One-step homogeneous C-reactive protein assay for saliva

Background: Cardiovascular disease is the leading cause of death in the world. Human C-reactive protein (CRP) has been used in the risk assessment of coronary events. Human saliva mirrors the body's health and well-being and is non-invasive, easy to collect and ideal for third world countries as well as for large patient screening. The aim was to establish a saliva CRP reference range and to demonstrate the clinical utility of salivary CRP levels in assessing the coronary events in a primary health care setting. Methods: We have used a homogeneous bead based assay to detect CRP levels in human saliva. We have developed a rapid 15 min (vs 90 min), sequential, one-step assay to detect CRP in saliva. Saliva was collected from healthy volunteers (n = 55, ages 20-70 years) as well as from cardiac patients (n = 28, ages 43-86 years). Results: The assay incubation time was optimised from 90 min to 15 mm and generated a positive correlation (n = 29, range 10-2189 pg/mL, r2 = 0.94; Passing Bablok slope 0.885. Intercept 0, p>0.10), meaning we could decrease the incubation time and produce equivalent results with confidence. The mean CRP level in the saliva of healthy human volunteers was 285 pg/mL and in cardiac patients was 1680 pg/mL (p<0.01). Analysis of CRP concentrations in paired serum and saliva samples from cardiac patients gave a positive correlation (r2 = 0.84, p<0.001) and the salivary CRP concentration capable of distinguishing healthy from diseased patients. Conclusions: The results suggest that this minimally invasive, rapid and sensitive assay will be useful in large patient screening studies for risk assessment of coronary events. (C) 2011 Elsevier B.V. All rights reserved.

An immunodiagnostic assay for quantitation of specific ige to the major pollen allergen component, pas n 1, of the subtropical bahia grass

Background Pollens of the Panicoideae subfamily of grasses including Bahia (Paspalum notatum) are important allergen sources in subtropical regions of the world. An assay for specific IgE to the major molecular allergenic component, Pas n 1, of Bahia grass pollen (BaGP) would have immunodiagnostic utility for patients with pollen allergy in these regions. Methods Biotinylated Pas n 1 purified from BaGP was coated onto streptavidin ImmunoCAPs. Subjects were assessed by clinical history of allergic rhinitis and skin prick test (SPT) to aeroallergens. Serum total, BaGP-specific and Pas n 1-specific IgE were measured. Results: Pas n 1 IgE concentrations were highly correlated with BaGP SPT (r = 0.795, p < 0.0001) and BaGP IgE (r = 0.915, p < 0.0001). At 0.23 kU/l Pas n 1 IgE, the diagnostic sensitivity (92.4%) and specificity (93.1%) for the detection of BaGP allergy was high (area under receiver operator curve 0.960, p < 0.0001). The median concentrations of Pas n 1 IgE in non-Atopic subjects (0.01 kU/l, n = 67) and those with other allergies (0.02 kU/l, n = 59) showed no inter-group difference, whilst grass pollen-Allergic patients with allergic rhinitis showed elevated Pas n 1 IgE (6.71 kU/l, n = 182, p < 0.0001). The inter-Assay coefficient of variation for the BaGP-Allergic serum pool was 6.92%. Conclusions Pas n 1 IgE appears to account for most of the BaGP-specific IgE. This molecular component immunoassay for Pas n 1 IgE has potential utility to improve the sensitivity and accuracy of diagnosis of BaGP allergy for patients in subtropical regions.

Conformational epitopes on the diabetes autoantigen GAD65 identified by peptide phage display and molecular modeling

The major diabetes autoantigen, glutamic acid decarboxylase (GAD65), contains a region of sequence similarity, including six identical residues PEVKEK, to the P2C protein of coxsackie B virus, suggesting that cross-reactivity between coxsackie B virus and GAD65 can initiate autoimmune diabetes. We used the human islet cell mAbs MICA3 and MICA4 to identify the Ab epitopes of GAD65 by screening phage-displayed random peptide libraries. The identified peptide sequences could be mapped to a homology model of the pyridoxal phosphate (PLP) binding domain of GAD65. For MICA3, a surface loop containing the sequence PEVKEK and two adjacent exposed helixes were identified in the PLP binding domain as well as a region of the C terminus of GAD65 that has previously been identified as critical for MICA3 binding. To confirm that the loop containing tile PEVKEK sequence contributes to the MICA3 epitope, this loop was deleted by mutagenesis. This reduced binding of MICA3 by 70%. Peptide sequences selected using MICA4 were rich in basic or hydroxyl-containing amino acids, and the surface of the GAD65 PLP-binding domain surrounding Lys358, which is known to be critical for MICA4 binding, was likewise rich in these amino acids. Also, the two phage most reactive width MICA4 encoded the motif VALxG, and the reverse of this sequence, LAV, was located in this same region. Thus, we have defined the MICA3 and MICA4 epitopes on GAD65 using the combination of phage display, molecular modeling, and mutagenesis and have provided compelling evidence for the involvement of the PEVKEK loop in the MICA3 epitope.

Use of phage display technology to investigate allergen-antibody interactions

Phage display is an advanced technology that can be used to characterize the interactions of antibody with antigen at the molecular level. It provides valuable data when applied to the investigation of IgE interaction with allergens. The aim of this rostrum article is to provide an explanation of the potential of phage display for increasing the understanding of allergen- IgE interaction, the discovery of diagnostic reagents, and the development of novel therapeutics for the treatment of allergic disease. The significance of initial studies that have applied phage display technology in allergy research will be highlighted. Phage display has been used to clone human IgE to timothy grass pollen allergen Phl p 5, to characterize the epitopes for murine and human antibodies to a birch pollen allergen Bet v 1, and to elucidate the epitopes of a murine mAb to the house dust mite allergen Der p 1. The technology has identified peptides that functionally mimic sites of human IgE constant domains and that were used to raise antiserum for blocking binding of IgE to the FcεRI on basophils and subsequent release of histamine. Phage display has also been used to characterize novel peanut and fungal allergens. The method has been used to increase our understanding of the molecular basis of allergen-IgE interactions and to develop clinically relevant reagents with the pharmacologic potential to block the effector phase of allergic reactions. Many advances from these early studies are likely as phage display technology evolves and allergists gain expertise in its research applications.

Characterization of epitopes for virus-neutralizing monoclonal antibodies to Ross River virus E2 using phage-displayed random peptide libraries

Ross River virus (RRV) is the predominant cause of epidemic polyarthritis in Australia, yet the antigenic determinants are not well defined. We aimed to characterize epitope(s) on RRV-E2 for a panel of monoclonal antibodies (MAbs) that recognize overlapping conformational epitopes on the E2 envelope protein of RRV and that neutralize virus infection of cells in vitro. Phage-displayed random peptide libraries were probed with the MAbs T1E7, NB3C4, and T10C9 using solution-phase and solid-phase biopanning methods. The peptides VSIFPPA and KTAISPT were selected 15 and 6 times, respectively, by all three of the MAbs using solution-phase biopanning. The peptide LRLPPAP was selected 8 times by NB3C4 using solid-phase biopanning; this peptide shares a trio of amino acids with the peptide VSIFPPA. Phage that expressed the peptides VSIFPPA and LRLPPAP were reactive with T1E7 and/or NB3C4, and phage that expressed the peptides VSIFPPA, LRLPPAP, and KTAISPT partially inhibited the reactivity of T1E7 with RRV. The selected peptides resemble regions of RRV-E2 adjacent to sites mutated in neutralization escape variants of RRV derived by culture in the presence of these MAbs (E2 210-219 and 238-245) and an additional region of E2 172-182. Together these sites represent a conformational epitope of E2 that is informative of cellular contact sites on RRV.

Towards the pathogenesis of autoimmune liver disease

There have been recent improvements in the clinical understanding and definition of the major types of autoimmune liver disease. However, still lacking is knowledge of their prevalence and pathogenesis. Three areas of study are in progress in our laboratory. First, in type 1 autoimmune hepatitis, the search continues to identify a liver/disease-specific autoantigenic reactant. Using hepatocyte membrane preparations, immunoblotting has underlined the problem of distinguishing, among multiple reactants, those that may be causally rather than consequentially related to hepatocellular damage. Second, in primary biliary cirrhosis (PBC), the need for population screening to ascertain prevalence and detect preclinical cases can be met by a rapid automated procedure for detection, by specific enzyme inhibition in microtitre wells, of antibody (anti-M2) to the pyruvate dehydrogenase complex E2 subunit (PDC-E2). Third, the structure of the conformational epitope within the inner lipoyl domain of PDC-E2 is being investigated by screening random phage-displayed peptide libraries using PBC sera. This has yielded phage clones in which the sequence of the peptide insert portrays the structure of this epitope, as judged by clustering of PBC-derived sequences to particular branches of a guide-tree that shows relatedness of peptides, and by reactivity of selected phage clones with anti-PDC-E2. Thus phage display identifies a peptide 'mimotope' of the antibody epitope in the inner lipoyl domain of PDC-E2.

Multiple alignment and sorting of peptides derived from phage-displayed random peptide libraries with polyclonal sera allows discrimination of relevant phagotopes

Biopanning of phage-displayed random peptide libraries is a powerful technique for identifying peptides that mimic epitopes (mimotopes) for monoclonal antibodies (mAbs). However, peptides derived using polyclonal antisera may represent epitopes for a diverse range of antibodies. Hence following screening of phage libraries with polyclonal antisera, including autoimmune disease sera, a procedure is required to distinguish relevant from irrelevant phagotopes. We therefore applied the multiple sequence alignment algorithm PILEUP together with a matrix for scoring amino acid substitutions based on physicochemical properties to generate guide trees depicting relatedness of selected peptides. A random heptapeptide library was biopanned nine times using no selecting antibodies, immunoglobulin G (IgG) from sera of subjects with autoimmune diseases (primary biliary cirrhosis (PBC) and type 1 diabetes) and three murine ascites fluids that contained mAbs to overlapping epitope(s) on the Ross River Virus envelope protein 2. Peptides randomly sampled from the library were distributed throughout the guide tree of the total set of peptides whilst many of the peptides derived in the absence of selecting antibody aligned to a single cluster. Moreover peptides selected by different sources of IgG aligned to separate clusters, each with a different amino acid motif. These alignments were validated by testing all of the 53 phagotopes derived using IgG from PBC sera for reactivity by capture ELISA with antibodies affinity purified on the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the major autoantigen in PBC: only those phagotopes that aligned to PBC-associated clusters were reactive. Hence the multiple sequence alignment procedure discriminates relevant from irrelevant phagotopes and thus a major difficulty with biopanning phage-displayed random peptide libraries with polyclonal antibodies is surmounted.

Prospective external validation of an accelerated (2-h) acute coronary syndrome rule-out process using a contemporary troponin assay

Background: Recently there have been efforts to derive safe, efficient processes to rule out acute coronary syndrome (ACS) in emergency department (ED) chest pain patients. We aimed to prospectively validate an ACS assessment pathway (the 2-Hour Accelerated Diagnostic Protocol to Assess Patients with Chest Pain Symptoms Using Contemporary Troponins as the Only Biomarker (ADAPT) pathway) under pragmatic ED working conditions. Methods: This prospective cohort study included patients with atraumatic chest pain in whom ACS was suspected but who did not have clear evidence of ischaemia on ECG. Thrombolysis in myocardial infarction (TIMI) score and troponin (TnI Ultra) were measured at ED presentation, 2 h later and according to current national recommendations. The primary outcome of interest was the occurrence of major adverse cardiac events (MACE) including prevalent myocardial infarction (MI) at 30 days in the group who had a TIMI score of 0 and had presentation and 2-h TnI assays <99th percentile. Results: Eight hundred and forty patients were studied of whom 177 (21%) had a TIMI score of 0. There were no MI, MACE or revascularization in the per protocol and intention-to-treat 2-h troponin groups (0%, 95% confidence interval (CI) 0% to 4.5% and 0%, 95% CI 0% to 3.8%, respectively). The negative predictive value (NPV) was 100% (95% CI 95.5% to 100%) and 100% (95% CI 96.2% to 100%), respectively. Conclusions: A 2-h accelerated rule-out process for ED chest pain patients using electrocardiography, a TIMI score of 0 and a contemporary sensitive troponin assay accurately identifies a group at very low risk of 30-day MI or MACE.

Estimation of mineral-adhered biomass of Thiobacillusferrooxidans by protein assay-some problems and remedies

Enumeration of adhered cells of Thiobacillus ferrooxidans on sulphide minerals through protein assay poses problems due to interference from dissolved mineral constituents. The manner in which sulphide minerals such as pyrite, chalcopyrite, sphalerite, arsenopyrite and pyrrhotite interfere with bacterial protein estimation is demonstrated. Such interferences can be minimised either through dilution or addition of H2O2 to the filtrate after hot alkaline digestion of the biotreated mineral samples.

Pasteurella multocida detection by 5' Taq nuclease assay: a new tool for use in diagnosing fowl cholera

A 5′ Taq nuclease assay utilising minor groove binder technology and targeting the 16S rRNA gene was designed to detect Pasteurella multocida (the causative agent of fowl cholera) in swabs collected from poultry. The assay was first evaluated using pure cultures. The assay correctly identified four P. multocida taxonomic type strains, 18 P. multocida serovar reference strains and 40 Australian field isolates (17 from poultry, 11 from pigs and 12 from cattle). Representatives of nine other Pasteurella species, 26 other bacterial species (18 being members of the family Pasteurellaceae) and four poultry virus isolates did not react in the assay. The assay detected a minimum of approximately 10 cfu of P. multocida per reaction. Of 79 poultry swabs submitted to the laboratory for routine bacteriological culture, 17 were positive in the 5′ Taq nuclease assay, but only 10 were positive by culture. The other 62 swabs were negative for P. multocida by both 5′ Taq nuclease assay and culture. The assay is suitable for use in diagnosing fowl cholera, is more rapid than bacteriological culture, and may also have application in diagnosing P. multocida infections in cattle and pigs.

Validation of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Babesia bigemina Antibodies in Cattle

A competitive enzyme-linked immunosorbent assay (cELISA) based on a broadly conserved, species-specific, B-cell epitope within the C terminus of Babesia bigemina rhoptry-associated protein 1a was validated for international use. Receiver operating characteristic analysis revealed 16% inhibition as the threshold for a negative result, with an associated specificity of 98.3% and sensitivity of 94.7%. Increasing the threshold to 21% increased the specificity to 100% but modestly decreased the sensitivity to 87.2%. By using 21% inhibition, the positive predictive values ranged from 90.7% (10% prevalence) to 100% (95% prevalence) and the negative predictive values ranged from 97.0% (10% prevalence) to 48.2% (95% prevalence). The assay was able to detect serum antibody as early as 7 days after intravenous inoculation. The cELISA was distributed to five different laboratories along with a reference set of 100 defined bovine serum samples, including known positive, known negative, and field samples. The pairwise concordance among the five laboratories ranged from 100% to 97%, and all kappa values were above 0.8, indicating a high degree of reliability. Overall, the cELISA appears to have the attributes necessary for international application.

A comparison of a blocking ELISA and a haemagglutination inhibition assay for the detection of antibodies to Avibacterium (Haemophilus) paragallinarum in sera from artificially infected chickens

The ability of blocking ELISAs and haemagglutination-inhibition (HI) tests to detect antibodies in sera from chickens challenged with either Avibacterium (Haemophilus) paragallinarum isolate Hp8 (serovar A) or H668 (serovar C) was compared. Serum samples were examined weekly over the 9 weeks following infection. The results showed that the positive rate of serovar A specific antibody in the B-ELISA remained at 100% from the second week to the ninth week. In chickens given the serovar C challenge, the highest positive rate of serovar C specific antibody in the B-ELISA appeared at the seventh week (60% positive) and was then followed by a rapid decrease. The B-ELISA gave significantly more positives at weeks 2, 3, 7, 8 and 9 post-infection for serovar A and at week 7 post-infection for serovar C. In qualitative terms, for both serovar A and serovar C infections, the HI tests gave a lower percentage of positive sera at all time points except at 9 weeks post-infection with serovar C. The highest positive rate for serovar A HI antibodies was 70% of sera at the fourth and fifth weeks post-infection. The highest rate of serovar C HI antibodies was 20% at the fifth and sixth weeks post-infection. The results have provided further evidence of the suitability of the serovar A and C B-ELISAs for the diagnosis of infectious coryza.

Identification and immunogenicity of an immunodominant mimotope of Avibacterium paragallinarum from a phage display peptide library

Avibacterium paragallinarum is the causative agent of infectious coryza. The protective antigens of this important pathogen have not yet been clearly identified. In this paper, we applied phage display technique to screen the immunodominant mimotopes of a serovar A strain of A. paragallinarum by using a random 12-peptide library, and evaluated the immunogenicity in chickens of the selected mimotope. Polyclonal antibody directed against A. paragallinarum strain 0083 (serovar A) was used as the target antibody and phage clones binding to this target were screened from the 12-mer random peptide library. More than 50% of the phage clones selected in the third round carried the consensus peptide motif sequence A-DP(M)L. The phage clones containing the peptide motif reacted with the target antibody and this interaction could be blocked, in a dose-dependent manner, by A. paragallinarum. One of the peptide sequences, YGLLAVDPLFKP, was selected and the corresponding oligonucleotide sequence was synthesized and then inserted into the expression vector pFliTrx. The recombinant plasmid was transferred into an expression host Escherichia coli GI826 by electroporation, resulting in a recombinant E. coli expressing the peptide on the bacterial surface. Intramuscular injection of the epitope-expressing recombinant bacteria into chickens induced a specific serological response to serovar A. A. paragallinarum. The chickens given the recombinant E. coli showed significant protection against challenge with A. paragallinarum 0083. These results indicated a potential for the use of the mimotope in the development of molecular vaccines for infectious coryza.

Rapid and sensitive detection of Avibacterium paragallinarum in the presence of other bacteria using a 5' Taq nuclease assay: A new tool for diagnosing infectious coryza

A 5' Taq nuclease assay specific for Avibacterium paragallinarum was designed and optimized for use in diagnosing infectious coryza. The region chosen for assay design was one of known specificity for Av. paragallinarum. The assay detected Av. paragallinarum reference strains representing the three Page and the eight Kume serovars, and field isolates from diverse geographical locations. No cross-reactions were observed with other Avibacterium species, with other bacteria taxonomically related to Av. paragallinarum nor with bacteria and viruses likely to be present in swabs collected from suspected infectious coryza cases. The detection limit for the assay was 6 to 60 colony-forming units per reaction. Twenty-two out of 53 swabs collected from sick birds reacted in the 5' Taq nuclease assay, whereas Av. paragallinarum was not isolated from any of the swabs. All of the 22 swabs yielded other bacteria in culture. The presence of Av. paragallinarum in the swabs was also demonstrated by sequencing, thereby confirming the ability of the assay to detect Av. paragallinarum in the presence of other bacteria. The ability to quantify bacterial load in the swabs using the 5' Taq nuclease assay was demonstrated.

A PCR assay for gender assignment in dugong (Dugong dugon) and West Indian manatee (Trichechus manatus)

Gender assignment for some aquatic mammals in the field is difficult. Molecular sexing from tissue biopsies is possible as males are heterogametic. Here we describe a multiplex PCR assay that amplifies the male specific SRY gene and differentiates ZFX and ZFY gametologues in two sirenian species, dugong (Dugong dugon) and West Indian manatee (Trichechus manatus). The assay was validated with animals of known gender and proved accurate and robust to experimental failure.