when compared to the unmodified protein. We discuss the suitability and limitations of the selected assays for the estimation of protein concentration in biopharmaceutical applications.     

Serum C-Reactive Protein Distribution in Minimally Invasive Total Knee Arthroplasty Do Not Differ with Distribution in Conventional Total Knee Arthroplasty

Minimally invasive total knee arthroplasty (MITKA) has been developed to reduce surgical trauma and facilitate rehabilitation after arthroplasty. A plausible hypothesis is that this reduced trauma results in lower concentrations of circulating inflammatory biomarkers, such as C-reactive protein (CRP). In this study, we compared CRP concentrations in patients undergoing MITKA to those undergoing conventional TKA (CTKA). Eight hundred and seven patients undergoing MITKA were prospectively recruited. CRP was measured before operation and on days 2, 4, 21, and 42 after operation. Two hundred and forty-seven patients who had CTKA were collected retrospectively, with the same inclusion and exclusion criteria as those who had MITKA. We found in both groups, that CRP values rose abruptly after operation, with peak values reached on day 2 or 4. Values then declined so that by days 21 and 42 they were only modestly above baseline values. Throughout the entire study period, mean CRP in MITKA patients did not differ significantly from those in CTKA patients. However, a significantly higher proportion of CTKA patients than of MITKA patients had peak CRP values at day 4 rather than at day 2 (76.8% vs 42.5%), a difference that was more pronounced in women. Also, by day 42, CRP values were still above baseline in 18.5% of MITKA patients and 28.8% of CTKA patients without known complications. In conclusion, CRP distribution pattern was similar in patients who received MITKA or CTKA,. CRP values remained slightly elevated in both MITKA and CTKA patients for as long 42 days after operation. These findings suggest that MITKA is no less traumatic than CTKA, as determined by CRP values, and the patterns of postoperative CRP may be useful in the management of TKA patients.