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α-conotoxin (α-CTX) MI is a small peptide toxin with 14 amino acids and two disulfide bonds. It potently inhibits muscle-type nicotinic acetylcholine receptors (nAChRs), and poses a threat as a toxin to tropical fishermen. However, there are currently no effective drugs for the treatment of MI envenomation due to the toxin's low immunogenicity. In this report, we generated neutralizing antiserum and F(ab') 2 to MI by synthesizing a new MI antigen through the coupling of alkynyl-modified MI and azide-modified bovine serum albumin (BSA), followed by immunization into mouse and horse. The new MI-BSA antigen generated high titers of mouse and horse antiserum (1:204,800 and 1:51,200, respectively), and both the antiserum as well as the horse F(ab') 2 displayed highly potent neutralization and detoxification efficacy. 12.5 μL of mouse or horse antiserum preincubated with MI could completely neutralize a lethal dose of the MI (0.4 μg, 1.7 × LD 50), while 6.25 μL (mouse) or 10.41 μL (horse) of the antiserum could exert complete detoxification of mice injected with 1.7 × LD 50 of MI. Moreover, the mouse and horse antiserum exhibited medium cross-reactivity for highly toxic α-CTX GI. These results demonstrate that the integrity of MI's antigen epitope and carrier effect of BSA can improve MI's immunogenicity, and provides an effective detoxification treatment for highly toxic α-conotoxins as well as an effective method for the preparation of antiserum of small peptide toxins. [Display omitted] • α-conotoxin MI-bovine serum albumin conjugate antigen was synthesized by click reaction. • This antigen maintains the integrity of MI's antigen epitope. • This antigen displays high immunogenicity. • This antigen produces antiserum with high neutralizing and detoxification activity. • The MI's antiserum possesses medium cross-reactivity to α-conotoxin GI. [ABSTRACT FROM AUTHOR]

Fungal cell surface carbohydrates and proteins are useful antigens for the development of antifungal vaccines. In this study, glycopeptides consisting of the β -1,2-mannan and N -terminal peptide epitopes of Candida albicans (C. albicans) cell wall phosphomannan complex and Als1p (rAls1p-N) protein, respectively, were synthesized and covalently conjugated with keyhole limpet hemocyanin (KLH) and human serum albumin (HSA) through homobifunctional disuccinimidyl glutarate. The resultant KLH-conjugates were immunologically evaluated using Balb/c mice to reveal that they induced high levels of IgG antibodies. Furthermore, these conjugates showed self-adjuvanting property, as they could promote robust antibody responses without the presence of an external adjuvant. More significantly, the obtained antisera could effectively recognize both the carbohydrate and the Als1 peptide epitopes and immunofluorescence and flow cytometry assays also demonstrated that the elicited antibodies could react with the cell surface of a number of fungi, including C. albicans , C. tropicalis , C. lustaniae and C. glabrata. These results suggested the great potential of these conjugates as antifungal vaccines. Image 1 • A series of β-1,2-mannan-peptide conjugates have been synthesized as antifungal vaccines. • The KLH-conjugates elicited high levels of IgG antibodies in mice even administered without complete Freund's adjuvant. • The obtained antisera could effectively recognize both the carbohydrate and the Als1 peptide epitopes. • 1b elicited antibodies could react with cell surface antigens of C. albicans, C.tropicalis, C. lustaniae , and C. glabrata. [ABSTRACT FROM AUTHOR]

To investigate the cross-reactivity between the sera collected from Vaccinia Virus Tiantan Strain vaccinated rabbits and viral antigens of monkeypox virus. Vaccinia viruses were prepared on chicken embryo fibroblasts (CEF) and Vero cells respectively named as CEF-VTT NVSI-1 and Vero-VTT NVSI-1. Rabbits were inoculated with a total of three doses of adjuvanted 1.3 × 108 PFU CEF-VTT NVSI-1 each dose or adjuvanted 3.9 × 107 PFU Vero-VTT NVSI-1 (Freunds complete adjuvant) via the subcutaneous route. We then performed the enzyme-linked immunosorbent assay (ELISA) and bio-layer interferometry (BLI) for determination of the binding activity and affinity of immune sera to five crucial surface antigens on monkeypox virus including A35, B6R, H3 and to corresponding homologous antigens A33R, B5 and L1R of vaccinia virus. For comparison, plaque reduction neutralizing tests were used to evaluate the neutralization of immune sera against vaccinia virus. Both CEF-VTT NVSI-1 and Vero-VTT NVSI-1 vaccinations following planned schedule could induce neutralizing antibody titers greater than 1:2048 in rabbit sera. Binding antibodies targeting monkeypox viral antigens were confirmed by both indirect ELISA and BLI methods. Indirect ELISA for rabbit sera revealed 1:51200 binding antibody titers to A35/B6R/H3 monkeypox virus antigens while BLI tests yielded affinities at 2 × 10−6 to 8 × 10−7 between the sera and the three antigens. Similarly, such sera showed binding strength to vaccinia virus antigens A33R/B5/L1R consistent with that to three preceding monkeypox virus antigens. These results demonstrated the cross-reactivity between the sera of vaccinia virus vaccinated animals and monkeypox virus antigens. Traditional ELISA test and BLI method displayed a high consistency in antigen screening and they were further proved to correlate to the results of plaque reduction neutralizing test, which indicates that BLI could be utilized as an indirect alternative for assessment of neutralizing activity of samples in response to live virus. Sera of vaccinia virus-vaccinated rabbits exhibited cross-reactivity with viral antigens of monkeypox virus. Potential in improving the accuracy of antigen discovery while reducing the lengthy work needed for the screening as BLI method possesses, it contributes greatly to the rapid preliminary evaluation of immune response generated by vaccines. • Vaccinia Virus elicited antibodies could effectively cross-react with MPXV antigens. • Affinity screening with Biolayer interferometry. • Vaccinia Virus elicited immune response reactive to diverse antigens. [ABSTRACT FROM AUTHOR]

For the detection of small hapten molecules, indirect competitive enzyme-linked immunosorbent assay (icELISA) is a preferred method. However, diverse coating antigen might bring different antiserum titer and sensitivity for the identical antiserum. In the present study, four AFB1-protein (aflatoxin B1-carrier protein) conjugates were prepared by activated ester method (AFB1O-BSA/AFB1O-OVA) and mannich method (AFB1-cBSA/AFB1-cOVA), and then applied as coating antigen for titer and sensitivity detection of the identical antiserum obtained from rabbit immunized by AFB1-KLH. Afterwards, the ultraviolet–visible, fluorescence and far-ultraviolet circular dichroism (far-UV CD) spectra were recorded for understanding the difference in titer and sensitivity obtained. Results revealed that AFB1O-BSA/AFB1O-OVA showed a strong intrinsic fluorescence band centered at 450 nm that originated from the emission of AFB1, which differed from AFB1-cBSA/AFB1-cOVA, while the decrease of α-helical and increase of β-sheet in AFB1-cBSA was the most remarkable. This indicated that the better sensitivity obtained by using AFB1O-BSA as coating antigen might be caused by its extended structure, because such structure affect the binding between AFB1 and antibody. The study might offer structural information for understanding the titer and sensitivity difference caused by coating antigen. [ABSTRACT FROM AUTHOR]

Background Haemagglutination has been the gold standard for defining the blood group status. However, these tests depend upon the availability of specific and reliable antisera. Potent antisera for extended phenotyping are very costly, weakly reacting or available in limited stocks and unavailable for some blood group systems like Indian, Dombrock, Coltan, Diego etc. The Indian blood group system consists of two antithetical antigens, In a and In b . The In a /In b polymorphism arises from 252C > G missense mutation in the CD44 gene. This knowledge has allowed the development of molecular methods for genotyping IN alleles. Material and methods Blood samples were collected from 715 blood donors from Mumbai. DNA was extracted using phenol-chloroform method and genotyping for Indian (In a / IN*01 , In b / IN*02 ) blood group alleles was done by Sequence Specific PCR. Results Seventeen donors among 715 were heterozygous for In a antigen i.e. In (a+b+). The In a antigen positivity was confirmed serologically, using anti-In a prepared in-house and the genotype-phenotype results were concordant. The frequency of In a (2.37%) was higher than Caucasians and comparable to those reported among Indians of Bombay. Conclusion This is the first study reporting molecular screening of Indian blood group antigens in Indian population. The frequency of In a and In b antigens was found to be 2.37% and 100% respectively. Red cells of In a positive donors can be used as in-house reagent red cells for screening and identification of corresponding antibodies. Thus, DNA based methods will help in large scale screening of donors to identify rare blood groups, when commercial antisera are unavailable. [ABSTRACT FROM AUTHOR]

• All these patients even at 12 months after onset had IgG and IgM antibodies. • Multiple peptides identified were able to be recognized by most of the sera isolated from both AS and S patients, and three peptides located in RBD. • •Five epitopes exhibited strong discriminatory ability between the AS and s patients, and were identified to be associated with the clinical adverse events. We know little about the antigen bias in SARS-CoV-2 humoral response and the epitopes of spike recognized by the immune system in asymptomatic (AS) patients and symptomatic (S) patients. Here, we used a microarray to evaluate the humoral immune response in the sera collected from 33 COVID-19-recovered patients up to 1 year. We found that the levels of IgG and IgM induced by the 23 proteins differed significantly in the same patients, and were able to distinguish AS and S patients. The N- and S-specific antibodies were detected even at 12 months after onset. Five epitopes were identified to be associated with the clinical adverse events, and three peptides located in RBD. Overall, this study presents a systemic view of the SARS-CoV-2 specific IgG and IgM responses between AS and S recovered patients and provide insights to promote precise development of SARS-CoV-2 vaccines. [ABSTRACT FROM AUTHOR]

• Ninety eight out of 680 E. necatrix protein spots were recognized by immune sera. • Out of the 56 spots selected for MS analysis, 50 proteins were identified. • Of the 50 proteins, 8 were previously characterized. • The proteins have a wide range of structures, cellular locations and functions. Eimeria necatrix , an apicomplexan protozoa of the genus Eimeria, causes intestinal coccidiosis that can reduce growth performance of poultry and result in high mortality in older chickens. In this report, the whole sporozoite proteins of E.necatrix were studied by two-dimensional electrophoresis (2-DE) and Western blotting using hyper-immune chicken serum containing E.necatrix -specific antibodies. Approximately 680 protein spots for E.necatrix sporozoite were detected by 2-DE with silver staining, where 98 spots were cross-reacted with the E. necatrix- specific immune sera. Out of the 56 spots that were selected for MALDI-TOF-MS/MS analysis, 50 unique proteins were identified using the MASCOT software, 8 proteins were identified as known E.necatrix proteins and the rest were all putative proteins. These proteins have a wide range of known or predicted structures, cellular locations and functions, including proteins in category nuclear location & function, multifunctional- or multifunctional motifs-containing proteins, cellular transport and structure-related proteins, proteins of enzymatic activities, motor proteins-related, cell surface and organelle-related proteins. These new findings will enhance our understandings of parasite immunogenicity and immune evasion mechanisms of E. necatrix and facilitate the discovery phase of highly effective vaccine candidates. [ABSTRACT FROM AUTHOR]

• Non-epidemic norovirus genotype GII.8 virus-like particles (VLP) were expressed using the baculovirus-insect cell system. • Binding of GII.8 VLP to tissue blood group antigens (HBGAs) was observed. • The cross-reactivity of GII.8 VLP antiserum with other 27 (9 GI genotypes and 18 GII genotypes) capsid protein was evaluated. • Norovirus GII.8 strain antiserum had no cross-blocking effect on the binding of epidemic genotypes (GII.4, GII.17) to HBGA. • The existing vaccine candidate strain GI.1 antiserum and GII.4 antiserum had no cross-blocking effect on the binding of NoV GII.8 to HBGA. Noroviruses are causative agents of acute nonbacterial gastroenteritis epidemics worldwide. There are various genotypes, among which the non-epidemic genotype GII.8 can cause norovirus outbreaks. We previously demonstrated that the immunogenicity of GII.8 differed from that of epidemic variants. This study aimed to comprehensively compare the receptor profile and immunogenicity of the GII.8 variant with those of the epidemic variants. Using the baculovirus-insect cell expression system, we observed that recombinant capsid protein VP1 of the norovirus GII.8 GZ2017-L601 strain formed virus-like particles (VLPs) with a diameter of approximately 30 nm, as evidenced by transmission electron microscopy analysis. The GII.8 VLPs showed weak or moderate binding with all secretor histo-blood group antigens (HBGAs), but not with non-secretors, as evidenced by the HBGA-VLP binding test. The GII.8 VLP antiserum obtained from immunized BALB/c mice was tested for cross-reactivity with other norovirus genotypes (n = 28). The results showed that this antiserum demonstrated moderate cross-reactivity with GI.1, GII.3, and GII.15; however, no cross-reactivity with the epidemic variants of GII.2, GII.4, and GII.17 was observed. Additionally, the blocking-antibody activity of GII.8 antisera against GII.4 VLP-HBGAs and GII.17 VLP-HBGAs interactions and the cross-blocking of GII.8 VLP-HBGAs interactions by GI.1 and GII.4 antisera were evaluated using the HBGAs-VLP blocking test. However, no cross-blocking effect was observed. In summary, the characterization of norovirus GII.8 VLPs and derived antisera revealed that the GII.8 immunogenicity differed from that of epidemic variants. [ABSTRACT FROM AUTHOR]

Polyagglutination is a rare entity in immunohematology and unusually presents in a healthy blood donor. The general presentation was described in the literature in association with bacterial infections, which result in the exposure of crypt antigens. Nowadays, polyagglutination is rarely detected due to the use of monoclonal antisera. Our case report describes the presence of Tn polyagglutination in a healthy adult blood donor with no prior history of any infection in the recent past. Immunohematology work-up for incompatible cross-match was done in the serology lab using commercially procured antisera and column agglutination gel card (Tulip Diagnostics India Pvt. Ltd, Goa, India). The three cell-screening panel was procured commercially (ID Dia cell I, II, III; Bio-Rad, Switzerland), and in-house lectin was prepared as per the standard method. We have come across a case of incompatible cross-match with negative antibody screen, auto-control, and Negative direct coombs test. Cross-match with multiple adult serum and cord serum gives us a clue towards polyagglutination. Further, Polyagglutination was confirmed serologically using anti-A1 lectin and later concludes of Tn type by lectin prepared in-house from Salvia Sclarea. Resolution of incompatible cross-match in a case of polyagglutination needs a skilled workforce and rare reagents. Identification of reason for incompatibility helps in an early issue of blood units. [ABSTRACT FROM AUTHOR]

Tumor-associated carbohydrate antigens Lewis X (Lex), Lewis Y (Ley), and KH-1 are useful targets for cancer immunotherapy. In this regard, an insight into the structure-immunogenicity relationships of these antigens is important but this has not been systematically investigated yet. In the current study, Lex, Ley, and KH-1 antigens with a lactose unit at the reducing end as a spacer were synthesized and coupled with keyhole limpet hemocyanin (KLH) protein. Immunological evaluations of the resultant conjugates revealed that they all could elicit robust immune responses whilst the Ley conjugate could provoke the highest titers of total and IgG antibodies. The binding assays of their antisera to each antigen and to cancer cells showed that each antiserum had extensive cross-reaction with all three antigens as protein conjugates and strong but somewhat antigen-selective binding towards MCF-7 cancer cell. Moreover, none of these antisera had obvious binding to SKMEL-28 cancer cell that does not express Lex, Ley and KH-1. The results of assays of these antisera to mediate complement-dependent cytotoxicity (CDC) to MCF-7 and SKMEL-28 cancer cells were very similar to the results of binding assays. Thus, it was concluded that all three antigens could form effective conjugate vaccines whereas the Ley conjugate induced the most robust immune responses and the antiserum of Lex had the highest binding and cytotoxicity to target cancer cells. In addition, as the antibodies induced by each antigen had extensive cross-reaction with other two antigens, either Lex or Ley or the two combined can be utilized to formulate effective conjugate vaccines for cancer immunotherapy. Another paradigm-shifting discovery of this study is that the presentation of Lex, Ley, and KH-1 antigens on cancer cell can be different from that in synthetic conjugates, which should be taken into consideration during the design and optimization of related cancer vaccines or immunotherapies. Image 1 [ABSTRACT FROM AUTHOR]

Aim Interaction between complementarity determining regions (CDR) of the antibody (Ab) and HLA (contact surface area of 700–900 Å 2 ) can be detected by mean fluorescence intensity (MFI) or lysis of cell membrane by complement dependent cytotoxicity (CDC). These biological functions of Ab depend on amino acid residue (AAR) composition of functional and structural epitopes (Ep) of the antigen. The aim of this study was to analyze Ep structure of HLA in order to interpret reactivity of Abs in the serum of kidney recipient (KR). Methods Ab analysis (IgG) and complement binding activity (C1q test) of alloantibodies were analyzed using solid phase single antigen (One Lambda, Inc.) (positive cut off MFI = 280–500) and cell based CDCx3 washes assays. Ep analysis was conducted by using HLAMatchmaker computer algorithm (MFI = 1000 was used as a positive cut off). Results Various combinations of four (63EE, 82LR, 127K, and 113YH) eplet-defined Eps were detected in HLAs Abs in KR serum reacted with, when SAA was applied. Consensus of AAR within 15 Å radius of aforementioned eplets comprised sixteen positions. The results of reactivity of specific eplet carrying alleles in three assays are presented in Fig. 1. Our findings indicate that Ab reactivity is associated with AAR composition of structural Eps involved in contact with CDR of IgG. Binding to HLA likely depends on AARs at positions 44, 152, 156, and 158, whereas C1q reactivity is affected by AARs at 9H/24T and 163E/167S positions, and CDC reactivity is abolished by tryptophan (W) at 95 position. Conclusions Biological function of anti-HLA Ab requires not only the specific recognition of eplets but also depends on the interactions of their CDRs with structural Eps within the contact surface area. C1q + test does not always predict a positive CDC assay. The results of this study represent a new insight into organ allocation, immunomodulation therapy, and post-transplant Ab monitoring in solid organ recipients. [ABSTRACT FROM AUTHOR]