Particle Counting Immunoassay Research Articles

"Lollipop" particle counting immunoassay based on antigen-powered CRISPR-Cas12a dual signal amplification for the sensitive detection of deoxynivalenol in the environment and food samples

Deoxynivalenol is one of the most widely distributed mycotoxins in cereals and poses tremendous threats to the agricultural environment and public health. Therefore, it is particularly important to develop sensitive and interference-resistant deoxynivalenol analysis methods. Here, we establish a “Lollipop” particle counting immunoassay (LPCI) based on antigen-powered CRISPR-Cas12a dual signal amplification. LPCI achieves high sensitivity and accuracy through antigen-powered CRISPR-Cas dual signal amplification combined with particle counting immunoassay. This strategy not only broadens the applicability of the CRISPR-Cas system in the field of non-nucleic acid target detection; it also improves the sensitivity of particle counting immunoassay. The introduction of a polystyrene "lollipop" immunoassay carrier further enables efficiently simultaneous pre-treatment of multiple samples and overcomes complex matrix interference in real samples. The linear detection range of LPCI for deoxynivalenol was 0.1–500 ng/mL with a detection limit of 0.061 ng/mL. The platform greatly broadens the scope of the CRISPR-Cas sensor for the detection of non-nucleic acid hazards in the environment and food samples.

Particle counting immunoassay system using a low-aspect-ratio pore

We have developed a new particle counting immunoassay system integrated with a resistive-pulse sensor with a low-aspect-ratio pore. The particle counting immunoassay is based on the principle of latex aggregation. The target single-molecule concentration is measured by counting aggregated latex particles that form multimers over the reaction time. To enable highly accurate identification of the number of aggregated particles, we designed and fabricated a low-aspect-ratio pore device to perform quantitative structural analysis of particles. The aggregation numbers of the conjugated particles were classified based on blockade current profiles when the particles translocated through the pore. Time-dependent agglutination reaction monitoring demonstrated that the reaction speed increased for a solution containing a higher concentration of antigens. The results prove the feasibility of using the proposed immunoassay system to count particles while determining the aggregation number based on their current profiles.

COVID-19 variants' cross-reactivity on the paper microfluidic particle counting immunoassay.

SARS-CoV-2 has mutated many times since the onset of the COVID-19 pandemic, and the omicron is currently the most dominant variant. Determining the specific strain of the virus is beneficial in providing proper care and containment of the disease. We have previously reported a novel method of counting the number of particle immunoagglutination on a paper microfluidic chip using a smartphone-based fluorescence microscope. A single-copy-level detection was demonstrated from clinical saline gargle samples. In this work, we further evaluated two different SARS-CoV-2 monoclonal antibodies to spike vs. nucleocapsid antigens for detecting omicron vs. delta and spike vs. nucleocapsid proteins. The SARS-CoV-2 monoclonal antibody to nucleocapsid proteins could distinguish omicron from delta variants and nucleocapsid from spike proteins. However, such distinction could not be found with the monoclonal antibody to spike proteins, despite the numerous mutations found in spike proteins among variants. This result may suggest a clue to the role of nucleocapsid proteins in recognizing different variants.

Plastic antibody for DNA damage: fluorescent imaging of BPDE–dG adducts in genomic DNA

Exogenous and endogenous genotoxic carcinogens and their in vivo metabolites may result in DNA damage and cause adverse health effects. It is very difficult to specifically detect trace DNA damage in the presence of a large excess of unmodified nucleosides. Here we report a molecularly imprinted polymer (MIP) nanocomposite, namely nanoMIP, which can be used as a "plastic antibody" for specific recognition of benzo[a]pyrene diol epoxide (BPDE)-DNA adduct. Enhanced binding affinity (880 nM) of nanoMIPs was achieved by using two tailor-made functional monomers. The property of binding kinetics was greatly improved in virtue of the well-defined nanostructure, which was fabricated by initiators for continuous activator regeneration-atom transfer radical polymerization (ICAR-ATRP). The BPDE-adducted DNA can be specifically captured by synthetic nanoMIP. By taking advantage of this antibody-mimicking behavior, we further developed a fluorescently imaged particle counting immunoassay (FIPCIA) method for ultrasensitive detection of BPDE-ssDNA adducts using a laser scanning confocal microscope (LSCM). The number of countable fluorescent dots is proportional to the content of BPDE-ssDNA adducts in the DNA sample. The proposed plastic antibody-based FIPCIA method can detect traces of BPDE-DNA adducts as low as 18 pM in human lung carcinoma A549 cells. This highly-sensitive detection of DNA lesions offers a promising alternative to immunogenic antibody-based immunoassays for genomics and DNA modification analysis.

Fluorescently Imaged Particle Counting Immunoassay for Sensitive Detection of DNA Modifications

Modifications of genomic DNA may change gene expression and cause adverse health effects. Here we for the first time demonstrate a particle counting immunoassay for rapid and sensitive detection of DNA modifications using benzo[a]pyrenediol epoxide (BPDE)-DNA adducts as an example. The BPDE-adducted DNA is specifically captured by immunomagnetic particles and then isolated from unmodified DNA by applying an external magnetic field. By taking advantage of the fluorescence signal amplification through multiple labeling of captured DNA by OliGreen dye, the captured BPDE-DNA adducts can be quantified by particle counting from fluorescence imaging. This clearly demonstrates that the number of fluorescently countable particles is proportional to the modification content in genomic DNA. It is interesting to note that the background fluorescence signal caused by nonspecific adsorption of OliGreen dye can be more effectively quenched than that induced by the binding of OliGreen dye to ssDNA, allowing for significant reduction in the background fluorescence and further enhancing the detection sensitivity. The developed method can detect trace BPDE-DNA adducts as low as 180 fM in the presence of 1 billion times more normal nucleotides in genomic DNA and has a dynamic range over 4 orders of magnitude. By using anti-5-methylcytosine antibody, the method is extended to the detection of global DNA methylation. With high sensitivity and specificity, this rapid and easy-to-perform analytical method for DNA modifications shows a broad spectrum of potential applications in genotoxical and epigenetic analysis.

Characterization of an anti-idiotypic MoAb bearing an internal image of the receptor-binding epitope of cholera toxin.

A mouse anti-cholera toxin (CT) MoAb, mAb1, specific for the GM1-binding epitope of CT, was used to raise a syngenic anti-idiotypic MoAb, mAb2. Purified mAb2 was specific for mAb1 as shown by latex particle counting immunoassay and ELISA. Several experiments of competition between mAb2 and CT for binding to mAb1 demonstrated that mAb2 bore an internal image of the GM1-binding epitope of CT. Binding of mAb2 to GM1 unambiguously corroborated the mAb1-paratopic specificity of mAb2. Furthermore, mAb2 acted as a CT-surrogate antigen: rabbits injected with mAb2 produced some anti-CT antibodies, Ab3, which resembled mAb1 in specificity as expected. The potential use of this mAb2 as vaccine or as prophylactic agent to prevent CT from binding to its cellular receptor is discussed.

056-PA10 A rapid and inexpensive method based on particle counting immunoassay (PACIA) for screening antituberculosis agents

056-PA10 A rapid and inexpensive method based on particle counting immunoassay (PACIA) for screening antituberculosis agents

Simultaneous Determination of Enzyme Activity and Enzyme Quantity in Single Human Erythrocytes

An integrated laser-based fluorescence enzyme assay and particle-counting immunoassay system with zeptomole sensitivity has been developed for simultaneous determinations of activity and concentration of enzymes in single erythrocytes. The product NADPH of the reaction between glucose-6-phosphate and NADP+ catalyzed by glucose-6-phosphate dehydrogenase (G6PDH) is monitored. Simultaneously, the agglutination of antibody-coated particles in the presence of G6PDH produces large particles which are counted by light scattering. The correlation between the two parameters indicates that on average only about 35% of the enzyme in individual cells is active. There is more than a 10-fold cell-to-cell variation in the ratios of the two parameters. This indicates that there are multiple mechanisms for the degradation of intracellular enzymes as a function of cell age.

Laser-Based Particle-Counting Microimmunoassay for the Analysis of Single Human Erythrocytes

A particle-counting immunoassay system for ultrasensitive analysis of proteins in a capillary environment has been developed. The assay is based on the agglutination of antibody-coated particles in the presence of an antigen (usually a protein). The particles were electrophoretically migrated in a 20-microns-i.d. capillary past a detection window where a laser beam irradiates continuously. The light scattering events generated by the agglutinated particles were counted while those produced by unreacted particles were electronically rejected. Glucose-6-phosphate dehydrogenase (G6PDH) was chosen as a test compound for the off-column as well as for the on-column versions of this method. A limit of detection of 620 molecules of G6PDH (1 zmol) was found in the on-column assay. The standard deviation between runs was approximately 6%, which is comparable to that of standard immunoassay methods. The application to the determination of G6PDH levels in individual human erythrocytes is presented. A 14-fold cell-to-cell variation was found which can be explained by the age distribution in the red blood cells.

A particle counting immunoassay for the direct detection of Clostridium difficile serogroup specific antigen in faecal specimens.

The potential of a particle counting immunoassay (PACIA) for the direct detection of Clostridium difficile serogroup G specific antigen in faecal specimens was evaluated. F(ab')2 fragments from a rabbit anti-serogroup G antiserum were covalently coupled to carboxylated latex beads. This reagent was mixed with acid extracts of faecal specimens and the reaction was assayed with an optical counter which discriminated unagglutinated from agglutinated latex particles. Culture for C. difficile, faecal cytotoxin detection, PACIA and serogrouping of C. difficile isolates were performed on 249 stools. Of the 71 culture-negative specimens, none gave a positive result in the cytotoxin assay or in PACIA. Faecal cytotoxin was detected in 100 of the 178 culture-positive specimens. PACIA was positive for 63 of the 71 faecal specimens that yielded serogroup G C. difficile on culture. PACIA gave negative results for all other culture-positive stools tested with one exception, from which a serogroup A7 C. difficile strain was isolated. PACIA detection of serogroup G antigen in faecal specimens showed a sensitivity of 88.7%, a specificity of 99.7%, a predictive value of a positive culture with a serogroup G strain of 98.4%, and a predictive value for specimens that were culture-negative for a serogroup G strain of 95.6%. The results indicate that PACIA with specific antiserum is a rapid and reliable method for detecting serogroup specific antigens of C. difficile in faecal specimens. Clinical applications of the method are discussed.

Detection of Mycobacterial Antigens Present in Short-term Culture Media Using Particle Counting Immunoassay

Particle counting immunoassay (PACIA) was compared with the BACTEC system for detecting mycobacterial growth after short-term culture and was used to identify M. tuberculosis. The latex particles were coated with polyclonal anti-BCG or with specific 2A1-2 monoclonal antibodies. Bottles containing nonradioactive Middlebrook 7H9 liquid medium and BACTEC 12B vials were inoculated with equal amounts of mycobacteria from four reference strains (M. tuberculosis, M. kansasii, M. avium, and M. xenopi). Using anti-BCG, PACIA detected mycobacterial antigens 3 to 6 days before the BACTEC system. M. tuberculosis was differentiated from the other mycobacteria using 2A1-2. Seventeen clinical samples were also studied. In the same 10, the two techniques detected mycobacteria, PACIA with anti-BCG after 9 days and BACTEC 1 to 5 days later. For 9 of the 10 samples, PACIA with 2A1-2 detected M. tuberculosis after 20 days, a result confirmed with the AccuProbe system. M. xenopi was biochemically identified in Specimen 10. Nonmycobacterial diseases were diagnosed in the 7 remaining unreactive specimens. We conclude that PACIA detects mycobacterial growth earlier than BACTEC and that M. tuberculosis can be distinguished from other mycobacteria in PACIA performed with specific monoclonal antibodies.

Particle counting immunoassay of choriogonadotropin using monoclonal antibodies

A latex particle immunoassay has been developed for the quantification of choriogonadotropin in human serum using two monoclonal antibodies specific for the ß-chain of the hormone. The assay, based on optical counting of monomeric particles, was achieved in 40 min and the calibration curve was linear between 10 and 200 IU/l. Intra- and interassay precisions at three different levels of the curve varied between 3.3 and 10.9%. The method was validated by comparison with two different radioimmunoassays and correlation coefficients of 0.97–0.99 were obtained.

Free kappa and lambda light chain levels in the cerebrospinal fluid of patients with multiple sclerosis and other neurological diseases

Free kappa and lambda light chains were assayed by particle-counting immunoassay in cerebrospinal fluid (CSF) from patients with various neurological disorders. Detection limits were 25 and 50 ng/ml, respectively. Values of free kappa chain were higher than 50 ng/ml (upper reference limit) in 155 of 191 (81%) multiple sclerosis (MS) patients, in 100 of 168 (60%) patients with central nervous system (CNS) infections but in 41 of 217 (19%) patients with other neurological disorders. Free kappa chains were also assayed in 273 matched sera. The mean concentration in the control group (1.58 μg/ml; SD: 0.41) did not differ significantly from those in MS sera (1.63 μg/ml; SD: 0.43). The free kappa chain index was increased in 86% of MS patients and in 40% of patients with CNS infections. Regarding free lambda chains, CSF values were higher than 240 ng/ml (upper reference limit) in most neurological disorders (50–100%). However, the use of a lambda chain index increased the specificity of the assay as this index was higher than the upper reference value in 86% of MS patients and in only 23% of patients with infectious diseases. In MS, high levels of free kappa and lambda indices correlated significantly ( P < 0.01) with either the presence of oligoclonal bands or a high IgG index. Local synthesis of free light chains is an additional marker of an ongoing immune response within the CNS, especially in MS.

Particle counting immunoassay of S100 protein in serum. Possible relevance in tumors and ischemic disorders of the central nervous system.

S100 protein (S100) was assayed by particle counting immunoassay in serum samples from 50 healthy individuals, 325 patients with various neurological disorders, and 20 patients with malignant melanoma. The detection limit for this protein was 0.3 microgram/L. We detected none in healthy individuals or in 50 patients with multiple sclerosis, 23 with dementia, or 20 with meningitis. S100 was detectable in serum of only a few patients with meningoradiculitis (2/20), peripheral neuropathy (2/30), encephalitis (1/14), Guillain-Barré syndrome (1/25), or AIDS (2/20). In contrast, we observed high concentrations in 29 of 75 patients with tumors of the central nervous system, especially in meningioma (6/9), glioblastoma (9/23), and neurinoma (5/5). Values for S100 were mainly abnormally high (greater than 0.3 microgram/L) in serum from patients with cerebrovascular disorders (43/48) or with metastases of melanoma (9/11).

Cord serum IgE in relation to family history and as predictor of atopic disease in early infancy.

Cord serum IgE was assayed by particle counting immunoassay (PACIA) in an unselected series of European newborns (n = 190; geom mean = 0.37 IU/ml) and a cut-off limit established (greater than or equal to 1.20 IU/ml) for prediction of atopy. At control follow-up by questionnaire 18 months after birth, 38 infants (20.0%) had developed definite (9.5%) or probable (10.5%) atopy with a significant predominance of boys (P less than 0.03). Infants with a positive immediate family history (IFH) had a higher risk of developing atopy (P less than 0.0025) and also had a higher incidence of elevated cord IgE (P less than 0.02) than infants with a negative IFH. Maternal atopy influenced cord IgE levels significantly (P less than 0.00005), whereas paternal atopy did not (P = 0.23). No fetal IgE antibodies against five common allergens could be demonstrated in 36 cord sera tested. Breast-feeding for 3 months was not sufficient to prevent atopic symptoms. The predictive value of cord IgE was high since 26 of 36 newborns (positive predictive value = 72.2%) with elevated cord IgE had developed atopic symptoms before follow-up. Of the 38 infants who developed atopic symptoms, 26 had elevated cord IgE (sensitivity = 68.4%) compared to only 10 (6.6%) of the 152 atopy-free infants (P less than 0.00005). The data indicate that elevated cord IgE as determined by PACIA is a good predictor of early-onset atopy, better than family history (P less than 0.008), and that primarily maternal atopy seems to affect fetal IgE synthesis.

Immunoassay of hepatitis b surface antigen by particle counting after pepsin digestion

Particle counting immunoassay is based on latex agglutination, the reaction being measured by instrument counting of the particles remaining unagglutinated. Most interference which generally affects latex agglutination can be avoided by pepsin digestion of the sample, provided the antigen (Ag) of interest resists pepsin, which is the case of the hepatitis B surface antigen (HBsAg). Pepsin treatment has the additional advantage of inactivating antibodies and so releasing the Ag from immune complexes. We have set up an assay of HBsAg, proceeding in a prototype of Impact Instrument (Acade Diagnostic Systems, Belgium) at a rate of 60 samples· h −1 and a total running time of 2 or 4 h. This assay was compared with Abbott radioimmunoassay (RIA) in 706 consecutive patients (A) and 31 selected sera for which values close to the cut-off had been obtained by RIA (B). In A, 38 sera were found positive and 668 negative by both methods. In B, RIA after neutralization classified the samples as positive ( n = 14), negative ( n = 14), or dubious ( n = 3). Complete agreement between latex and RIA was achieved for nine positive, 12 negative, and two dubious samples. Of five RIA-positive samples, two were classified as latex-negative and three as dubious in the latex assay. One sample dubious in RIA was found latex-positive and two RIA-negative samples were found, respectively, latex-positive and dubious; when retested after pepsin digestion, the first of them became RIA-positive. That pepsin is useful to release antigen from immune complexes was also shown by addition of rabbit anti-HBs antibodies to five positive serum samples and by the recovery of the antigen both in RIA and latex agglutination after pepsin digestion.

The use of particle counting immunoassay for the diagnosis of neurological disease

Particle counting immunoassay (PACIA) is applied to the determination of proteins at trace levels in normal and pathological cerebrospinal fluid (CSF) samples. Performance characteristics for the assays are given and the results for immunoglobulins (Igs) expressed as Ig indices. Ig profiles characteristic of various neurological disorders have been studied, and anti-herpes antibodies detected using an agglutination inhibition method. Ferritin, C-reactive protein and the brain protein S-100 have also been studied.

Pregnancy-specific beta 1-glycoprotein in serum in monoclonal gammopathies: relationship with serum beta 2-microglobulin, and cellular origin.

Pregnancy-specific beta 1-glycoprotein (SP1) was assayed by particle-counting immunoassay in serum from 86 healthy blood donors and 236 patients with various types of gammopathy. A concentration of 1 microgram/L was taken as the upper normal limit. Abnormally high values were found in one of 10 patients with monoclonal gammopathy of undetermined significance, in 65% of 152 patients with multiple myeloma, in 84% of 64 patients with Waldenström's macroglobulinemia, and in seven of 10 patients with monoclonal gammopathies associated with other myeloproliferative disorders. In a study of 90 myeloma patients, the SP1 value correlated (p less than 0.001) with the concentration of beta 2-microglobulin in serum, a value which had been corrected for possible renal dysfunction, but not with the concentration of the monoclonal component. SP1 was detected by direct immunofluorescence in myeloma cells of bone-marrow smears from six of 10 patients with myelomatosis. These six patients had serum SP1 values greater than 1 microgram/L, whereas the four patients with fluorescence-negative myeloma cells had SP1 values less than 1 microgram/L.

Autoantibodies of the IgM Class against a Human Myeloma Protein IgE(DES). II. Specificity

Five different monoclonal IgE proteins, papain and pepsin fragments of myeloma protein IgE(DES) and particle-counting immunoassay were used to study in detail the epitope(s) of IgE(DES) involved in the agglutination with IgM anti-IgE(DES) antibodies. The specificity of these autoantibodies was restricted to IgE(DES) as they did not react with latex particles coated with four other IgE myeloma proteins. Antibodies reactive with latex-IgE other than latex-IgE(DES), when present, were also of restricted specificity as shown by criss-cross absorption experiments. These differences between IgE myeloma proteins could not be attributed to the light chain type nor to the Em(1)-allotype. The epitope(s) of IgE(DES) participating in the reaction was heat-resistant (56 degrees C, 2 h) and localized in the pepsin F(ab')2 epsilon fragment, but was absent in the papain Fc epsilon and Fab epsilon fragments. Further degradation by pepsin of the F(ab')2 epsilon fragment (molecular weight 145,000 daltons) to 5S epsilon (90,000 daltons) and Fc" epsilon fragments (30,000 daltons) destroyed the reacting epitope. These results indicate that the IgM antibodies were not directed against kappa chain or idiotypic determinants of IgE(DES), but to some epitope(s) in the undegraded hinge region. Therefore, it seems that some kind of antigenic heterogeneity, perhaps related to the carbohydrate moieties, is present in IgE myeloma proteins besides the known idiotypic, allotypic and isotypic ones.

Monoclonal Antibodies against Human IgE

The specificity of five monoclonal anti-IgE antibodies (Mabs) was studied in direct latex agglutination and agglutination-inhibition experiments by particle-counting immunoassay. Twenty IgE myeloma proteins and several purified D epsilon O-, D epsilon 2-containing pepsin and papain fragments of IgE-DES(kappa) were used in the evaluation. The results demonstrate two Mabs with isotypic specificity for two distinct epitopes of the Fc epsilon-fragment within the D epsilon 1- and D epsilon 2-determinants. One Mab recognized only the immunizing IgE protein and was directed against determinants on the Fd epsilon-fragment probably related to the idiotype. Anti-Em(1) allotypic Mabs recognized all 20 IgE myeloma proteins including two of Japanese origin and the Em(1)-allotype was confined to D epsilon-determinants. Interestingly, one Mab (ALE) reacted with all 8 IgE myeloma proteins of the lambda light-chain type but none out of 12 bearing kappa chains. ALE seems therefore to recognize a new marker on IgE besides the known idiotypic, allotypic and isotypic ones. These results illustrate that a critical specificity control of Mabs is always warranted. Moreover, one should be aware of possible interference in IgE assays from the kind of determinants recognized by ALE whenever intact IgE myeloma proteins are used to raise polyclonal antisera, to get immunosorbent-purified anti-IgE antibodies or when used as tracers and standards.