Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics

BACKGROUND: Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification. RESULTS: Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies. CONCLUSION: The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings.     

Construction of linker-scanning mutations using a kanamycin-resistance cassette with multiple symmetric restriction sites

We demonstrate how a kanamycin-resistance (KmR) cassette flanked by polylinkers with multiple restriction sites can be used to introduce nucleotide (nt) sequence replacements into a region of interest. This method differs in two significant ways from traditional methods of linker mutagenesis. First, the presence of the KmR gene allows for selection of the polylinker, greatly facilitating formation of linker-containing molecules. Second, the polylinker with multiple restriction sites allows a given linker insertion to be combined with a second linker insertion in a variety of different ways and makes possible a range of novel nt to remain in the resulting linker replacement. The result of this flexibility is that fewer different molecules are needed to cover a region, and that relatively large replacements (greater than 40 nt) are possible. We have used this method to introduce a series of sequence replacements that span the mouse dihydrofolate reductase promoter region.     

Fetal microchimerism in kidney biopsies of lupus nephritis patients may be associated with a beneficial effect

IntroductionMicrochimeric male fetal cells (MFCs) have been associated with systemic lupus erythematosus, and published studies have further correlated MFC with lupus nephritis (LN). In the present study, we evaluated the frequency of MFC in the renal tissue of patients with LN.MethodsTwenty-seven renal biopsies were evaluated: Fourteen were from women with clinical and laboratory findings of LN, and thirteen were from controls. Genomic DNA was extracted from kidney biopsies, and the male fetal DNA was quantified using real-time quantitative polymerase chain reactions for the detection of specific Y chromosome sequences.ResultsMFCs were detected in 9 (64%) of 14 of patients with LN, whereas no MFCs were found in the control group (P = 0.0006). No differences in pregnancy history were found between patients with LN and the control group. Significantly higher amounts of MFCs were found in patients with LN with serum creatinine ≤1.5 mg/dl. Furthermore, women with MFCs had significantly better renal function at the time of biopsy (P = 0.03). In contrast, patients with LN without MFCs presented with more severe forms of glomerulonephritis (World Health Organization class IV = 60% and class V = 40%).ConclusionsOur data indicate a high prevalence of MFCs in renal biopsy specimens from women with LN, suggesting a role for MFCs in the etiology of LN. The present report also provides some evidence that MFCs could have a beneficial effect in this disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0615-4) contains supplementary material, which is available to authorized users.     

Comparison of cardiorespiratory responses of moderately trained men and women using two different treadmill protocols

In practice, the Bruce protocol is the most commonly used treadmill protocol to assess maximal oxygen consumption (V(.-)O2max). It has been suggested that a running protocol (e.g., Astrand) may elicit a comparatively higher V(.-)O2max and different cardiorespiratory responses when applied to moderately trained runners. Thus, the purpose of this study was to compare V(.-)O2max and other cardiorespiratory responses as elicited by the standard Bruce and a modified Astrand treadmill protocol in moderately trained runners. Fifteen women (age = 21 years, height = 171.5 cm, weight = 63 kg, and body fat = 18%) and 15 men (age = 26 years, height = 177 cm, weight = 72 kg, and body fat = 9%) who were moderately trained runners completed a standard Bruce and modified Astrand protocol (random order), separated by approximately 7 days. Heart rate, Borg ratings of perceived exertion, blood pressure, and pulmonary gas exchange variables were measured during the exercise tests using standard laboratory procedures. This study revealed V(.-)O2max values between the Bruce protocol (51.3 +/- 11.6 ml x kg(-1) x min(-1)) and modified Astrand (51.5 +/- 10.9 ml x kg(-1) x min(-1)) were not significantly different in either the men or the women. However, the Bruce protocol elicited significantly higher maximum treadmill time in men and maximum respiratory exchange ratio (RERmax) and maximum minute ventilation (VEmax) values in both genders. Conversely, the modified Astrand elicited a higher HRmax. These data suggest that V(.-)O2max in both moderately trained men and women runners is independent of treadmill protocol despite differences in HRmax, RERmax, and VEmax.     

Transformation with oligonucleotides creating clustered changes in the yeast genome

We have studied single-strand oligonucleotide (oligo) transformation of yeast by using 40-nt long oligos that create multiple base changes to the yeast genome spread throughout the length of the oligos, making it possible to measure the portions of an oligo that are incorporated during transformation. Although the transformation process is greatly inhibited by DNA mismatch repair (MMR), the pattern of incorporation is essentially the same in the presence or absence of MMR, whether the oligo anneals to the leading or lagging strand of DNA replication, or whether phosphorothioate linkages are used at either end. A central core of approximately 15 nt is incorporated with a frequency of >90%; the ends are incorporated with a lower frequency, and loss of the two ends appears to be by different mechanisms. Bases that are 5-10 nt from the 5' end are generally lost with a frequency of >95%, likely through a process involving flap excision. On the 3' end, bases 5-10 nt from the 3' end are lost about 1/3 of the time. These results indicate that oligos can be used to create multiple simultaneous changes to the yeast genome, even in the presence of MMR.     

Thematic review series: patient-oriented research. Imaging atherosclerosis: state of the art

The ability to image obstructive arterial disease brought about a revolution in clinical cardiovascular care; the development of newer technologies that image arterial wall thicknesses, areas, volumes, and composition allows valid imaging of atherosclerosis for the first time. Development of noninvasive imaging of atherosclerosis has further led to a quantum shift in research in the field by enabling the study of asymptomatic populations and thus allowing investigators to focus on preclinical disease without the many biases associated with the study of symptomatic patients. These noninvasive investigations have broad implications for clinical care as well. Coronary angiography, computed tomographic (CT) imaging of coronary calcium, intravascular ultrasound, multidetector CT angiography, B mode ultrasound of the carotid arteries, and MRI of the carotid arteries all have unique strengths and weaknesses for imaging atherosclerosis. Certain of these techniques are extremely useful as outcome variables for clinical trials, and others are uniquely useful as predictors of the risk of cardiovascular disease. All are informative in one way or another with regard to the role of plaque remodeling and composition in disease causation. CT and MRI technology are advancing very rapidly, and research and clinical uses of these imaging modalities promise to further advance our understanding of atherosclerosis and its prevention.     

Effects of nisin on growth of bacteria attached to meat

Nisin had an inhibitory effect on gram-positive bacteria (Listeria monocytogenes, Staphylococcus aureus, and Streptococcus lactis) but did not have an inhibitory effect on gram-negative bacteria (Serratia marcescens, Salmonella typhimurium, and Pseudomonas aeruginosa) attached to meat. Nisin delayed bacterial growth on meats which were artificially inoculated with L. monocytogenes or Staphylococcus aureus for at least 1 day at room temperature. If the incubation temperature was 5 degrees C, growth of L. monocytogenes was delayed for more than 2 weeks, and growth of Staphylococcus aureus did not occur. We also found that the extractable activity of nisin decreased rapidly when the meats were incubated at ambient temperatures and that this decrease was inversely related to the observed inhibitory effect. These findings disclosed that nisin delays the growth of some gram-positive bacteria attached to meat. However, nisin alone may not be sufficient to prevent meat spoilage because of the presence of gram-negative and other nisin-resistant gram-positive bacteria.     

X heterochromatin subdivision and cytogenetic analysis in Sciara coprophila (Diptera,Sciaridae)

Five new translocations recovered from irradiated sperm, each having a break-point in the proximal X heterochromatin, have been designated T23, T29, T32, T40, and T70. These translocations, together with Oak Ridge T1, make possible the precise localization of several genetic components including the X centromere, the controlling element, and the ribosomal cistrons. Only the data pertaining to centromere location are presented here. The ribosomal cistrons and controlling element will be dealt with separately. Full cytological details are given for each of the five translocations. The break-points on the X define three blocks of heterochromatin designated H₁, H₂, and H₃. Together they comprise the right arm of the X. H₃ is the smallest and forms the very end of the chromosome. H₁ lies immediately to the right of the centromere. In T23 and T70 the breakpoints are located between H₂ and H₃; in T29 and T32 between H₂ and H₁. In Oak Ridge T1 the break-point lies between H₁ and the centromere and in T40 between the centromere and the whole left arm of the chromosome. For the first time it has been possible to determine the exact breakpoints of the long paracentric inversion that is found on the X homologue.This series of papers is dedicated to Professor Sally Hughes-Schrader —cytologist, naturalist, scholar — who in her eighty-second year is still exulting in the wonders of chromosome behavior and still endowed with that understanding and grace which have heightened immeasurably the lives of those who have known her