Name: characterization of thymus-dependent and thymus-independent immunoglobulin isotype responses in mice using enzyme-linked immunosorbent assay
Uploaded: 2018-09-07T14:30:00
Description: 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

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...

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.

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Research

JoVE Journal

Immunology and Infection

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

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 ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...