The detection of influenza A and B viruses in clinical specimens using a quartz crystal microbalance

Detection of human immunodeficiency virus (HIV) infections has been enhanced by incorporating p24 antigen detection with current HIV antibody detection using enzyme immunoassays (EIAs). However, screening for HIV antibodies has increased through the use of rapid, lateral-flow HIV antibody detection assays that currently do not have the capability to detect HIV p24 antigen. In this report, a lateral-flow based assay using super-paramagnetic particles as the detection marker was developed for the detection of HIV-1 p24 antigen. This magnetic immuno-chromatographic test (MICT) uses an inexpensive, low-maintenance instrument that detects the magnetic moment of the super-paramagnetic particles in a magnetic field. MICT is simple to perform, provides a numerical output for easier determination of reactive results and can be completed in 40 min. The lower limit of detection for HIV-1 p24 spiked into assay sample buffer and 50% plasma was 30 pg/ml for both. Detection of HIV-1 p24 antigen at 50 pg/ml was reproducible in both inter-run and intra-run assays with coefficients of variation of <13%. Furthermore, the MICT p24 assay was able to detect intact virus spiked into 50% plasma (lower detection limit of 250,000 viral RNA copies/ml). MICT detection of increasing HIV-1 p24 levels in commercially available seroconversion panels by MICT was only slightly later than that detected by much more complex EIAs. MICT could provide a simple, low-cost, and portable method for rapid HIV-1 p24 detection in a variety of testing environments.     

Diagnostic ability of different human milk fat globule antigens in breast cancer

Human mammary epithelial antigens (HME-Ags) are released into the circulation by breast tumors and not by normal breast tissue (Proc. Natl. Acad. Sci. USA 74: 582–586, 1977). This characteristic made them valuable, together with other breast cancer related antigens later identified, to develop immunoassays useful in the follow-up of breast cancer. Assays for these antigens in serum have less than complete sensitivity and partial specificity, and as a result of this have not been totally successful in studying the relapsing breast cancer patient. In the present work, correlations are made among 3 assays available for breast cancer disease follow-up. They detect HME-Ags, CEA, and the heavy molecular weight mucin of the human milk fat globule (HMFG). Values for sensitivity and specificity for the 3 assays were obtained from approximately 300 samples of patients whose clinical diagnosis at the time of blood drawing was rigorously established. A small but definite advantage in sensitivity is demonstrated for the HME-Ags assay over the other two. A similar advantage is also demonstrated in the sequential follow-up of breast cancer patients, where HME-Ags respond more rapidly in most instances to changes in tumor burden. Further, the ability of increases in levels of these assays to predict relapse was studied in 15 patients who relapsed. HME-Ags demonstrated a predictive value of 73%, while CEA and the heavy molecular weight mucin remained at 47%.The present study exemplifies the search for novel antigens (Ags) with maximal ability to detect breast cancer relapse and with improved sensitivity to monitor tumor burden changes. Here, assays for different antigens to be compared are tested in the same serum samples obtained from carefully staged patients. The results suggest a role as breast cancer markers for antigens of lower molecular weight than the epithelial mucin-like components studied previously.     

A prospective study of protein-specific assays used to investigate idiopathic thrombocytopenic purpura

Idiopathic thrombocytopenic purpura (ITP) is a disorder in which platelets, sensitized by autoantibodies, are destroyed by the reticuloendothelial system. The diagnosis of ITP has been a clinical one because assays measuring platelet-associated IgG (PAIgG) have low specificity. The recently introduced assays that measure antibodies against specific platelet glycoproteins (GP) offer the possibility of improved specificity. In this report we describe two prospective studies. In the first study we compared two protein-specific assays (AC and MAIPA) looking for the presence of autoantibodies against GP IIb/IIIa in 81 patient samples. These results were compared with an immunoradiometric assay for PAIgG. The second study investigated the enhanced sensitivity of measuring anti-GP Ib/IX autoantibodies in 76 patient samples. The protein-specific assays were able to differentiate immune from non-immune thrombocytopenia (specificity 91%, sensitivity 39%), whereas the PAIgG assay could not (specificity 19%, sensitivity 78%). The addition of the Ib/IX AC assay maintained a specificity of 92% while increasing the diagnostic sensitivity to 66%. In contrast to the PAIgG assay, there was no correlation between the platelet count and the likelihood or degree of positivity within the control samples using the glycoprotein assays. These studies confirm that glycoprotein assays can be used as diagnostic tests for ITP.     

Comparison of four Mycoplasma pneumoniae IgM-, IgG- and IgA-specific enzyme immunoassays in blood donors and patients

Mycoplasma pneumoniae antibodies were studied in 504 blood donors and 102 patients with infections not caused by M. pneumoniae with the use of enzyme immunoassay kits from ThermoLabsystems (L), Savyon (S), Bio-Rad (B) and Novitec (N). Detection frequencies of M. pneumoniae IgM in blood donors were 14.9% (L), 16.0% (S), 2.8% (B) and 3.8% (N), and in patients were 40.2% (L), 42.2% (S), 9.8% (B) and 16.7% (N). Detection frequencies of M. pneumoniae IgA were 68.5% (L) and 22.8% (S), and in 65 respiratory disease patients were 100% (L) and 53.8% (S). Thus, use of some kits may lead to overdiagnosis of M. pneumoniae infections.     

Heparin-induced thrombocytopenia: evaluation of IgG and IgGAM ELISA assays

Background: Heparin‐induced thrombocytopenia (HIT) is a rare complication of heparin therapy resulting from antibody production to platelet factor 4 and heparin complexes (H‐PF4). Methods: We have evaluated four enzyme‐linked immunosorbent assay (ELISA)‐based screening tests to identify the best assay(s) with the highest specificity but without underdiagnosis of HIT. As functional assays are difficult to perform, ELISAs are useful to provide clinicians with a timely answer. Over a 10‐month period, all samples (N = 107) referred to our laboratory were tested for HIT antibodies using four commercially available ELISA kits, two detecting IgG/A/M anti‐H‐PF4 antibodies and the other two IgG specific. Results: Twenty‐eight samples were positive by at least one assay; IgGAM ELISAs were found to be more sensitive with 24 samples positive by Asserachrom IgGAM and 23 by Zymutest IgGAM. Only 18 samples were positive by GTI‐PF4‐IgG and Zymutest IgG. The gold standard serotonin release assay (SRA) was used as a confirmation assay, and 11/28 samples tested positive. All these SRA‐positive samples were positive by all four assays. None of the IgGAM‐only‐positive samples was found to be positive by SRA suggesting a better specificity for the IgG‐only assays. Conclusion: Our data strongly support the use of IgG‐only assays for the detection of HIT antibodies.     

Two new types of assays to determine protein concentrations in biological fluids using mass spectrometry of intact proteins with cystatin C in spinal fluid as an example

Abstract

There is no reference method that is generally acknowledged to be unbiased for the determination of the concentration of any protein in biological fluids. This is probably because mass spectrometry (MS) methods acknowledged as reference methods for determination of low molecular mass substances in biological fluids, e.g. creatinine, have been difficult to adapt for proteins. Here we suggest two tentative MS methods, which might be used as reference methods for the determination of protein concentrations in biological fluids. One is based upon the addition to the fluid of a non-proteome reference protein, very similar to the one to be measured, and analyzing the ratio between the corresponding peaks in a selected ion monitoring (SIM) chromatogram. We call this method LC-MS-NPRP (NPRP, Non-Proteome Reference Protein). The other method is based upon the classical standard addition assay for low molecular mass substances. The results of these assays for cystatin C in spinal fluid were compared to those obtained by an immunoassay. Both methods indicated lower concentration than the immunoassay. This was found to be due to the presence of a significant fraction of monohydroxylated cystatin C in spinal fluid. It turned out that the sum of the unhydroxylated and hydroxylated cystatin C concentrations, determined by either of the two MS methods, were close to the results obtained by the immunoassay. These MS-based methods analyze intact proteins and therefore seem more suitable for the determination of protein concentrations in biological fluids than other MS-based methods requiring proteolytic degradation with its inherent lack of precision.     

肿瘤标志物检测技术的应用和进展

肿瘤尤其是恶性肿瘤严重威胁着人类的健康,肿瘤标志物(TM)的检测对恶性肿瘤的早期诊断、后期治疗有着重要的意义。随着细胞生物学和分子生物学的发展,TM及其应用已成为临床肿瘤诊断与研究的热点。TM检测技术的合理应用和结果是否准确,可直接影响肿瘤诊断和治疗的效果。文章就TM检测技术的应用和进展进行综述。     

The sera of patients with acquired immunodeficiency syndrome contain specific antibodies in a latent state

Latent antibodies reacting with antigens of human immunodeficiency virus are detected in patients with acquired immunodeficiency syndrome. Antibodies are detected by ion-exchange chromatography on QAE-Sephadex. A relationship is noted between the disease stage and the level of latent antibodies. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny Vol. 121, N ^o 3, pp. 315–316, March, 1996 Presented by A. F. Tsyb, Member of the Russian Academy of Medical Sciences