This work was supported in part by the Tsinghua University-Gates Foundation (no. OPP1021992), the National Natural Science Foundation of China (no. 21502103, 21877068 and 041301475), and the National Key Research and Development Program of China (no. 2017YFA0505103).

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	<li>Rhoades, R. A., Pflanzer, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Physiology (4th ed.). , (2003).</li><li>Halstead, S. B., O&#8217;Rourke, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-enhanced+dengue+virus+infection+in+primate+leukocytes.">Antibody-enhanced dengue virus infection in primate leukocytes.</a> Nature. 265, 739-741 (1977).</li><li>Gammon, G., Sercarz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+some+T+cells+escape+tolerance+induction.">How some T cells escape tolerance induction.</a> Nature. 342, 183-185 (1989).</li><li>Takada, A., Kawaoka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-dependent+enhancement+of+viral+infection:+molecular+mechanisms+and+in+vivo+implications.">Antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications.</a> Reviews in Medical Virology. 13, 387-398 (2003).</li><li>Boonnak, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Dendritic+Cells+in+Antibody-Dependent+Enhancement+of+Dengue+Virus+Infection.">Role of Dendritic Cells in Antibody-Dependent Enhancement of Dengue Virus Infection.</a> Journal of Virology. 82, 3939-3951 (2008).</li><li>Gilhus, N. E., Skeie, G. O., Romi, F., Lazaridis, K., Zisimopoulou, P., Tzartos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myasthenia+gravis+-+autoantibody+characteristics+and+their+implications+for+therapy.">Myasthenia gravis - autoantibody characteristics and their implications for therapy.</a> Nature Reviews neurology. 12, 259 (2016).</li><li>Contifine, B. M., Milani, M., Kaminski, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myasthenia+gravis:+past,+present,+and+future.">Myasthenia gravis: past, present, and future.</a> Journal of Clinical Investigation. 116, 2843-2854 (2006).</li><li>Webster, D. P., Farrar, J., Rowland-Jones, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+towards+a+dengue+vaccine.">Progress towards a dengue vaccine.</a> The Lancet Infectious Diseases. 9, 678-687 (2009).</li><li>Vikas, K., Kaminski, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+Myasthenia+Gravis.">Treatment of Myasthenia Gravis.</a> Current Neurology and Neuroscience Reports. 11, 89-96 (2011).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+dual-functional+conjugate+of+antigenic+peptide+and+Fc-III+mimetics+(DCAF)+for+targeted+antibody+blocking.">Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) for targeted antibody blocking.</a> Chemical Science. 10, 3271-3280 (2019).</li><li>Bello, M. S., Rezzonico, R., Righetti, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+taylor-aris+dispersion+for+measurement+of+a+solute+diffusion+coefficient+in+thin+capillaries.">Use of taylor-aris dispersion for measurement of a solute diffusion coefficient in thin capillaries.</a> Science. 266, 773-776 (1994).</li><li>Fang, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+chemical+synthesis+by+ligation+of+peptide+hydrazides.">Protein chemical synthesis by ligation of peptide hydrazides.</a> Angewandte Chemie. International Edition. 50, 7645-7649 (2011).</li><li>Fang, G. M., Wang, J. X., Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+chemical+synthesis+of+proteins+by+ligation+of+peptide+hydrazides.">Convergent chemical synthesis of proteins by ligation of peptide hydrazides.</a> Angewandte Chemie. International Edition. 51, 10347-10350 (2012).</li><li>Zheng, J. S., Tang, S., Qi, Y. K., Wang, Z. P., Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+synthesis+of+proteins+using+peptide+hydrazides+as+thioester+surrogates.">Chemical synthesis of proteins using peptide hydrazides as thioester surrogates.</a> Nature Protocols. 8, 2483-2495 (2013).</li></ol>

The authors have nothing to disclose.

The protocol here describes the semi-synthesis and detection of DCAF1 by using NCL approach, which is shown in Figure 1. Briefly, the two fragments of DCAF1 are chemical synthesized and recombinantly expressed, respectively; then, the full length DCAF1 molecule is assembled, modified and purified. For the hydrazine derived Fc-III fragment synthesis, using low-capacity 2-Cl resin is quite important, because high-capacity has a negative effect for hydrazine generation and leads to low yield of...

Antibodies play important roles in humoral immune response for the neutralization of pathogenic bacteria and viruses1. However, some antibodies exhibit harmful impacts to the organisms, such as cross-reactive antibodies in the ADE effect during DENV infection and over-reactive antibodies in myasthenia gravis, which is an autoimmune diseases2,3. ADE effect is mediated by the cross-reactive antibodies that make the bridge to connect DENV and Fc receptor presenting cells4,5, while myasthenia gravis is caused by the excessive antibodies that attack acetylcholine receptors between the cell-cell junctions in muscle tissue6,7. Although partially effective approaches have been developed to treat these diseases8,9, undoubtedly direct elimination of these harmful antibodies would make progress for the interventions.
Recently, DCAF molecules, which have dual-functional groups, have been developed for targeted antibody blocking10. DCAF is a long peptide that is composed of 3 parts: 1) an antigen part that can specific recognize the cognate antibody, 2) an Fc-III or Fc-III-4C tag for strongly binding to the Fc region of the antibody to inhibit either Fc receptor or complement component proteins, 3) a long &#945;-helical linker that conjugates these two functional groups10. The linker part, designed from Moesin FERM domain, was optimized by Rosseta software to ensure the antigen part and Fc-III part in a DCAF molecule can bind to the Fab and Fc regions of IgG simultaneously. Four DCAF molecules have been synthesized to target 4 different antibodies, among them DCAF1 was used to eliminate 4G2 antibody, which is a cross-reactive antibody during DENV infection to contribute to ADE effect; and DACF4 was designed for the rescue of acetylcholine receptors by blocking mab35 antibody in myasthenia gravis10.
In the present study, taken DCAF1 as the example, we showed the protocols for the synthesis of DCAF molecule and the detection of the interaction between a DCAF and its cognate antibody. The DCAF1 is semi-synthesized by NCL approach11,12,13,14, which conjugates the hydrazine derivative of a Fc-III peptide and the expressed linker-antigen parts together. The NCL approach has significant advantages over fully chemical synthesis and fully recombinant expression for DCAF1 synthesis, because both these methods lead to low yield and high cost. The current approach is not only the most cost-effective way to get the full-length DCAF, but also can maintain the conformation of the linker part similar as its native form. Since different DCAF molecules have similar sequences except for the antigen parts, our methods for DCAF1 synthesis and the interaction assay between DCAF1 and 4G2 antibody can be applied to other DCAF molecules to targeted block their cognate antibodies as well.

<table>
	<tbody>
		<tr>
			<td>2-Chlorotrityl resin</td>
			<td>Tianjin Nankai HECHENG S&#38;T</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5-b]pyridinium hexafluorophosphate 3-oxide</td>
			<td>GL Biochem</td>
			<td>00703</td>
			<td></td>
		</tr>
		<tr>
			<td>2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate</td>
			<td>GL Biochem</td>
			<td>00706</td>
			<td></td>
		</tr>
		<tr>
			<td>2,2&#8242;-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride</td>
			<td>J&#38;K Scientific</td>
			<td>503236</td>
			<td></td>
		</tr>
		<tr>
			<td>4G2 antibody</td>
			<td>Thermo</td>
			<td>MA5-24387</td>
			<td></td>
		</tr>
		<tr>
			<td>4-mercaptophenylacetic acid</td>
			<td>Alfa Aesar</td>
			<td>H27658</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well microtiter plates</td>
			<td>NEST</td>
			<td>701001</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Thermo-Fisher</td>
			<td>A955</td>
			<td>MS Grade</td>
		</tr>
		<tr>
			<td>AgOAc</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>30164324</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GST antibody</td>
			<td>Abclonal</td>
			<td>AE001</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IgG, HRP-linked Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>7076P2</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Beijing DINGGUO CHANGSHENG BOITECHNOL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD spectrometer</td>
			<td>Applied Photophysics Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis bag</td>
			<td>Sbjbio</td>
			<td>SBJ132636</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>80047360</td>
			<td></td>
		</tr>
		<tr>
			<td>diethyl ether</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10009318</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Gel Extraction Kit</td>
			<td>Beyotime</td>
			<td>D0056</td>
			<td></td>
		</tr>
		<tr>
			<td>Fusion Lumos mass spectrometer</td>
			<td>Thermo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GSH Sepharose</td>
			<td>GE Lifesciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Guanidine hydrochloride</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>30095516</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrazine hydrate</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>80070418</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10011018</td>
			<td></td>
		</tr>
		<tr>
			<td>imidazole</td>
			<td>SIGMA</td>
			<td>12399-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-Thiogalactoside</td>
			<td>SIGMA</td>
			<td>5502-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>kanamycin</td>
			<td>Beyotime</td>
			<td>ST101</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Thermo-Fisher</td>
			<td>A456</td>
			<td>MS Grade</td>
		</tr>
		<tr>
			<td>N, N-Diisopropylethylamine</td>
			<td>GL Biochem</td>
			<td>90600</td>
			<td></td>
		</tr>
		<tr>
			<td>N, N-Dimethylformamide</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>8100771933</td>
			<td></td>
		</tr>
		<tr>
			<td>NcoI</td>
			<td>Thermo</td>
			<td>ER0571</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer</td>
			<td>Solarbio</td>
			<td>P1022</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptide BEH C18 Column</td>
			<td>Waters</td>
			<td>186003625</td>
			<td></td>
		</tr>
		<tr>
			<td>piperidine</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>80104216</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Extraction Kit</td>
			<td>Sangon Biotech</td>
			<td>B611253-0002</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAexpress Kit</td>
			<td>QIAGEN</td>
			<td>32149</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapid DNA Ligation Kit</td>
			<td>Beyotime</td>
			<td>D7002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate dihydrate</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>20040718</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10019762</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium nitrite</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10020018</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium&#160;chloride</td>
			<td>Sinopharm Chemical Reagent</td>
			<td>10019318</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Fmoc-protected amino acids</td>
			<td>GL Biochem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sterilizing pot</td>
			<td>Tomy</td>
			<td>SX-700</td>
			<td></td>
		</tr>
		<tr>
			<td>SUMO Protease</td>
			<td>Thermo Fisher</td>
			<td>12588018</td>
			<td></td>
		</tr>
		<tr>
			<td>stop solution</td>
			<td>Biolegend</td>
			<td>423001</td>
			<td></td>
		</tr>
		<tr>
			<td>the whole gene sequence that can express SUMO-linker-antigen</td>
			<td>Taihe Biotechnology Compay</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TMB reagent</td>
			<td>Biolegend</td>
			<td>421101</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>SIGMA</td>
			<td>T6508</td>
			<td></td>
		</tr>
		<tr>
			<td>Triisopropylsilane</td>
			<td>GL Biochem</td>
			<td>91100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphine hydrochloride</td>
			<td>Aladdin</td>
			<td>T107252-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>tryptone</td>
			<td>OXOID</td>
			<td>LP0042</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Solarbio</td>
			<td>T8220</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultimate 3000 HPLC</td>
			<td>Thermo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>vacuum pump</td>
			<td>YUHUA</td>
			<td>SHZ-95B</td>
			<td></td>
		</tr>
		<tr>
			<td>XhoI</td>
			<td>Thermo</td>
			<td>IVGN0086</td>
			<td></td>
		</tr>
		<tr>
			<td>yeast extract</td>
			<td>OXOID</td>
			<td>LP0021</td>
			<td></td>
		</tr>
	</tbody>
</table>

targeted antibody blocking by a dual-functional conjugate of antigenic peptide and fc-iii mimetics (dcaf)

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic fever and autoimmune diseases. Since thousands of antibodies with different epitopes are circulating in blood, no universal method, except for the dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF), was reported to target specific harmful antibodies. The development of DCAF molecules makes significant contribution to the progress of targeted therapy, which were demonstrated to eliminate the antibody dependent enhancement (ADE) effect in a Dengue virus (DENV) infection model and to boost the acetylcholine receptor activity in a myasthenia gravis model. Here, we describe a protocol for the synthesis of a DCAF molecule (DCAF1), which can selectively block 4G2 antibody to attenuate ADE effect during Dengue virus infection, and illustrate the binding of DCAF1 to 4G2 antibody by an ELISA assay. In our method, DCAF1 is synthesized by the conjugation of a hydrazine derivative of a Fc-III peptide and a recombinant expressed long &#945;-helix with antigenic sequence through native chemical ligation (NCL). This protocol has been successfully applied to DCAF1 as well as other DCAF molecules for targeting their cognate antibodies.

The flowchart for the synthesis route by native chemical ligation in this article is shown in Figure 1. Figures 2-6 show the chromatograms (A) and mass spectra (B) of chemical synthesized hydrazine derivative of a Fc-III peptide, recombinant expressed linker and antigen part, the purified product from NCL reaction, the purified product from desulfurization reaction and the purified final product DCAF1, respectively. The chromatograms show th...

Watch this Scientific Journal Video about Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics DCAF at JoVE.com

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics DCAF

Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) is novel for the elimination of harmful antibodies. Here, we describe a detailed protocol for the synthesis of DCAF1 molecule, which can selectively block 4G2 antibody to eliminate antibody dependent enhancement effect during Dengue virus infection.

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic ...

targeted-antibody-blocking-dual-functional-conjugate-antigenic

Research

JoVE Journal

Immunology and Infection

6.7K Views.  Westlake University. Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) is novel for the elimination of harmful antibodies. Here, we describe a detailed protocol for the synthesis of DCAF1 molecule, which can selectively block 4G2 antibody to eliminate antibody dependent enhancement effect during Dengue virus infection.

Video: Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics DCAF

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles coupled to antibodies recognizing surface antigens. The captured virions or vesicles were labeled with fluorescent antibodies against other surface antigens. The resultant complexes were separated in high magnetic field and analyzed with conventional flow cytometers triggered on fluorescence.

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

A Method for Targeted 16S Sequencing of Human Milk Samples

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as with all these technologies, reproducible results depend on a laboratory workflow that incorporates appropriate precautions and controls. This is particularly important with low-biomass samples where contaminating bacterial DNA can generate misleading results. This article details a semi-automated workflow to identify microbes from human breast milk samples using targeted sequencing of the 16S ribosomal RNA (rRNA) V4 region on a low- to mid-throughput scale. The protocol describes sample preparation from whole milk including: sample lysis, nucleic acid extraction, amplification of the V4 region of the 16S rRNA gene, and library preparation with quality control measures. Importantly, the protocol and discussion consider issues that are salient to the preparation and analysis of low-biomass samples including appropriate positive and negative controls, PCR inhibitor removal, sample contamination by environmental, reagent, or experimental sources, and experimental best practices designed to ensure reproducibility. While the protocol as described is specific to human milk samples, it is adaptable to numerous low- and high-biomass sample types, including samples collected on swabs, frozen neat, or stabilized in a preservation buffer.

A semi-automated workflow is presented for targeted sequencing of 16S rRNA from human milk and other low-biomass sample types.

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as ...

Antigenic Liposomes for Generation of Disease-specific Antibodies

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a na&#239;ve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5&#39;-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird&#39;s-eye view of the antibody repertoire, and our computational methods.

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...