This study was supported by the National Institutes of Health grants R01 CA158402 (P. Xie) and R21 AI128264 (P. Xie), the Department of Defense grant W81XWH-13-1-0242 (P. Xie), a Pilot Award from the Cancer Institute of New Jersey through Grant Number P30CA072720 from the National Cancer Institute (P. Xie), a Busch Biomedical Grant (P. Xie), a Victor Stollar Fellowship (A. Lalani), and an Anne B. and James B. Leathem Fellowship (S. Zhu).

This protocol follows the guidelines of institutional animal research ethics committee of Rutgers University. All mice are used in accordance with NIH guidelines and under an animal protocol approved by the Institutional Animal Care and Use Committee.
1. Preparation of Mice and Collection of Na&#239;ve Mouse Sera
<ol>
	<li>Keep all mice for immunization experiments in a specific pathogen-free animal facility.</li>
	<li>Use gender-matched, young adult (8&#8211;12 weeks old) knockout and litte...

The authors have no competing financial interests.

Here, we describe the protocol for the characterization of TD and TI Ig isotype responses in mice using ELISA. Successful implementation of this protocol requires the use of materials specified in Table 1, including ELISA assay plates, immunization Ags, mouse Ig isotype-specific antibodies and standards. Care should be taken to avoid using tissue culture treated plates for ELISA. Dilutions of the standards and serum samples should be done in separate untreated plates (round-bottom) and then added into th...

B lymphocytes are the principal player in humoral immunity and the only cell type in mammals that are capable of producing antibodies, also termed as immunoglobulins (Ig)1,2. Antibodies secreted by B cells provide a formidable defense against invading pathogens via diverse mechanisms including neutralization, opsonization and complement activation, leading to protective immunity3. Secretion of antibodies by B cells is only achieved after full activation of specific B cells, which normally requires two distinct signals3. Signal 1 is relayed by direct binding of the antigen (Ag) to the B cell receptor (BCR) expressed on the surface of specific na&#239;ve B cells3. Depending on the source of Signal 2, B cell activation can be divided into thymus-dependent (TD) or thymus-independent (TI)3,4. In a TD antigen response, Signal 2 is provided by activated cognate CD4 T helper (TH) cells, which express CD154, the ligand for the co-stimulatory receptor CD40 expressed on B cells1,2,3. In a TI antigen response, Signal 2 comes from either engagement of Toll-like receptors (TLRs in the case of type 1 TI Ag) or extensive cross-linking of the BCRs (in the case of type 2 TI Ag) on the B cells3,4. Type 1 TI (TI-1) antigens are microbial ligands of TLRs, including bacterial lipopolysaccharides (LPS), viral RNAs, and microbial CpG DNA4,5. Type 2 TI (TI-2) antigens have highly repetitive structure, and are able to deliver prolonged and persistent signaling to the B cell by multiple cross-linking of the BCRs4,6. Typical examples of TI-2 antigens include pneumococcal polysaccharides and hapten-conjugated polysaccharide6,7. Both TD and TI antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) with the help provided by antigen presenting cells (APCs) such as dendritic cells (DCs)1,2,3. Furthermore, both TD and TI antigens are able to induce memory responses with the help of APCs, but TD antigens are more efficient at inducing memory B cell generation3,8.
In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens 2,4,6-trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH) and TNP-polysaccharide (neutral, highly branched and high-mass), respectively9,10,11. TD antigens are usually used with an adjuvant to enhance the production of antibodies12. Here in our protocol, TNP-KLH is injected with alum, a commonly used adjuvant in immunization studies12. Other examples of adjuvants that can be used include complete or incomplete Freund&#39;s adjuvant (CFA or IFA), monophosphoryl-lipid A/trehalose dicorynomycolate (&#34;Ribi&#34; adjuvant), and CpG oligodeoxynucleotides, etc.13,14. After immunization, mouse sera are harvested at different time points and TNP-specific antibodies in sera are quantified using Ig isotype-specific enzyme-linked immunosorbent assay (ELISA)9,10,11.
ELISA is a plate-based assay that is widely used as a diagnostic tool in medicine and also as an analytical tool in biomedical research15,16. It is used to detect and quantify analytes including antibodies, hormones, cytokines, chemokines, and various antigens, etc. ELISA can be performed in several different formats, including direct, indirect, sandwich and competitive ELISA15,16. In general, it involves the immobilization of the antigen to a solid surface, usually a 96-well microtiter plate, which is incubated with a primary antibody. After incubation, the unbound antibody is washed away. In a direct ELISA, the primary antibody is directly conjugated to an enzyme (typically horseradish peroxidase or alkaline phosphatase), which can cleave a chromogenic substrate to yield a visible color change detected by a signal-detection instrument such as a spectrophotometer15,16. In contrast, if an enzyme-linked secondary antibody is used to bind the primary antibody, then this is considered as an indirect ELISA15,16. Direct ELISA is faster whereas indirect ELISA is more sensitive15,16. In a sandwich ELISA, the plates are coated with a &#34;capture&#34; antibody used to immobilize the antigen of interest in the samples, and then the captured antigen can be detected by another &#34;detection&#34; antibody in a direct or indirect manner15,16. Sandwich ELISA offers high specificity since the antigen is detected by two different antibodies of the antigen. In a competitive ELISA, the competition is established between the sample antigen and the plate-bound antigen for binding to the primary antibody, and then the antigen concentration in sample is quantified by measuring the reduction in signal from the substrate15,16. Competitive ELISA can be performed using the above mentioned direct or indirect format and is useful for the detection of small antigens with only one epitope15,16.
Alternative techniques for the measurement of antibodies include radio-immunoassay (RIA), electrochemiluminescence (ECL) assay and surface plasmon resonance (SPR) assay17. RIA was the first immunoassay developed that measures the presence of an antigen (or antibody) with high specificity and sensitivity using radiolabeled reagents18,19. However, due to the concerns of radioactive toxicity, disposal costs, shelf-life and special licenses to work with radioactive materials, ELISA is a better and more convenient technique for common uses20,21. ECL is a highly sensitive assay in which chemiluminescent reactions are initiated using electricity to generate highly reactive species from stable precursors on the surface of an electrode, and can be used to measure the amount of analytes (such as antigens or antibodies)22. However, ECL requires a special instrument and thus is not as broadly used as ELISA23. SPR is a direct assay that can be used to measure the binding of ligands (e.g., antibodies) to immobilized molecules (e.g., antigens) on a sensor chip surface24. SPR detects the interactions in real time very specifically and does not require the use of labelled reagents as in ELISA. However, SPR also requires a special equipment and has lower sensitivity than ELISA17. Given the limitations of the alternative methods, ELISA is the most suitable and convenient technique for our purpose in this protocol. Here, we describe the use of sandwich ELISA for the analysis of total Ig isotype levels and the procedures of indirect ELISA for the analysis of antigen-specific Ig isotypes.

<table border="0" cellpadding="0" cellspacing="0" style="width:577px;" width="576">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">VersaMax Tunable Microplate Reader</td>
			<td style="width:111px;">MDS Analytical Technologies</td>
			<td style="width:109px;">VERSAMAX</td>
			<td style="width:201px;">Equipment to read the plates</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">SOFTmax PRO 5.3</td>
			<td style="width:111px;">MDS Analytical Technologies</td>
			<td style="width:109px;">SOFTmax PRO 5.3</td>
			<td style="width:201px;">Software for the plate reader</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">GraphPad Prism</td>
			<td style="width:111px;">GraphPad</td>
			<td style="width:109px;">Prism</td>
			<td style="width:201px;">Software for graphing and statistics</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">TNP-AECM-polysaccharide (FICOLL)</td>
			<td style="width:111px;">Biosearch Technologies</td>
			<td style="width:109px;">F-1300-10</td>
			<td style="width:201px;">A TI Ag for immunization</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">TNP-KLH</td>
			<td style="width:111px;">Biosearch Technologies</td>
			<td style="width:109px;">T-5060-5</td>
			<td style="width:201px;">A TD Ag for immunization</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:156px;">TNP(38)-BSA</td>
			<td style="width:111px;">Biosearch Technologies</td>
			<td style="width:109px;">T-5050-10 (conjugation ratio: 38)</td>
			<td style="width:201px;">Coating Ag for TNP-specific ELISA</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:156px;">TNP(3)-BSA</td>
			<td style="width:111px;">Biosearch Technologies</td>
			<td style="width:109px;">T-5050-10 (conjugation ratio: 3)</td>
			<td style="width:201px;">Coating Ag for high affinity TNP-specific Ig</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Imject Alum</td>
			<td style="width:111px;">Fisher Scientific&#160;</td>
			<td style="width:109px;">PI-77161</td>
			<td style="width:201px;">Alum adjuvant for immunization</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">Falcon Polypropylene tubes</td>
			<td style="width:111px;">Fisher Scientific&#160;</td>
			<td style="width:109px;">14-959-11A</td>
			<td style="width:201px;">For incubation of TNP-KLH/alum</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">BD Insulin Syringe</td>
			<td style="width:111px;">Fisher Scientific&#160;</td>
			<td style="width:109px;">14-829-1B</td>
			<td style="width:201px;">For i.p. injection of mice</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">Immuno 96-Well Plates, Flat-Bottom</td>
			<td style="width:111px;">Fisher Scientific&#160;</td>
			<td style="width:109px;">14-245-61</td>
			<td style="width:201px;">For ELISA</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:156px;">Untreated 96-Well Microplates, Round-Bottom</td>
			<td style="width:111px;">VWR</td>
			<td style="width:109px;">82050-622</td>
			<td style="width:201px;">For serial dilutions of standards and samples</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">Phosphatase substrate, 5 mg Tablets</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:109px;">S0942-200TAB</td>
			<td style="width:201px;">AP substrate</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">Diethanolamine</td>
			<td style="width:111px;">VWR</td>
			<td style="width:109px;">IC15251690</td>
			<td style="width:201px;">A component of AP substrate buffer</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Goat anti-mouse IgM</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1020-01</td>
			<td style="width:201px;">Capture Ab for mouse IgM</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Goat anti-mouse IgG1</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1070-01</td>
			<td style="width:201px;">Capture Ab for mouse IgG1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Goat anti-mouse IgG2a</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1080-01</td>
			<td style="width:201px;">Capture Ab for mouse IgG2a</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Goat anti-mouse IgG2b</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1090-01</td>
			<td style="width:201px;">Capture Ab for mouse IgG2b</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Goat anti-mouse IgG3</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1100-01</td>
			<td style="width:201px;">Capture Ab for mouse IgG3</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Goat anti-mouse IgA</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1040-01</td>
			<td style="width:201px;">Capture Ab for mouse IgA</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Goat anti-mouse IgE</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1110-01</td>
			<td style="width:201px;">Capture Ab for mouse IgE</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">AP-Goat anti-mouse IgM</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1020-04</td>
			<td style="width:201px;">Detection Ab for mouse IgM</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">AP-Goat anti-mouse IgG1</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1070-04</td>
			<td style="width:201px;">Detection Ab for mouse IgG1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">AP-Goat anti-mouse IgG2a</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1080-04</td>
			<td style="width:201px;">Detection Ab for mouse IgG2a</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">AP-Goat anti-mouse IgG2b</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1090-04</td>
			<td style="width:201px;">Detection Ab for mouse IgG2b</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">AP-Goat anti-mouse IgG3</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1100-04</td>
			<td style="width:201px;">Detection Ab for mouse IgG3</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">AP-Goat anti-mouse IgA</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1040-04</td>
			<td style="width:201px;">Detection Ab for mouse IgA</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">AP-Goat anti-mouse IgE</td>
			<td style="width:111px;">SouthernBiotech</td>
			<td style="width:109px;">1110-04</td>
			<td style="width:201px;">Detection Ab for mouse IgE</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Mouse IgM standard</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:109px;">553472</td>
			<td style="width:201px;">TNP-specific IgM, Clone&#160; G155-228</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Mouse IgG1 standard</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:109px;">554054</td>
			<td style="width:201px;">TNP-specific IgG1, Clone&#160; 107.3</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">Mouse IgG2a standard</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:109px;">556651</td>
			<td style="width:201px;">TNP-specific IgG2a, Clone&#160; G155-178</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Mouse IgG2b standard</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:109px;">554055</td>
			<td style="width:201px;">TNP-specific IgG2b, Clone&#160; 49.2</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Mouse IgG3 standard</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:109px;">553486</td>
			<td style="width:201px;">KLH-specific IgG3, Clone&#160; A112-3</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:156px;">Mouse IgA standard</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:109px;">550924</td>
			<td style="width:201px;">Mineral oil-induced IgA, Clone&#160; MOPC-320</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:156px;">Mouse IgE standard</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:109px;">557079</td>
			<td style="width:201px;">TNP-specific IgE, Clone&#160; C38-2</td>
		</tr>
	</tbody>
</table>

characterization of thymus-dependent and thymus-independent immunoglobulin isotype responses in mice using enzyme-linked immunosorbent assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

We have used this protocol to investigate the roles of a critical regulator of the immune system, TRAF3, in TI and TD Ig isotype responses9,10,11. TRAF3 directly or indirectly regulates the signal transduction of a number of innate and adaptive immune receptors, including the TNF receptor superfamily, Toll-like receptors and T cell receptor/CD28, among others27,...

Watch this Scientific Journal Video about Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay at JoVE.com

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

This method can help answer key questions in the immunology field about antigen immunization induced dependent and independent antibody responses as well as B cell activation and germinal center reactions. The main advantage of this technique is that it provides reliable and quantitative Ig isotype response measurements with a direct comparison of B cell activation and humoral immunity in mice. For TNP-polysaccharide immunization load one milliliter insulin syringe per mouse with 100 microliters of freshly prepared antigen solution and insert the needle into the lower right quadrant of each animal at an approximately 30 degree angle to avoid lower organ injection.When all the mice have been injected, return the animals to their cages for housing in a specific pathogen-free facility. At the appropriate experimental time points collect 150 to 200 microliters of blood from each immunized animal by retro-orbital bleeding and allow the samples to coagulate at room temperature for one to two hours. At the end of the incubation, centrifuge the coagulated blood samples and transfer the clear serum from the top of each blood clot into a sterile 1.5 milliliter microcentrifuge tube.After a second centrifugation to remove any residual blood clot, transfer 50 microliters of serum into individual sterile 1.5 milliliter microcentrifuge tubes for storage at minus 80 degrees Celsius. Before plating, coat individual 96 well immunoplates with the appropriate concentration of mouse Ig isotype specific capturing antibodies for total serum Ig isotype TNP-38BSA antigen for TNP-specific Ig isotype or TNP-3BSA antigen for high affinity TNP-specific Ig isotype and incubate the coated plates overnight at four degrees Celsius. The next morning wash the plates two times with 200 microliters of wash buffer per well.Discarding the wash buffer and patting the plates dry on a stack of paper towels after each wash. Next, block the plates with 200 microliters of blocking buffer per well for a one hour incubation at room temperature. While the plates are being blocked prepare the appropriate standard and serum dilutions and blocking buffer in a separate untreated 96 well plate at 150 microliters per well as indicated.Then wash the ELISA plate with wash buffer as demonstrated and add 100 microliters of each diluted Ig isotype standard and serum sample to the appropriate wells of the ELISA plate. After an overnight incubation at four degrees Celsius wash the plates as demonstrated and add 100 microliters of an appropriate alkaline phosphatase conjugated isotype-specific antibody diluted in blocking buffer to each well for a one to two hour incubation at room temperature. At the end of the incubation, wash the plates and add 100 microliters of freshly prepared substrate solution to each well.Monitoring the reaction as it develops over the next few minutes at room temperature. When the reaction is robust enough to analyze place the plate in a microplate reader and click settings to set the wavelength to 405 nanometers. Click ok then click template to assign the appropriate blank, standard, and unknown wells according to the 96 well plate set up.Then when the most concentrated standard reaches an optical density of about one, 1.5, two, and 2.5 click read to read the plate and save the resulting data file after each analysis. In the absence of immunization, an increase in basal serum levels of IgM, IgG2a, IgG2b, IgG3, and IgA is observed in B cell specific TNF receptor associated factor three or B-TRAF3 knockout mice, compared to gender and age matched B-TRAF3 sufficient litter mate control animals. Seven days after immunization with TNP-polysaccharide or TNP-KLH as just demonstrated analysis of the thymus-independent Ig isotype responses to immunization reveals significantly higher thymus-independent TNP-specific IgG3 levels as well as elevated thymus-dependent TNP-specific IgG2b levels in myeloid cell-specific TRAF3 knockout mice compared to litter mate controls.Further, evaluation of thymus-dependent mouse primary and memory responses to TNP-KLH immunization indicates the presence of partially decreased thymus-dependent IgM primary responses and defective IgG1 primary and memory responses in T cell specific TRAF3 knockout mice. While attempting this procedure, it's important to remember that ELISA have detection limits that are usually defined by the linear range of the standard curves and therefore multiple dilutions of your sample might be required. Following this procedure, other complementary methods like flow cytometry or L spot can be performed to gain a comprehensive understanding of antibody responses in a given experimental setting.

characterization-thymus-dependent-thymus-independent-immunoglobulin

Research

JoVE Journal

Immunology and Infection

9.2K Görüntüleme.  Rutgers University. This method can help answer key questions in the immunology field about antigen immunization induced dependent and independent antibody responses as well as B cell activation and germinal center reactions. The main advantage of this technique is that it provides reliable and quantitative Ig isotype response measurements with a direct comparison of B cell activation and humoral immunity in mice. For TNP-polysaccharide immunization load one milliliter insulin syringe per mouse with 100 microlit...