Plant bioreactor is a new production platform for expression of recombinant protein, which is one of the cores of molecular farming. In this study, the anti DYKDDDDK (FLAG) antibody was recombinantly expressed in tobacco (Nicotiana benthamiana) and purified. FLAG antibody with high affinity was obtained after immunizing mice for several times and its sequence was determined. Based on this, virus vectors expressing heavy chain (HC) and light chain (LC) inoculated into Nicotiana benthamiana leaves by using Agrobacterium-mediated delivery. Accumulation of the HC and LC was analyzed by SDS/PAGE followed by Western blotting probed with specific antibodies from 2 to 9 days postinfiltration (dpi). Accumulation of the FLAG antibody displayed at 3 dpi, and reached a maximum at 5 dpi. It was estimated that 66 mg of antibody per kilogram of fresh leaves could be obtained. After separation and purification, the antibody was concentrated to 1 mg/mL. The 1:10 000 diluted antibody can probe with 1 ng/mL FLAG fused antigen well, indicating the high affinity of the FLAG antibody produced in plants. In conclusion, the plant bioreactor is able to produce high affinity FLAG antibodies, with the characteristics of simplicity, low cost and highly added value, which contains enormous potential for the rapid and abundant biosynthesis of antibodies.
Animals ; Mice ; Antibodies ; Nicotiana/genetics* ; Agrobacterium/genetics* ; Bioreactors ; Blotting, Western

Objective To prepare mouse monoclonal antibodies against the ectodomain of E2 (E2ecto) glycoprotein of Western equine encephalitis virus (WEEV). Methods A prokaryotic expression plasmid pET-28a-WEEV E2ecto was constructed and transformed into BL21 (DE3) competent cells. E2ecto protein was expressed by IPTG induction and presented mainly as inclusion bodies. Then the purified E2ecto protein was prepared by denaturation, renaturation and ultrafiltration. BALB/c mice were immunized with the formulated E2ecto protein using QuickAntibody-Mouse5W as an adjuvant via intramuscular route, boosted once at an interval of 21 days. At 35 days post-immunization, mice with antibody titer above 1×10⁴ were inoculated with E2ecto intraperitoneally, and spleen cells were fused with SP2/0 cells three days later. Hybridoma cells secreting specific monoclonal antibodies were screened by the limited dilution method, and ascites were prepared after intraperitoneal inoculation of hybridoma cells. The subtypes and titers of the antibodies in ascites were assayed by ELISA. The biological activity of the mAb was identified by immunofluorescence assay(IFA) on BHK-21 cells which were transfected with eukaryotic expression plasmid pCAGGS-WEEV-CE3E2E1. The specificity of the antibodies were evaluated with E2ecto proteins from EEEV and VEEV. Results Purified WEEV E2ecto protein was successfully expressed and obtained. Four monoclonal antibodies, 3G6G10, 3D7G2, 3B9E8 and 3D5B7, were prepared, and their subtypes were IgG2c(κ), IgM(κ), IgM(κ) and IgG1(κ), respectively. The titers of ascites antibodies 3G6G10, 3B9E8 and 3D7G2 were 10⁵, and 3D5B7 reached 10⁷. None of the four antibody strains cross-reacted with other encephalitis alphavirus such as VEEV and EEEV. Conclusion Four strains of mouse mAb specifically binding WEEV E2ecto are successfully prepared.
Horses ; Animals ; Mice ; Encephalitis Virus, Western Equine ; Ascites ; Immunosuppressive Agents ; Antibodies, Monoclonal ; Immunoglobulin M

BACKGROUND

Pre-transfusion testing is done to avoid transfusion morbidity from unexpected RBC antibodies. Available commercial kits from Western brands may not consider racial differences in antibody frequencies between East/Southeast Asians and Western populations. The limited number of blood banks in the Philippines precludes research on RBC antibody screening and identification in the country.

This study aimed to compare RBC antibody screening and identification methods in patients at a tertiary hospital in the Philippines, assess the frequency of major blood group antibodies using both techniques, and review clinical histories of discrepant and nonspecific cases.

Retrospective review showed 118 cases with both screening and identification tests using both conventional tube-based technique and column agglutination or gel-based technique. Antibody frequencies and discrepant or nonspecific results were recorded. Concordance rates were calculated, and differences between the two methods were analyzed using 95% confidence interval (95% CI). Clinical histories of discrepant and nonspecific cases were also reviewed.

The most frequent major blood group was Rh (41 cases or 34.7%), followed by MNS (34 cases or 28.8%) and Kidd (15 cases or 12.7%). The most common antibody was Anti-E (24 cases or 20.3%), followed by Anti-Mia (19 cases or 16.1%), and Anti-M and Anti-c (12 cases each, or 10.2% each). The concordance rate for screening was statistically significant at 72%. Concordance rate for identification was 59.3%, with significant difference in identifying Anti-Mia. Clinical histories for discrepant or nonspecific cases showed previous transfusions, pregnancy, lymphoproliferative conditions, and certain medications.

Statistically significant differences between the two methods were found, with the gel-based technique identifying more Anti-Mia cases. Negative results from the tube-based method do not fully exclude Anti-Mia. These discrepancies highlight the benefit of using both methods for comprehensive RBC antibody screening and identification, done as a complement to the other.

Hypoglycemic disorders are rare in persons without diabetes, and clinical evaluation to identify its etiology can be challenging. We present a case of insulin autoimmune syndrome induced by carbimazole in a middle-aged Chinese man with underlying Graves’ disease, which was managed conservatively with a combination of dietary modification and alpha-glucosidase inhibitor.
Hypoglycemia ; Hyperinsulinism ; Insulin Antibodies

BACKGROUND

The Philippines is hyperendemic for hepatitis B infection. Vaccination is crucial for protection. Local data on the antibody response after completion of the primary vaccination series is limited.

This study aimed to measure the anti-HBs levels among college students who completed primary hepatitis B vaccination series, compare seropositivity across stratified groups and correlate anti-HBs levels with the time elapsed since the last vaccine dose.

This cross-sectional study included 111 college students in health-related courses with immunization record showing the complete primary Hepatitis B vaccination series. Participants were stratified based on the following vaccination schedules: 0-1-6-month group; 0-1-2-month group; and booster group. Anti-HBs titers were determined.

Statistical analyses included One-way ANOVA, Kruskal-Wallis test, Fisher’s Exact test and Shapiro-Wilk normality test. Kaplan-Meier Survival Estimate assessed the probability of anti-HBs seropositivity over time. Data were analyzed using STATA 13.1.

The baseline characteristics of the study population were homogenous. The median anti-HBs titer several years after primary vaccination was low at 2.9 mIU/mL. Participants in the booster group had the highest seropositivity rate (57.14%) with a median titer of 30.16 mIU/mL. There was an inverse relationship between anti-HBs titer and elapsed time since the last vaccine dose. Kaplan-Meier Survival Estimate showed that the seropositivity decreases to 90.56% after 15.8 years, 51.3% after 17.5 years, and 2.97% at 18 years.

This study revealed low anti-HBs titers among students who previously completed primary vaccination series, with no significant difference between two schedules. Booster doses resulted in the highest seropositivity. Over-all, seropositivity declines over time.

Porcine epidemic diarrhea (PED) is a highly contagious disease that causes high mortality in suckling piglets. Although several licensed inactivated and live attenuated vaccines were widely used, the infection rate remains high due to unsatisfactory protective efficacy. In this study, mRNA vaccine candidates against PED were prepared, and their immunogenicity was evaluated in mice and pregnant sows. The mRNA PED vaccine based on heterodimer of viral receptor binding region (RBD) showed good immunogenicity. It elicited robust humoral and cellular immune responses in mice, and the neutralizing antibody titer reached 1:300 after a single vaccination. Furthermore, it induced neutralizing antibody level similar to that of the inactivated vaccine in pregnant sows. This study developed a new design of PED vaccine based on the mRNA-RBD strategy and demonstrated the potential for clinical application.
Pregnancy ; Swine ; Animals ; Female ; Mice ; Antibodies, Viral ; Swine Diseases/epidemiology* ; Viral Vaccines/genetics* ; Antibodies, Neutralizing ; Vaccines, Attenuated ; Diarrhea/veterinary*

So far,the coronavirus disease 2019(COVID-19)has been persisting for nearly three years,infecting about 700 million people and causing more than 6 million deaths,which has seriously affected the human society.According to Global Initiative on Sharing All Influenza Data,there are more than 12 million SARS-CoV-2 variants,of which the five major variants of concern are Alpha,Beta,Gamma,Delta and Omicron.Their infectivity,pathogencity,and neutralization resistance have changed greatly compared with the original strain,which has brought great pressure to the prevention and control of the pandemic.Antibody level testing is critical for confirming infection,epidemiological investigation,vaccine development,and neutralizing drug preparation.Focusing on the humoral immunity against SARS-CoV-2,this paper introduces the mutation sites,neutralization resistance,and vaccination efficacy of the five variants of concern,and briefly summarizes the evolutionary characteristics,future mutation directions,and host immunity.
Humans ; SARS-CoV-2/genetics* ; Antibody Formation ; COVID-19 ; Gamma Rays ; Antibodies, Neutralizing ; Antibodies, Viral

Zearalenone(ZEN) is a toxic metabolite produced by Fusarium culmorum, F. graminearum, F. tricinctum, and other fungi, with estrogenic characteristics. Exposure to or ingestion of ZEN during pregnancy can cause reproductive dysfunction, miscarriage, stillbirth, and malformation, and seriously endanger human life and health. The detection methods for ZEN in the Chinese Pharmacopoeia(2020 edition) are liquid chromatography(LC) and liquid chromatography-mass spectrometry(LC-MS), and it is stipulated that ZEN should not exceed 500 μg in 1 000 g of Coicis Semen. Although these detection methods by instruments can achieve the qualitative and quantitative analysis of ZEN in Coicis Semen, their high detection cost and long periods hinder the rapid screening of a large number of samples in the field. In this study, the synthesized ZEN hapten was conjugated with bovine serum albumin(BSA) and ovalbumin(OVA) to obtain the complete ZEN antigen. By virtue of antibody preparation techniques, ZEN monoclonal antibody 4F6 was prepared, which showed 177.5%, 137.1%, and 109.7% cross-reactivity with ZEN structural analogs zearalanol, zearalenone, and α-zearalenol, respectively, and no cross-reactivity with other fungal toxins such as aflatoxin. Direct competitive enzyme-linked immunosorbent assay(dcELISA) based on ZEN monoclonal antibody 4F6 was developed for the determination of ZEN in Coicis Semen with an IC_(50) of 1.3 μg·L~(-1) and a detection range of 0.22-21.92 μg·L~(-1). The recoveries were 83.91%-105.3% and the RSD was 4.4%-8.0%. The established dcELISA method was used to determine the ZEN residuals in nine batches of Coicis Semen samples, and the results were validated by LC-MS. The correlation between the two detection methods was found to be 0.993 9, indicating that the established dcELISA could be used for the rapid qualitative and quantitative detection of ZEN residuals in Coicis Semen.
Humans ; Female ; Pregnancy ; Zearalenone ; Coix ; Enzyme-Linked Immunosorbent Assay ; Mycotoxins ; Antibodies, Monoclonal

Both anti-glomerular basement membrane (GBM) disease and the anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) are common causes of pulmonary-renal syndrome. Organizing pneumonia (OP), a special pattern of interstitial lung disease, is extremely rare either in AAV or anti-GBM disease. We report an old woman presented with OP on a background of co-presentation with both ANCA and anti-GBM antibodies.
Female ; Humans ; Antibodies, Antineutrophil Cytoplasmic ; Organizing Pneumonia ; Autoantibodies ; Glomerulonephritis ; Anti-Glomerular Basement Membrane Disease ; Pneumonia ; Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/complications*

Food-dependent, exercise-induced anaphylaxis (FDEIA) is a potentially life-threatening disorder that often occurs with exercise, and patients typically have eaten a specific food within hours before disease onset. This disease is exceedingly rare, with a prevalence of 0.02%. No well-recognized prevention or treatment strategy has been available for FDEIA except avoiding triggers strictly. Here we report an 11-year-old boy with a history of recurrent anaphylaxis of unknown etiology more than 10 times within two years. As the anaphylactic symptoms had not been controlled after traditional treatments, the patient was given subcutaneous injection of dupilumab seven times within 33 weeks. During dupilumab treatments, the patient was exposed to culprit mushrooms plus exercises at least twice a month but without notable anaphylaxis. Thus, Dupilumab may improve the allergic reactions in FDEIA patients.
Male ; Humans ; Child ; Anaphylaxis/etiology* ; Food Hypersensitivity/diagnosis* ; Exercise-Induced Allergies ; Antibodies, Monoclonal, Humanized/therapeutic use*