Experimental optimization of the detection limit of one-step solid-phase radioimmunoassay

The minimal detection limit and the conditions of maximal sensitivity of a one-step solid-phase inhibition radioimmunoassay for human immunoglobulin A have been determined by application of statistical methods of experimental optimization. The choice of the optimal combination of qualitative variables, such as the origin of the antibody and the nature of the solid phase, was made by the study of a covariable under non-optimal conditions of the quantitative variables, such as the amount of antibody. The covariable was the avidity of the antibody, which is expected to have a large influence on the sensitivity. Only the difference in avidity between two immunosorbents with cellulose or Sepharose as solid-phase material proved to be statistically significant, and further study was done with cellulose. The experimental optimization of the sensitivity as a function of five quantitative variables yielded a reduction of the detection limit by a factor 5.6 (from 23.5 to 4.2 ng IgA). The variables determining the amount of insolubilized antibody in the assay had the largest influence on the value of the detection limit. The conditions of optimal sensitivity did agree with the predictions by a physical model of radioimmunoassay. The results are discussed in relation to the assay parameters such as the amount and the avidity of the insolubilized antibody and the initial percentage of binding, and in relation with theoretical optimization of the sensitivity.     

Experimental optimization of the detection limit of two-step solid-phase radioimmunoassay

For two-step inhibition radioimmunoassay (sequential saturation or delayed addition of labeled antigen) generally a higher sensitivity than for one-step inhibition radioimmunoassay (equilibrium assay) is expected. The detection limit of a two-step solid phase inhibition radioimmunoassay for human serum immunoglobulin. A was minimized by statistical methods of experimental optimization. Under optimal conditions the detection limit was 2.3 ng IgA. This is about 1.8 times lower than the minimal detection limit of the one-step assay under similar conditions of the qualitative variables such as the origin of the antibody. This increase in sensitivity was associated with a decrease in the precision of the assay. The results are discussed with respect to the comparison of the one-step and the two-step assay and the usefulness of a sensitive radioimmunoassay in practice.     

Prospective evaluation of bacterial antigen detection in cerebral spinal fluid in the diagnosis of bacterial meningitis in a predominantly adult hospital

The results of all CSF latex agglutination (LA) tests performed in a large predominantly adult hospital over 1 yr are reviewed. Eight cases of bacterial meningitis were recognized during this time, and of these only three were due to organisms detectable by commercially available LA kits. In one case, the LA was uninterpretable, and in the second the LA was not performed because less than 5 x 10(6) WBC/L were seen in the CSF. In the third case the Gram stain was positive. No cases of partially treated meningitis were diagnosed. In this setting, the LA test did not contribute significantly to patient management.     

Extraction of human plasma or sera by heat treatment for a solid-phase radioimmunoassay of carcinoembryonic antigen.

Heat treatment and a solid-phase radioimmunoassay are combined to give a relatively simple and rapid procedure for assay of carcinoembryonic antigen in plasma or serum. The new way we describe to extract this antigen is an alternative to the conventional method of extraction with perchloric acid. Heating plasma or serum samples in acetate buffer (0.16 mol/L, pH 5.0) at 70 degrees C for 15 min precipitates out most of the heat-labile, nonspecific plasma proteins, but leaves most of the antigen in solution, with its immunochemical properties apparently unaffected. Comparison between the heat treatment and the perchloric acid extraction yielded comparable values when tested either by solid-phase radioimmunoassay or by the zirconyl phosphate precipitation method. An added advantage of our method is that it gives the same assay values for both plasma and serum. Results for a group of pathological plasma samples, assayed by both our method and the perchloric acid-zirconyl phosphate precipitation method, gave a correlation coefficient of 0.90.     

Comparison of a within-day solid-phase immunoradiometric assay with a solution-phase radioimmunoassay for the measurement of alpha-fetoprotein in amniotic fluid

Using 231 amniotic fluid samples from a Regional Screening Service, the performance of a monoclonal antibody-based solid-phase immunoradiometric assay (IRMA) for alpha-fetoprotein was compared with that of a polyclonal antiserum-based solution phase radioimmunoassay (RIA). alpha-Fetoprotein values determined from these samples by the two methods were in excellent agreement (r = 0.992) and the diagnostic performance of the two assays was identical. However, the IRMA assay displayed a greater working range than the RIA, and in addition was more rapid to perform, allowing within-day turnaround of laboratory results.     

Use of a solid-phase fluorescent cytometric technique for the detection of bacteria in platelet concentrates

Blood services worldwide are now striving to reduce the risk of transmission of bacteria by transfusion. The BacT/ALERT microbial detection system (bioMerieux, Basingstoke, Hants, UK) is currently regarded as the 'gold standard' for bacterial screening of platelet concentrates. The BacT/ALERT is a culture system and will not generate an 'instant' (within 2 h) determination. We report on the Scansystem (Hemosystem, Marseille, France), a solid-phase fluorescent cytometric technique, which enables the rapid detection of bacteria (within 90 min) in platelet concentrates. The study was performed in two parts - one involving the routine screening of platelet concentrates and the other determining the sensitivity of the system. In both arms of the study, the BacT/ALERT was used for comparative purposes. In total, 900 platelet concentrates were screened (63 apheresis and 837 buffy coat pooled). No bacteria were detected in any of the platelet concentrates tested by means of either the Scansystem or the BacT/ALERT. The sensitivity of the Scansystem was in the order of 10(3) cfu mL(-1). Escherichia coli and Staphylococcus aureus were detected by using the Scansystem at 1 cfu mL(-1). The BacT/ALERT detected all organisms tested (n = 6) at 1 cfu mL(-1). The Scansystem offers a sensitive alternative technology to bacterial culture, with the benefit of a rapid test time.     

Use of a pH meter for bacterial screening of whole blood platelets

BACKGROUND: Bacterial contamination of blood products is a leading cause of transfusion-related morbidity and mortality. Transfusion services are now compelled to employ methods of detecting bacteria in platelet (PLT) components. The use of pH screening of whole-blood PLTs (WBPs) was evaluated with a pH meter at the time of issue as a surrogate test for bacterial contamination. STUDY DESIGN AND METHODS: All WBPs selected for transfusion in May through September 2004 were tested individually for pH at time of issue. Those with a pH value of less than 7.0 were cultured in an automated culture system for 5 days. The white blood cell (WBC) and PLT counts in 56 representative WBP units that failed pH screening were compared to WBP units with acceptable pH values. RESULTS: Of the 37,060 WBP units that underwent pH screening, 405 had a pH value of less than 7.0 (1.1%). Four of those units were culture positive (1.0%) for Staphylococcus aureus, Bacillus subtilis, diphtheroids, and coagulase-negative Staphylococcus. Only one cocomponent red blood cell (RBC) unit was culture-positive and grew the same bacteria (S. aureus) as the WBP unit. The rate of pH failure increased with WBP storage length with the greatest rate of pH failures occurring in 5-day-old WBPs. The units that failed pH screening had significantly more WBCs and PLTs than units with acceptable pH values. CONCLUSION: pH screening of WBPs at issue prevented transfusion of bacterially contaminated WBPs and RBCs. This method, however, results in significant PLT wastage. Higher WBC and PLT content likely explains pH failures not due to bacterial contamination.     

PSA检测前列腺癌临床应用的建议

前列腺癌是许多西方国家最常见的男性癌症，在我国老年男性中也有一定的发病率。前列腺特异性抗原(prostate specific antigen，PSA)是前列腺癌最重要的标志物，具有高度脏器特异性，并不具有肿瘤特异性。尽管PSA在临床应用中具有局限性，但仍是目前前列腺癌筛查、辅助诊断和监测疗效的最好指标。     

前列腺癌特异性抗原基因检测研究进展

前列腺癌是男性常见肿瘤之一 ,且前列腺癌患者逐年增加。为了适应临床早期检出原发性和潜在的转移性肿瘤的要求 ,前列腺癌特异性抗原 (PSA)的基因检测方法发展很快。而前列腺癌微转移检测在肿瘤早期诊断、治疗监测及预后判断方面具有重要意义。本文就PSA基因分子生物学、检测方法学以及在前列腺癌微转移检测中的应用价值作一综述     

Spontaneous in vitro differentiation of antigen-specific lymphocytes from a patient with immunoglobulin M gammopathy.

Recently we have identified two monoclonal immunoglobulin M (IgM) proteins that bind Klebsiella polysaccharides. The lymphocytes of one of these patients (M.A.Y.) were available for study. A substantial proportion of the B lymphocytes isolated from this patient's peripheral blood also bound Klebsiella polysaccharides with a pattern of specificity identical to that of the monoclonal IgM, and reacted with an anti-idiotypic antiserum directed against this IgM. Stripping the surface immunoglobulin from these lymphocytes eliminated this reactivity. Although no plasma cells were detected in the freshly isolated peripheral blood lymphocytes of this patient, plasma cells binding Klebsiella polysaccharide appeared after 7 d of in vitro culture. This occurred regardless of whether the cultures were supplemented with autologous plasma, normal human plasma, or fetal calf serum. Pokeweed mitogen neither stimulated nor inhibited the in vitro differentiation of the monoclonal B lymphocytes into plasma cells. This differentiation was, however, abrogated by F(ab')2 fragments of anti-human IgM and by anti-idiotypic antibodies, as well as by the Klebsiella polysaccharide with which the monoclonal IgM reacted.     

Evaluation of pooled cultures for bacterial detection in whole blood-derived platelets

BACKGROUND: The BacT/ALERT (bioMérieux) system is highly efficient for bacterial detection in apheresis platelets (PLTs) and whole blood-derived PLTs produced by the buffy-coat method. Detection of bacterial contamination in whole blood-derived PLTs produced by the PLT-rich plasma (PRP) method, however, is problematic. Prestorage pooling of these PLTs is not permitted in some countries including Canada and the United States, and culturing individual units is costly and may significantly reduce the PLT unit content. In this study, the sensitivity and specificity of BacT/ALERT cultures performed on pools derived from PRP PLTs are reported. STUDY DESIGN AND METHODS: The sensitivity of the BacT/ALERT system was evaluated for bacterial detection in PRP PLTs with a dilution effect. Thirty PLT pools were produced with 1 PLT unit previously spiked with bacteria and then pooled with other four nonspiked PLT units. Three bacteria, usually associated with PLT contamination, were selected for spiking. The specificity of this method was evaluated in 40 nonspiked PLT pools. RESULTS: The method was found to be 100 percent specific and 97 percent sensitive. Of the five spiked pools with Streptococcus pneumoniae at levels of less than 2 colony-forming units (CFUs) per mL, four were found to be positive whereas all 25 spiked pools with greater than 9 CFUs per mL of any of the chosen bacteria gave positive results. The mean time of detection was 17 to 19 hours for Staphylococcus epidermidis and 14 to 15 hours for S. pneumoniae and Pseudomonas aeruginosa when spiked with similar bacterial inocula. CONCLUSION: The evaluated system is highly sensitive and specific and may be a feasible method for bacterial detection in PRP PLTs.     

Bacterial antigen detection in cerebrospinal fluid of patients with meningitis

This study compared the sensitivity and specificity of four test systems in detecting Haemophilus influenzae type b, Neisseria meningitidis, Streptococcus pneumoniae, and gram-negative organisms in cerebrospinal fluid (CSF), versus culture. The tests used on CSF from 155 patients with meningitis were the Phadebact coagglutination (CoA) test, the Directigen latex agglutination (LA) test, counterimmunoelectrophoresis (CIE), and the Limulus amebocyte lysate (LAL) test. The sensitivity for patients with bacterial meningitis was 78% (18/23) for LA, 78% (25/32) for CoA, and 67% (18/27) for CIE for detection of H. influenzae type b; 71% (10/14) for CoA, 100% (6/6) for LA, and 50% (6/13) for CIE in detecting S. pneumoniae; and 33% (1/3) for LA and 50% (2/4) for CIE in detecting N. meningitidis. LAL had a sensitivity of 77% (37/48) in detecting CSF gram-negative endotoxin. The specificities of those with bacterial meningitis for H. influenzae, S. pneumoniae, and N. meningitidis tested by LA were, respectively, 100% (35/35), 96% (50/52), and 100% (54/54); for H. influenzae and S. pneumoniae using CoA 97% (62/64) and 96% (80/83); for H. influenzae, S. pneumoniae, and N. meningitidis using CIE 67% (18/27), 50% (6/12), and 50% (2/4). The specificity of LAL was 86% (38/44). The detection of bacterial antigen from CSF in patients with meningitis by commercial agglutination tests is more sensitive than CIE and is highly specific.     

Use of a solid-phase extraction with radioimmunoassay to identify the proportional bias of clinical B-type natriuretic peptide immunoassay: the impact of plasma matrix and antibody multispecificity.

BACKGROUND: Circulating immunoreactive B-type natriuretic peptide-32 (ir-BNP-32) has diagnostic and prognostic values in heart failure. We compared, in parallel, a point-of-care (POC) test (Triage((R)) BNP Test) of whole plasma and radioimmunoassay (RIA) of solid-phase extracted (SPE) plasma (SPE/RIA) utilizing a novel copolymer column, in the measurement of patient ir-BNP-32 concentrations. METHODS: Approximately 0.25 mL thawed plasma was transferred to a BNP test device and inserted in a Triage Meter Plus, which gave ir-BNP-32 concentration in pg/mL. Concurrently, for the SPE/RIA measurement, 1.0 mL plasma was acidified and extracted with an OASIS column; eluate dried, reconstituted and quantified by RIA. RESULTS: Inter-day coefficient of variation for both methods were <15%. Plasma SPE recovery was 75.2%. POC correlated with recovery corrected SPE/RIA for ir-BNP-32, r=0.843 (p<0.0001) and the Passing-Bablok model was POC ir-BNP-32=1.43x (recovery corrected SPE/RIA ir-BNP-32)+9.75 ng/L (n=81). A proportional bias was also evident from the Bland-Altman plot, r=0.716 (p<0.0001). CONCLUSIONS: A proportional bias is responsible for plasma ir-BNP-32 concentration differences between whole plasma POC test and recovery corrected SPE/RIA measurements. Ir-BNP-32 assays are influenced by plasma matrix and antibody multispecificity. Consequently, consistent analytical accuracy between immunoassays is necessary to attain a single ir-BNP-32 concentration threshold for diagnosis. Clin Chem Lab Med 2007;45:1353-9.     

Presentation of antigen in immune complexes is boosted by soluble bacterial immunoglobulin binding proteins

Using a snake toxin as a proteic antigen (Ag), two murine toxin-specific monoclonal antibodies (mAbs), splenocytes, and two murine Ag-specific T cell hybridomas, we showed that soluble protein A (SpA) from Staphylococcus aureus and protein G from Streptococcus subspecies, two Ig binding proteins (IBPs), not only abolish the capacity of the mAbs to decrease Ag presentation but also increase Ag presentation 20-100-fold. Five lines of evidence suggest that this phenomenon results from binding of an IBP-Ab-Ag complex to B cells possessing IBP receptors. First, we showed that SpA is likely to boost presentation of a free mAb, suggesting that the IBP-boosted presentation of an Ag in an immune complex results from the binding of IBP to the mAb. Second, FACS analyses showed that an Ag-Ab complex is preferentially targeted by SpA to a subpopulation of splenocytes mainly composed of B cells. Third, SpA-dependent boosted presentation of an Ag-Ab complex is further enhanced when splenocytes are enriched in cells containing SpA receptors. Fourth, the boosting effect largely diminishes when splenocytes are depleted of cells containing SpA receptors. Fifth, the boosting effect occurs only when IBP simultaneously contains a Fab and an Fc binding site. Altogether, our data suggest that soluble IBPs can bridge immune complexes to APCs containing IBP receptors, raising the possibility that during an infection process by bacteria secreting these IBPs, Ag-specific T cells may activate IBP receptor-containing B cells by a mechanism of intermolecular help, thus leading to a nonspecific immune response.     

LSPR based on-chip detection of dengue NS1 antigen in whole blood

The development of a biosensor for rapid and quantitative detection of the dengue virus continues to remain a challenge. We report a lab-on-chip device that combines membrane-based blood plasma separation and a localized surface plasmon resonance (LSPR) based biosensor for on-chip detection of dengue NS1 antigen from a few drops of blood. The LSPR effect is realized by irradiating UV-NIR light having a spectral peak at 655 nm onto nanostructures fabricated via thermal annealing of a thin metal film. We study the effect of the resulting metal nanostructures on the LSPR performance in terms of sensitivity and limit of detection, by annealing silver films at temperatures ranging from 100 to 500 °C. The effect of annealing temperature on the nanostructure size and uniformity and the resulting optical characteristics are investigated. Further, the binding between non-targeted blood plasma proteins and NS1-antibody-functionalized nanostructures on the LSPR performance is studied by considering different blocking mechanisms. Using a nanostructure annealed at 200 °C and 2X-phosphate buffer saline with 0.05% Tween-20 as the blocking buffer, from 10 μL of whole blood, the device can detect NS1 antigen at a concentration as low as 0.047 μg mL⁻¹ within 30 min. Finally, we demonstrate the detection of NS1 in the blood samples of dengue-infected patients and validate our results with those obtained from the gold-standard ELISA test.

A lab-on-chip device that combines membrane-based blood plasma separation and a localized surface plasmon resonance (LSPR) based biosensor for on-chip detection of dengue NS1 antigen from a few drops of blood.     

Development of a radioimmunoassay for a high molecular mass tubular antigen in urine--its application for early detection of tubular damage

In order to isolate urinary kidney antigens, the gammaglobulins fraction of an antiserum against human kidney cortex plasma membranes was coupled to Sepharose 4B. By immunospecific affinity chromatography an antigen fraction was obtained from the urine of a patient suffering from severe kidney disease. After gel filtration, the main fraction, eluted with the exclusion volume of a Sephadex G-200 column and enriched 16 000-fold, was labelled with 131I and used in a radioimmunoassay system. Soluble kidney antigens, presumably of proximal tubular origin, could be detected and quantified by the assay system in urine samples of patients with various diseases. The samples did not need to be treated, either concentrated or dialyzed, before application. The results of our experiments show a correlation between antigen excretion and kidney damage. Rejection episodes in patients with kidney transplants could be recognized early by enhanced antigen excretion. Potentially nephrotoxic drugs caused antigen excretion as well. In normal, healthy subjects output of the antigen was very low. The assay system might be of value for monitoring renal diseases.     

Characterization of a serum factor interfering with the radioimmunoassay of prostatic acid phosphatase

Storage of serum at temperatures above zero for short periods results in the spurious formation of a factor interfering with the radioimmunoassay of prostatic acid phosphatase. This factor acts by inhibiting the binding of radiolabelled tracer to the specific primary antiserum. The inhibiting factor is a protein of molecular mass 40 000 which is derived from an inactive high molecular mass precursor. Formation of the inhibiting factor is apparently reversible and its action can be neutralized by adding fresh serum to the incubation mixture. Serine-protease inhibitors of synthetic or natural origin did not influence the activity of the inhibitor. Since incubations for long periods at temperatures above freezing are standard procedures in radioimmunoassays, formation of this inhibitor may occur in susceptible sera. It is suggested that problems with establishing specific assays for prostatic acid phosphatase may be explained by the generation of this inhibiting factor.