Different fragments of SARS-CoV-2 nucleocapsid (N) protein were expressed and purified, and a fluorescence immunochromatography method for detection of SARS-CoV-2 total antibody was established. The effect of different protein fragments on the performance of the method was evaluated. The N protein sequence was analyzed by bioinformatics technology, expressed in prokaryotic cell and purified by metal ion affinity chromatography column. Different N protein fragments were prepared for comparison. EDC reaction was used to label fluorescence microsphere on the synthesized antigen to construct sandwich fluorescence chromatography antibody detection assay, and the performance was systemically evaluated. Among the 4 prepared N protein fragments, the full-length N protein (N419) was selected as the optimized coating antigen, N412 with 0.5 mol/L NaCl was used as the optimal combination; deleting 91-120 amino acids from the N-terminal of N412 reduced non-specific signal by 87.5%. the linear range of detection was 0.312-80 U/L, the limit of detection was 0.165 U/L, and the accuracy was more than 95%. A fluorescence immunochromatographic detection method for analysis of SARS-CoV-2 total antibody was established by pairing N protein fragments. The detection result achieved 98% concordance with the commercially available Guangzhou Wanfu test strip, which is expected to be used as a supplementary approach for detection of SARS-CoV-2. The assay could also provide experimental reference for improving the performance of COVID-19 antibody detection reagents.
Antibodies, Viral ; COVID-19 ; Chromatography, Affinity ; Fluorescent Antibody Technique ; Humans ; Microspheres ; SARS-CoV-2 ; Sensitivity and Specificity

Reslizumab and mepolizumab are recently approved monoclonal antibodies for the treatment of severe (uncontrolled) eosinophilic asthma. Both are effective in neutralizing the function of interleukin-5 (IL-5). This study is the first to compare the binding affinity and in vitro potency of both antibodies in head-to-head assays. Two assays assessed binding affinity (using the equilibrium dissociation constant [K(D)]) of each drug for human IL-5. In the Biacore surface plasmon resonance assay, the association constant (k(on)) values for human IL-5 for reslizumab and mepolizumab were 3.93 × 10⁶ and 1.83 × 10⁵, respectively. The dissociation constant (k(off)) values were 4.29 × 10⁻⁴ and 2.14 × 10⁻⁴, respectively. Calculated K(D) values for human IL-5 for reslizumab and mepolizumab were 109 and 1,170 pM, respectively, representing an approximately 11-fold stronger binding affinity with reslizumab. In the Kinetic Exclusion Assay, the k(on) values for human IL-5 for reslizumab and mepolizumab were 3.17 × 10⁶ and 1.32 × 10⁵, respectively. The k(off) values were 1.36 × 10⁻⁵ and 1.48 × 10⁻⁵, respectively. Measured K(D) values for human IL-5 for reslizumab and mepolizumab were 4.3 and 112 pM, respectively, representing an approximately 26-fold stronger binding affinity for reslizumab. A human-IL-5-dependent cell proliferation assay was developed to assess in vitro potency, based on a human cell line selected for enhanced surface expression of IL-5 receptor-alpha and consistent proliferation response to IL-5. The concentration at which 50% inhibition occurred (IC₅₀) was determined for both antibodies. Reslizumab and mepolizumab inhibited IL-5-dependent cell proliferation, with IC₅₀ values of approximately 91.1 and 286.5 pM, respectively, representing on average 3.1-fold higher potency with reslizumab. In conclusion, comparative assays show that reslizumab has higher affinity binding for and in vitro potency against human IL-5 compared with mepolizumab. However, these results do not take into consideration the different methods of administration of reslizumab and mepolizumab.
Antibodies ; Antibodies, Monoclonal ; Antibody Affinity ; Asthma ; Cell Line ; Cell Proliferation ; Drug Evaluation, Preclinical ; Eosinophils ; Humans ; In Vitro Techniques ; Interleukin-5 ; Surface Plasmon Resonance

The present study aimed at evaluating serum immunoglobulin G (IgG) avidity to Porphyromonas gingivalis in elderly patients with mild and severe chronic periodontitis. The avidity of antibodies against P. gingivalis present in the sera of 18 patients with mild chronic periodontitis and 18 patients with severe chronic periodontitis was evaluated using an ammonium thiocyanate-dissociated enzyme-linked immunosorbent assay (ELISA). The results showed that the mean absorbance value in serum IgG antibody titers was significantly higher in the severe chronic periodontitis group than in the mild chronic periodontitis group (198 ± 35 ELISA unit [EU] vs. 142 ± 32 EU, p < 0.01). However, there was no significant difference between the two groups in antibody avidity (65 ± 57 EU vs. 54 ± 27 EU). These findings suggest that humoral immune responses to P. gingivalis between mild and severe chronic periodontitis in elderly patients are characterized by the differences in the quantity rather than the quality of the antibodies.
Aged ; Ammonium Compounds ; Antibodies ; Antibody Affinity ; Chronic Periodontitis ; Enzyme-Linked Immunosorbent Assay ; Geriatrics ; Humans ; Immunity, Humoral ; Immunoglobulin G ; Immunoglobulins ; Porphyromonas gingivalis ; Porphyromonas

PURPOSE: High-affinity receptor I (FcεRI) on mast cells and basophils plays a key role in the immunoglobulin E (IgE)-mediated type I hypersensitivity mediated by allergen cross-linking of the specific IgE-FcεRI complex. Thus, prevention of IgE binding to FcεRI on these cells is an effective therapy for allergic disease. We have developed a strategy to disrupt IgE binding to FcεRI using an antibody targeting FcεRIα. MATERIALS AND METHODS: Fab fragment antibodies, which lack the Fc domain, with high affinity and specificity for FcεRIα and effective inhibitory activity against IgE-FcεRI binding were screened. IgE-induced histamine, β-hexosaminidase and Ca2+ release in basophils were determined by ELISA. A B6.Cg-Fcer1a(tm1Knt) Tg(FCER1A)1Bhk/J mouse model of passive cutaneous anaphylaxis (PCA) was used to examine the inhibitory effect of NPB311 on allergic skin inflammation. RESULTS: NPB311 exhibited high affinity to human FcεRIα (KD=4 nM) and inhibited histamine, β-hexosaminidase and Ca2+ release in a concentration-dependent manner in hFcεRI-expressing cells. In hFcεRIα-expressing mice, dye leakage was higher in the PCA group than in controls, but decreased after NPB311 treatment. NPB311 could form a complex with FcεRIα and inhibit the release of inflammation mediators. CONCLUSION: Our approach for producing anti-FcεRIα Fab fragment antibody NPB311 may enable clinical application to a therapeutic pathway in IgE/FcεRI-mediated diseases.
Animals ; Antibodies ; Antibody Affinity ; Basophils ; Enzyme-Linked Immunosorbent Assay ; Histamine ; Humans ; Hypersensitivity, Immediate ; Immunoglobulin E* ; Immunoglobulins* ; Inflammation Mediators ; Inflammation* ; Mast Cells ; Mice ; Passive Cutaneous Anaphylaxis ; Sensitivity and Specificity ; Skin

PURPOSE: High-affinity receptor I (FcεRI) on mast cells and basophils plays a key role in the immunoglobulin E (IgE)-mediated type I hypersensitivity mediated by allergen cross-linking of the specific IgE-FcεRI complex. Thus, prevention of IgE binding to FcεRI on these cells is an effective therapy for allergic disease. We have developed a strategy to disrupt IgE binding to FcεRI using an antibody targeting FcεRIα. MATERIALS AND METHODS: Fab fragment antibodies, which lack the Fc domain, with high affinity and specificity for FcεRIα and effective inhibitory activity against IgE-FcεRI binding were screened. IgE-induced histamine, β-hexosaminidase and Ca2+ release in basophils were determined by ELISA. A B6.Cg-Fcer1a(tm1Knt) Tg(FCER1A)1Bhk/J mouse model of passive cutaneous anaphylaxis (PCA) was used to examine the inhibitory effect of NPB311 on allergic skin inflammation. RESULTS: NPB311 exhibited high affinity to human FcεRIα (KD=4 nM) and inhibited histamine, β-hexosaminidase and Ca2+ release in a concentration-dependent manner in hFcεRI-expressing cells. In hFcεRIα-expressing mice, dye leakage was higher in the PCA group than in controls, but decreased after NPB311 treatment. NPB311 could form a complex with FcεRIα and inhibit the release of inflammation mediators. CONCLUSION: Our approach for producing anti-FcεRIα Fab fragment antibody NPB311 may enable clinical application to a therapeutic pathway in IgE/FcεRI-mediated diseases.
Animals ; Antibodies ; Antibody Affinity ; Basophils ; Enzyme-Linked Immunosorbent Assay ; Histamine ; Humans ; Hypersensitivity, Immediate ; Immunoglobulin E* ; Immunoglobulins* ; Inflammation Mediators ; Inflammation* ; Mast Cells ; Mice ; Passive Cutaneous Anaphylaxis ; Sensitivity and Specificity ; Skin

Recombinant antigenic proteins of Toxoplasma gondii are alternative source of antigens which are easily obtainable for serodiagnosis of toxoplasmosis. In this study, highly antigenic secretory organellar proteins, dense granular GRA2 and GRA3, rhoptrial ROP2, and micronemal MIC2, were analyzed by bioinformatics approach to express as water-soluble forms of antigenic domains. The transmembrane region and disorder tendency of 4 secretory proteins were predicted to clone the genes into pGEX-4T-1 vector. Recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli, and GST fusion proteins were expressed with IPTG. As a result, GST fusion proteins with GRA225-105, GRA339-138, ROP2324-561, and MIC21-284 domains had respectively higher value of IgG avidity. The rGST-GRA225-105 and rGST-GRA339-138 were soluble, while rGST-ROP2324-561 and rGST-MIC21-284 were not. GRA231-71, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The rGST-GRA231-71-ROP2324-561, a chimeric protein, appeared to be soluble. Moreover, rGST-GRA231-71-MIC21-284 was also soluble and had higher IgG avidity comparing to rGST-MIC21-284. These 4 highly expressed and water-soluble recombinant antigenic proteins may be promising candidates to improve the serodiagnosis of toxoplasmosis in addition to the major surface antigen of SAG1.
Animals ; Antibodies, Protozoan/immunology ; Antibody Affinity ; Antigens, Protozoan/chemistry/*diagnostic use/genetics/immunology ; *Gene Expression ; Immunoglobulin G/blood/immunology ; Mice, Inbred BALB C ; Recombinant Proteins/chemistry/*diagnostic use/genetics/immunology ; Serologic Tests/methods ; Solubility ; Toxoplasma/genetics/immunology/*metabolism ; Toxoplasmosis/diagnosis

Toxoplasma gondii is an obligate intracellular protozoan that is distributed worldwide. Recently, several tests for avidity of Toxoplasma IgG antibodies have been introduced to help discriminate between recently acquired and distant infections. The study was conducted in Jawaharlal Nehru Medical College and Hospital, India from February 2011 to September 2012. Serum specimens were subjected to Toxoplasma IgM ELISA and IgG avidity ELISA test. Out of 48 patients with abortions, 17 (35.4%) were positive for IgM ELISA, and 8 (16.6%) had low IgG avidity antibodies. Out of 48 patients with other obstetric problems, 23 (47.9%) were positive for IgM ELISA, and 17 (35.4%) had low IgG avidity antibodies. Combining both groups on avidity test, only 25 of 40 (62.5%) IgM-positive women had low-avidity IgG antibodies suggesting a recent T. gondii infection in these women. More importantly, 15 (37.5%) of the IgM-positive women had high-avidity antibodies suggesting that the infection was acquired before gestation The relation of IgM seropositivity with the following risk factors was not found to be statistically significant; contact with cats (0.13), non-vegetarian food habits (0.05), and low socio-economic status (0.49). While, for IgG avidity ELISA, only contact with cats (0.01) was significantly associated with seropositivity. All other risk factors have P-values of >0.05 (not significant). IgG avidity test when used in combination with IgM test was a valuable assay for diagnosis of ongoing or recently acquired T. gondii infection in India.
Abortion, Spontaneous/immunology ; Adolescent ; Adult ; Animals ; Antibodies, Protozoan/*immunology ; *Antibody Affinity ; Cats ; Enzyme-Linked Immunosorbent Assay ; Female ; Food Contamination ; Humans ; Immunoglobulin G/blood/immunology/*physiology ; Immunoglobulin M/blood ; India/epidemiology ; Risk Factors ; Seroepidemiologic Studies ; Toxoplasma/*immunology ; Toxoplasmosis/epidemiology/*immunology ; Young Adult

OBJECTIVETo identify the self-preparation monoclonal antibody which target to clenbuterol, and set up the standard curve to clenbuterol (CL) detection.

METHODSThe affinity constants and activity of the monoclonal antibody which target to CL were determined by ELISA. ELISA was also used to confirm whether the monoclonal antibody had any across-reaction with BSA and CL analogues. The rat ascites which contains the monoclonal antibody target to CL was purified by (NH4)2SO4 salt-out method and further by affinity column. At last, the CL detection standard curve which based on indirect competition ELISA was established.

RESULTSThe ELISA experiment showed that the antibody titer was 10(6) and the monoclonal antibody affinity constants was 2.90 x 10(10) L/mol. The result of the indirect competition ELISA confirmed that the monoclonal antibody had no cross-reaction with BSA and a few kind of CL analogue. CL detection standard curve based on indirect competition ELISA was established, which R2 was 0.9812, and the lowest detectable limit was 1.0 ng/ml.

CONCLUSIONThe standard curve based on indirectly competitioning ELISA was established. The self-preparation monoclonal antibody which target to CL has high affinity and high specific to CL, which had established the foundation to the advanced development of the CL immune test paper and CL ELISA kit.

Animals ; Antibodies, Monoclonal ; chemistry ; isolation & purification ; Antibody Affinity ; Clenbuterol ; immunology ; Cross Reactions ; Enzyme-Linked Immunosorbent Assay ; Limit of Detection ; Rats

Epidermal growth factor receptor (EGFR) is an attractive target for tumor therapy because it is overexpressed in the majority of solid tumors and the increase in receptor expression levels has been linked with a poor clinical prognosis. Also it is well established that blocking the interaction of EGFR and the growth factors could lead to the arrest of tumor growth and possibly result in tumor cell death. A13 is a murine monoclonal antibody (mAb) that specifically binds to various sets of EGFR-expressing tumor cells and inhibits EGF-induced EGFR phosphorylation. We isolated human immunoglobulin genes by guided selection based on the mAb A13. Four different human single chain Fvs (scFvs) were isolated from from hybrid scFv libraries containing a human VH repertoire with the VL of mAb A13 and a human VL repertoire with the VH of mAb A13. All the 4 scFvs bound to EGFR-expressing A431 cells. One scFv (SC414) with the highest affinity was converted to IgG1 (ER414). The ER414 exhibited ~17 fold lower affinity compared to the A13 mAb. In addition the ER414 inhibited an EGF-induced tyrosine phosphorylation of EGFR with much lower efficacy compared to the A13 mAb and Cetuximab (Merck KgaA, Germany). We identified that the epitope of A13 mAb is retained in ER414. This approach will provide an efficient way of converting a murine mAb to a human mAb.
Animals ; Antibodies, Monoclonal, Humanized/*genetics/immunology/therapeutic use ; Antibody Affinity ; Cell Line, Tumor ; Directed Molecular Evolution/*methods ; Epitope Mapping ; Epitopes/genetics/immunology/therapeutic use ; Humans ; *Immunotherapy ; Mice ; Neoplasms/*therapy ; Phosphorylation/drug effects ; Protein Binding ; Receptor, Epidermal Growth Factor/*antagonists & inhibitors/immunology ; Selection, Genetic ; Single-Chain Antibodies/*genetics/immunology/therapeutic use

Serum samples, 100 in the total number, were collected from different laboratories in Tehran, Iran and tested for anti-Toxoplasma specific IgG and IgM antibodies using indirect immunofluorescent antibody test (IFAT). Using the IgG (chronic) and IgM (acute) positive samples, the IgG avidity test was performed by ELISA in duplicate rows of 96-well microtiter plates. One row was washed with 6 M urea and the other with PBS (pH 7.2), then the avidity index (AI) was calculated. Sixteen out of 18 (88.9%) sera with acute toxoplasmosis showed low avidity levels (AI< or =50), and 76 out of 82 (92.7%) sera in chronic phase of infection showed high avidity index (AI>60). Six sera had borderline ranges of AI. The results showed that the IgG avidity test by ELISA could distinguish the acute and chronic stages of toxoplasmosis in humans.
Antibodies, Protozoan/*blood ; *Antibody Affinity ; Clinical Laboratory Techniques/*methods ; Enzyme-Linked Immunosorbent Assay/methods ; Fluorescent Antibody Technique, Indirect ; Humans ; Immunoglobulin G/*blood ; Immunoglobulin M/blood ; Iran ; Parasitology/*methods ; Toxoplasmosis/*diagnosis