Enzyme-linked Immunosorbent Assay Approach Research Articles

Comparative detection of goat's milk casein through various enzyme‐linked immunosorbent assay (ELISA) approaches using caprine alpha‐S1 casein peptides and bovine casein polyclonal antibodies

SummaryCasein causes allergic reactions in cow's milk protein allergy (CMPA) individuals. Casein can cross‐react with cows and goats. Hence, a rapid and highly specific technique is required for its detection. The study aims to evaluate and optimise multiple Enzyme‐Linked Immunosorbent Assay (ELISA) approaches for the detection of casein isolated from goat's milk. The study utilised and optimised various ELISA approaches, including direct, indirect and sandwich with various antibodies. Several parameters of ELISA including pre‐treatments of casein, blocking reagent, concentrations of antibodies and background signal were evaluated. The results showed that sandwich ELISA revealed a good signal‐to‐noise ratio at 1 mg mL−1. Indirect ELISA showed a higher signal‐to‐noise ratio without any heat treatment in comparison to the direct ELISA despite utilising an equivalent concentration (i.e., 10 μg mL−1) of goat's milk casein. The findings provide a theoretical foundation for obtaining a high signal‐to‐noise ratio for antigenicity studies of goat's milk casein to minimise its immunological response.

Evidence-based comparism of conventional sandwich enzyme-linked immunosorbent assay (ELISA) and lateral flow (LAT) method in the detection of human rotavirus pathogen for prompt monitoring and robust surveillance outcome

Introduction: Rotavirus has been known to be the causative agent of some cases of gastroenteritis across the world. A disease common in infants and young children ≤5 years of age, mostly in developing countries. This study was aimed at comparing the effectiveness of the sandwich ELISA and lateral flow method for the detection of rotavirus infection. Materials and methods: The human rotavirus sample (Code Number: BO218) at a concentration of 1x108/ml) was obtained from Dako A/S, Denmark and subsequently, a 1/4 serial dilution was performed to obtain varying concentrations. The already confirmed positive rotavirus samples obtained from the Nimi Briggs Hospital of the Rivers State University, Port Harcourt were used to evaluate and validate the sensitivity of both methods. Data obtained from ELISA method were analysed statistically using Microsoft excel and the spearman test to analyse the linear relationship between the absorbance and concentration of rotavirus expressed in number of rotavirus particles per ml. Results: The evidence-based results outcome showed that the ELISA approach was more sensitive to detecting the presence of rotavirus in the samples at the concentration of 381.5 rotavirus particle per ml, while that of LAT detected the presence of rotavirus at the concentration of 1,562,500 rotavirus particle per ml. The plot of absorbance and rotavirus particles following the serial dilution was plotted using Microsoft excel and the relationship between concentration and rotavirus was established. The result of the plot of the mean absorbance values against the rotavirus concentration (number of rotavirus per ml) showed an exponential increase in the absorbance value until a plateau was established as the concentration approached 1.0 × 108/ml of rotavirus in the solution. Also, the correlation of the absorbance and rotavirus concentration and a straight-line graph was plotted showing a correlation value of R2 =0.4505 with an intercept at 1.4236. Discussion: However, the ELISA is more robust, though very expensive for monitoring and surveillance of diarrhoea cases unlike the LAT method which is very cheap, with less expertise required, thus would be recommended for health care facilities in our remote communities.

Development and validation of ELISA for screening of Kratom (Mitragyna speciosa) habitual users using urinary AZ122 biomarker.

Kratom (Mitragyna speciosa Korth) has been used traditionally in Southeast Asia for its therapeutic properties. The major alkaloid of kratom, mitragynine, binds to opioid receptors to give opioid-like effects that causes addiction. In our previous study, we have identified AZ122 as a unique biomarker in habitual or regular kratom users through analysis of their urinary protein profiles. We aimed to develop and validate a screening method by means of enzyme-linked immunosorbent assay (ELISA) for detection of kratom habitual users. An ELISA approach was applied for the development of a screening method using urinary AZ122 as biomarker. Method validation was carried out using three quality control materials at different concentration of AZ122. The data was analyzed statistically using SPSS (Version 25). The ELISA was presented with Pearson correlation coefficient of 0.9993. The repeatability and reproducibility were presented at CV <7%, while the accuracy ranged from 78 to 96% at various AZ112 concentrations. Upon testing on 176 male respondents (n=88 regular kratom users and n=88 healthy controls), the specificity and sensitivity of the assay were both 100%. The ELISA has been validated and can be potentially used as a reliable screening test for detection of kratom habitual users.

Sensitive plasmonic ELISA assay based on butyrylcholinesterase catalyzed hydrolysis for the detection of Staphylococcus aureus

Enzyme-linked immunosorbent assay (ELISA) is one of the most powerful techniques for biomolecule detection, but suffers from insufficient sensitivity and relatively tedious operation. In this paper, we present a highly sensitive ELISA approach for Staphylococcus aureus (S. aureus) determination. S. aureus is first captured by vancomycin (Van) and butyrylcholinesterase (BChE) co-functionalized magnetic beads (MBs), and then immobilized on a 96-well plate through the binding with the precoated IgG. Afterwards, gold nanoparticles (AuNPs) and butyrylthiocholine (BTCh) are added, allowing BChE to catalyze BTCh into thiocholine (TCh). The presence of TCh triggers the aggregation of AuNPs, resulting in distinct color change from red to purple. Therefore, a sensitive quantification method for S. aureus can be developed based on the correlation between the amount of S. aureus and the color change of AuNPs. Under optimal conditions, naked eye detection can be realized at S. aureus down to 100 CFU mL−1. A limit of detection of 5 CFU mL−1 can be reached with microplate reader measurement, along with a dynamic range of 5–102 CFU mL−1 and 102-107 CFU mL−1. The system is selective towards S. aureus and shows resistance to other bacteria. By taking advantage of the high sensitivity of enzymatic reaction and AuNPs-based colorimetric method, this method greatly improves the dynamic range and sensitivity, providing alternative approach for the sensitive detection of S. aureus.

Highly accurate multiprotein detection on a digital ELISA platform.

The emerging single-molecule detection platform digital enzyme-linked immunosorbent assay (ELISA) can detect numerous proteins simultaneously at serum concentrations as low as picograms per milliliter. We sought to improve cytokine detection with this platform to aid diagnosis of conditions such as allergy and asthma. We developed a multiple single-molecule detection digital ELISA approach, through the application of encoded magnetic microbeads to simultaneously detect three cytokines in one serum sample. We tested the approach's utility to distinguish asthma-related cytokines in children. Concentrations of interleukin-4 (IL-4) and IL-6 were significantly higher in children with asthma than in healthy controls, while the concentration of interferon-γ (IFN-γ) was significantly lower. Our method has higher accuracy than conventional methods, and our results indicate that the proposed improved high-sensitivity digital ELISA-based diagnosis approach can facilitate early detection and treatment of childhood asthma or related diseases.

Development of a Dual ELISA for the Detection of CD2v-Unexpressed Lower-Virulence Mutational ASFV.

African swine fever virus (ASFV) is an important viral pathogen infecting pigs worldwide throughout the pig industry. CD2v (an outer-membrane glycosylated protein of ASFV)-unexpressed lower-virulence mutants have appeared in China and other countries in recent years. Using OIE-recommended quantitative PCR and ELISA methods, people can accurately judge whether pigs are infected with wild-type ASFV. However, the strategy has failed to distinguish ΔCD2v lower-virulence mutants and wild-type ASFV infection. Here, we expressed and purified the CD2v and p30 proteins via CHO cells and successfully established a dual enzyme-linked immunosorbent assay (ELISA), which can be used to differentiate pigs infected with wild-type ASFV or with CD2v-unexpressed lower-virulence mutants. The dual ELISA showed excellent specificity without cross-reactions with antibodies of PRRSV, CSFV, JEV, PRV, or PPV. The dual ELISA could detect ASFV-infected positive serum samples up to dilutions of 5120 times, possessing high sensitivity. Therefore, the application of this dual ELISA approach can play an important role in ASFV epidemiology study and fill the gaps in differential diagnosis.

Development of an enzyme-linked immunosorbent assay for Keap1-Nrf2 interaction inhibitors identification

Development of Keap1–Nrf2 interaction inhibitors is a promising strategy for the discovery of therapeutic agents against oxidative stress-mediated diseases. Two motifs of Nrf2, ETGE and DLG motif, are responsible for Keap1-Nrf2 binding. Previously, ETGE peptide or ETGE-derived peptide-based approaches were used to detect Keap1-Nrf2 interaction; however, these approaches are not able to monitor Keap1-DLG motif binding. We first report here a novel Enzyme-linked Immunosorbent Assay (ELISA) approach to detect the protein-protein interaction of full length Keap1 and Nrf2. In our assay, the test compounds can target either ETGE or DLG binding site, therefore facilitating the exploration of diverse Keap1-Nrf2 inhibitors. Three FDA-approved drugs, zafirlukast, dutasteride and ketoconazole, were found to inhibit the Keap1-Nrf2 interaction with IC50 of 5.87, 2.81 and 1.67 μM, respectively. Additionally, these three drugs also activated Nrf2 pathway in neuroblasts and lipopolysaccharide (LPS)-challenged mice. The results presented here indicate that the ELISA approach has the capacity to identify Keap1-Nrf2 inhibitors.

Evaluation of biobanked blood spot cards to detect cytokines in blood

Background & Aim Biobanks are routinely tasked with investigating new ways to store biospecimens without restricting which analytes are available. We sought to verify if very low concentration, physiologically important proteins, such as TNFα could be eluted from dried blood spot cards. While DNA and proteins are commonly extracted from biobanked samples stored as dried blood spots, it is unclear if such very low concentration proteins can be detected in suitable quality and quantity for further analysis. We chose to detect using an enzyme-linked immunosorbent assay (ELISA) approach, as ELISA is a more cost-effective approach than mass spectrometry and multiplex proximity extension assays. Methods, Results & Conclusion Different known concentrations of TNFα were loaded onto blood spot cards. After the card dried, protein was eluted from the card and quantified using an ELISA approach. It was determined that TNFα could be eluted from dried blood spot cards and quantified using ELISA. Samples eluted with a recovery of 30.1- 72.6% at concentrations on a dried blood spot between 78.13-312.5pg/ml. However, the assay's ability to detect TNFα as concentrations decreased, resulting in a decrease in the efficiency of protein recovery amount. Further refinement continues to confirm if this methodology can detect protein concentrations at common physiologic and pathologic levels of 5-27pg/ml.

Development, Optimization and Validation of a Sandwich ELISA for Detection of Recombinant Human Factor VII

Recombinant human factor VII (rh-FVII) is produced by engineered BHK cells at low dose. Accordingly, establishment of a precise method is crucial to reliably measuring expression level of this protein during manufacturing pro-cesses. We developed and established a reproducible sandwich enzyme-linked immunosorbent assay (ELISA) method for measuring amount of FVII during biopharmaceutical in upstream and downstream processes of Aryo-Seven. A sandwich ELISA was designed using two different high affinity mon-oclonal antibodies (mAb1 and mAb2) against h-FVII. The bounded FVII to the first antibody was revealed by the use of a second mouse anti-FVII monoclo-nal antibody (1F1-B11), labeled with HRP that binds to another antigenic determinant of the FVII. Then, validation was done by determination of spec-ificity, linearity, accuracy, precision, reproducibility, detection and quantifica-tion limit and robustness according to ICH Q2 (R1) guideline. In developed ELISA, no interference was found between FVII and proteins derived from BHK which commonly exist in the supernatant. Linear range of detection was from 25-1.56 ng/mL with P-Value &lt;0.001. In accuracy, spiked samples showed 109±2% recovery. Intra and intermediate precision assays showed %RSD not more than20. Detection limit of this assay was 0.99 ng /mL and limit of quantification was 2.99 ng/ml. The sandwich ELISA was found to be useful tool for measuring FVII/ FVIIa. The ELISA approach was precise, repro-ducible, and accurate. The ELISA therefore, is offered as an assured kit for detection of recombinant human factor.

Cold-microwave enhanced enzyme-linked immunosorbent assays—A path to high-throughput clinical diagnostics

The enzyme-linked immunosorbent assay (ELISA) constitutes an important clinical diagnostic approach. However, the prolonged incubation times involved lead to turnaround times of typically ⩾1day, potentially delaying a definitive diagnosis or an adequate treatment plan for individual patients. Here cold-microwave technology (CMT) was employed to significantly reduce the times required for diagnostic ELISAs. The new approach was validated and compared to a conventional ELISA setup measuring canine calprotectin (cCP). Canine serum and fecal specimens were used for the analytical validation of cCP ELISA by conventional and CMT–ELISA. Cross-validation of both ELISA methods consisted of the determination of analytic sensitivity, linearity, accuracy, precision, and reproducibility. The long-term stability of antibody-coated ELISA plates was also evaluated up to 33days. The ELISA approaches were comparable to each other. The observed-to-expected ratios for linearity and accuracy were 100.2±11.8 and 98.1±10.8% (mean±standard deviation), respectively. Precision and reproducibility were ⩽17.2%. For samples run on precoated ELISA plates over 33days %CVs were ⩽12.5%. While both ELISA approaches were analytically sensitive, linear, accurate, precise, and reproducible with measurements of cCP concentrations, CMT–ELISA offered a reduction in incubation times by 90–95%, facilitating a very fast turnaround time and suggesting CMT–ELISA for improved human and veterinary clinical diagnostics.

Renal Heparan Sulfate Proteoglycans Modulate Fibroblast Growth Factor 2 Signaling in Experimental Chronic Transplant Dysfunction

Depending on the glycan structure, proteoglycans can act as coreceptors for growth factors. We hypothesized that proteoglycans and their growth factor ligands orchestrate tissue remodeling in chronic transplant dysfunction. We have previously shown perlecan to be selectively up-regulated in the glomeruli and arteries in a rat renal transplantation model. Using the same model, here we present quantitative RT-PCR profiling data on proteoglycans and growth factors from laser-microdissected glomeruli, arterial tunicae mediae, and neointimae at 12 weeks after transplantation. In glomeruli and neointimae of allografts, selective induction of the matrix heparan sulfate proteoglycan perlecan was observed, along with massive accumulation of fibroblast growth factor 2 (FGF2). Profiling the heparan sulfate polysaccharide side chains revealed conversion from a non-FGF2-binding heparan sulfate phenotype in control and isografted kidneys toward a FGF2-binding phenotype in allografts. In vitro experiments with perlecan-positive rat mesangial cells showed that FGF2-induced proliferation is dependent on sulfation and can be inhibited by exogenously added heparan sulfate. These findings indicate that matrix proteoglycans such as perlecan serve as functional docking platforms for FGF2 in chronic transplant dysfunction. We speculate that heparin-like glycomimetics could be a promising intervention to retard development of glomerulosclerosis and neointima formation in chronic transplant dysfunction.

Determination of polychlorinated biphenyls in soil and sediment by selective pressurized liquid extraction with immunochemical detection

A selective pressurized liquid extraction (SPLE) method was developed for a streamlined sample preparation/cleanup to determine Aroclors and coplanar polychlorinated biphenyls (PCBs) in soil and sediment. The SPLE was coupled with an enzyme-linked immunosorbent assay (ELISA) for an effective analytical approach for environmental monitoring. Sediment or soil samples were extracted with alumina, 10% AgNO3 in silica, and sulfuric acid impregnated silica with dichloromethane at 100°C and 2000psi. The SPLE offered simultaneous extraction and cleanup of the PCBs and Aroclors, eliminating the need for a post-extraction cleanup prior to ELISA. Two different ELISA methods: (1) an Aroclor ELISA and (2) a coplanar PCB ELISA were evaluated. The Aroclor ELISA utilized a polyclonal antibody (Ab) with Aroclor 1254 as the calibrant and the coplanar PCB ELISA kit used a rabbit coplanar PCB Ab with PCB-126 as the calibrant. Recoveries of Aroclor 1254 in two reference soil samples were 92±2% and 106±5% by off-line coupling of SPLE with ELISA. The average recovery of Aroclor 1254 in spiked soil and sediment samples was 92±17%. Quantitative recoveries of coplanar PCBs (107–117%) in spiked samples were obtained with the combined SPLE–ELISA. The estimated method detection limit was 10ngg−1for Aroclor 1254 and 125pgg−1 for PCB-126. Estimated sample throughput for the SPLE–ELISA was about twice that of the stepwise extraction/cleanup needed for gas chromatography (GC) or GC/mass spectrometry (MS) detection. ELISA-derived uncorrected and corrected Aroclor 1254 levels correlated well (r=0.9973 and 0.9996) with the total Aroclor concentrations as measured by GC for samples from five different contaminated sites. ELISA-derived PCB-126 concentrations were higher than the sums of the 12 coplanar PCBs generated by GC/MS with a positive correlation (r=0.9441). Results indicate that the SPLE–ELISA approach can be used for quantitative or qualitative analysis of PCBs in soil and sediments. To our knowledge, this is the first report of an SPLE–ELISA approach not requiring a post-extraction cleanup step for detecting Aroclors and coplanar PCBs in soil and sediment.

Circulating autoantibody to FOXP3 may be a potential biomarker for esophageal squamous cell carcinoma

The present study was undertaken to develop a relatively quantitative enzyme-linked immunosorbent assay (ELISA) in-house using human leukocyte antigen class II-restricted epitopes in order to test circulating autoantibodies to human forkhead/winged helix transcription factor (FOXP3) as a biomarker for esophageal cancer. A total of 97 patients with esophageal squamous cell carcinoma (ESCC) and 227 healthy subjects were recruited for this study, and their plasma samples were collected for antibody analysis with the ELISA approach. Student's t test showed that the anti-FOXP3 IgG antibody levels were significantly higher in the patient group than the control group (t=6.23, P<0.0001). Based on a cutoff value determined by the mean+3SD of control IgG levels, the positive rate was 5.15 % in patients with ESCC as compared to 0.88 % in control subjects (X (2) =6.53, P=0.019, OR=5.85, 95 % CI 1.12-30.67), in which patients at stage I had the highest positivity (11.54 %, X (2) =12.15, P=0.0005, OR=13.10, 95 % CI 2.09-82.04). The sensitivity against >95 % specificity was 22.7 % for the IgG assay with an inter-assay deviation of 13.35 %. This work suggests that circulating IgG autoantibody to FOXP3 may be a potential biomarker for early diagnosis of esophageal cancer.

Circulating autoantibody to CD25 may be a potential biomarker for early diagnosis of esophageal squamous cell carcinoma

The present study was undertaken to develop an in-house enzyme-linked immunosorbent assay (ELISA) using human leukocyte antigen class II restricted epitopes to test circulating autoantibodies to CD25 as a biomarker for esophageal cancer. A total of 97 patients with esophageal squamous cell carcinoma (ESCC) and 226 healthy subjects were recruited for this study and their plasma samples were collected for antibody analysis with the ELISA approach. Mann-Whitney U test showed that the IgG anti-CD25 antibody level was significantly higher in the patient group than the control group (P < 0.001) while the IgA antibody level was not significantly different between these two groups (P = 0.361). Spearman correlation analysis failed to reveal a significant correlation between the levels of anti-CD25 IgG and IgA antibodies in either the patient group (r = -0.027, P = 0.797, n = 94) or the control group (r = 0.055, P = 0.429, n = 209). The sensitivity against >90 % specificity was 37.2 % for the IgG assay with an inter-assay deviation of 9.4 %, and 8.2 % for the IgA assay with an inter-assay deviation of 13.0 %. Based on a cut-off value determined by the 99th percentile of control IgG levels, the positive rate was 7.4 % in patients with ESCC, in which patients at stage I had the highest positivity (11.5 %) (χ (2) = 11.10, P = 0.001, OR = 12.12, 95 % CI 1.93-75.94). This work suggests that circulating IgG autoantibody to CD25 may be a potential biomarker for early diagnosis of esophageal cancer.

Validation of cold plasma treatment for protein inactivation: a surface plasmon resonance-based biosensor study

Gas plasma is being proposed as an interesting and promising tool to achieve sterilization. The efficacy of gas plasma to destroy bacterial spores (the most resistant living microorganisms) has been demonstrated and documented over the last ten years. In addition to causing damage to deoxyribonucleic acid by UV radiation emitted by excited species originating from the plasma, gas plasma has been shown to promote erosion of the microorganism in addition to possible oxidation reactions within the microorganism. In this work, we used lysozyme as a protein model to assess the effect of gas plasma on protein inactivation. Lysozyme samples have been subjected to the flowing afterglow of a gas discharge achieved in a nitrogen–oxygen mixture. The efficiency of this plasma treatment on lysozyme has been tested by two different assays. These are an enzyme-linked immunosorbent assay (ELISA) and a surface plasmon resonance (SPR)-based biosensor assay. The two methods showed that exposure to gas plasma can abrogate lysozyme interactions with lysozyme-specific antibodies, more likely by destroying the epitopes responsible for the interaction. More specifically, two SPR-based assays were developed since our ELISA approach did not allow us to discriminate between background and low, but still intact, quantities of lysozyme epitope after plasma treatment. Our SPR results clearly demonstrated that significant protein destruction or desorption was achieved when amounts of lysozyme less than 12.5 ng had been deposited in polystyrene 96-well ELISA plates. At higher lysozyme amounts, traces of available lysozyme epitopes were detected by SPR through indirect measurements. Finally, we demonstrated that a direct SPR approach in which biosensor-immobilized lysozyme activity is directly measured prior and after plasma treatment is more sensitive, and thus, more appropriate to define plasma treatment efficacy with more certainty.

Blood platelets activate the classical pathway of human complement.

Activation of the complement system plays a key role in inflammation associated with vascular injury. Recently, platelet P-selectin was shown to activate C3 via the alternative pathway of human complement. As platelets also posses binding sites for C1q, the recognition unit of the classical complement pathway, the present study examined classical pathway activation on platelets. Complement activation was assessed by either a solid phase enzyme-linked immunosorbent assay (ELISA) or flow cytometry. Using the ELISA approach, 2- to 10-fold increases (P < 0.001) in C1q and C4d deposition were demonstrated on adherent platelets following exposure (60 min 37 degrees C) to diluted (1/10) human plasma or serum. Similar results were obtained by flow cytometry using activated platelets in suspension. C1q and C4d deposition on platelets was accompanied by an approximately 4-fold increase in fluid phase C4d and C3a generation. Consistent with activation of the classical complement pathway, C4 cleavage failed to occur in serum depleted of C1q but was unchanged in factor B deficient serum. C4 activation was enhanced by platelet stimulation using chemical (SFLLRN peptide) or mechanical (shear) means, and decreased following platelet exposure to plasmin. These treatments were accompanied by changes in platelet surface gC1qR/p33 expression, a cellular C1q binding protein. In purified systems, recombinant gC1qR/p33 supported C4 activation, in a C1q dependent manner. These data provide the first evidence for C1q dependent classical complement pathway activation on platelets, and support a role for gC1qR/p33 in this process. However, monoclonal antibodies (mAb) to gC1qR/p33 produced only modest (20% +/- 8%, mean +/- SD, n = 5) reductions in C4 activation on platelets. Thus, further studies are required to investigate the involvement of additional platelet membrane constituents in classical complement pathway activation.

A novel sandwich ELISA for α1 domain based detection of soluble HLA-G heavy chains

The detection of soluble human leukocyte antigen G (HLA-G) has been a technically demanding task for several years now and various enzyme linked immunosorbent assay (ELISA) formats have been designed. However, no ELISA test has been described so far which is able to detect all possible kinds of soluble HLA-G (sHLA-G) molecules that might occur in bio fluids. Here we describe a new ELISA approach able to recognize soluble α1 domain containing heavy chains of all HLA-G isoforms. The detection limit is shown to be at about 150 pg soluble recombinant HLA-G1 heavy chain per milliliters. Detectable HLA-G fragments are shown to occur in the supernatants of different HLA-G transfected cell lines and appear to be particularly abundant in supernatant of trophoblast derived choriocarcinoma cell lines. The novel ELISA employs the well characterized HLA-G mAbs 4H84 and MEM-G1 which ensure high HLA-G specificity. A negative control ELISA format, designed against non-existing analytes, has been established to reveal non-specific signal interference.

Interaction of actin with the capping protein, CapZ from sea bass ( Dicentrarchus labrax) white skeletal muscle

We have compared the functional properties of CapZ from fish white skeletal muscle with those of CapZ from chicken muscle. CapZ is a heterodimer, which enhances actin nucleation and inhibits the depolymerization process by binding to the barbed ends of microfilaments. Here, we report the interaction of CapZ not only with F-actin, but also with monomeric actin. The affinity of sea bass CapZ for G-actin estimated by enzyme-linked immunosorbent assay (ELISA) was in the μM range. This association was PIP 2 dependent. Binding contacts with the barbed end of actin were delimited by both ELISA and fluorescence approaches. One site (actin sequence 338–348) was located in a helical region of the subdomain 1, region already implicated in the interaction with other actin binding proteins such as gelsolin. Another site implicates the C-terminal region (sequence 360–372) of actin. Finally, the partial competition of antibodies directed against CapZ α or β-subunits towards CapZ interaction with actin filaments suggests both subunits participate in the complex with actin.

Improved detection of mycobacterial antigens in clinical specimens by combined enzyme-linked immunosorbent assays

Three types of antibodies against cellular and secretory-excretory protein antigens were simultaneously used for the direct detection of mycobacterial antigens in sputum and cerebrospinal fluid (CSF) specimens, using enzyme-linked immunosorbent assay (ELISA). The antibodies consisted of in-house raised and prepared anti-whole-cell, heat-killed, and sonicated Mycobacterium tuberculosis, anti-secretory-excretory protein extract of bacilli Calmette-Guerin (BCG) strain, and commercially available anti-BCG. Sputum specimens comprised 24 smear positive, culture positive, and 47 smear-negative, culture positive (SNCP), from patients with pulmonary tuberculosis, as well as 45 smear-negative, culture-negative (SNCN) control samples. The CSF specimens included 18 SNCPs from patients with tuberculous meningitis and 18 SNCN controls. The sensitivity of the individual tests for sputum and CSF specimens ranged from 70% to 79% and 72% to 89%, respectively, whereas in the combined tests it reached 86%–96% for sputum specimens and 100% for CSF specimens. The specificity of ELISAs for sputum specimens was lower in the combined (73%–87%) than in the individual (87%–98%) tests, whereas for CSF specimens it was 100% in all tests. Thus, the combined ELISA approach for mycobacterial antigen detection provides a rapid and reliable laboratory adjunct in the diagnosis of patients with tuberculosis.