Experimental methods in immunology - Revision history

wikademia>Wikademia: moved Immunology - Experimental Methods in Immunology to Experimental methods in immunology

2009-05-11T21:27:17Z

moved Immunology - Experimental Methods in Immunology to Experimental methods in immunology

← Older revision	Revision as of 21:27, 11 May 2009
(No difference)

wikademia>Wikademia: 9 revisions

2009-05-11T21:26:59Z

9 revisions

← Older revision	Revision as of 21:26, 11 May 2009
(No difference)

wikademia>Mike.lifeguard: Reverted edits by Thommiddleton (Talk) to last version by Dallas1278

2009-04-23T19:12:46Z

Reverted edits by Thommiddleton (Talk) to last version by Dallas1278

← Older revision		Revision as of 19:12, 23 April 2009
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	The technique is both extremely sensitive, and specific, if costly, and it is especially useful in diagnosing and treating autoimmune diseases such as Hashimoto's thyroiditis and Systemic Lupus Erythematosus.		The technique is both extremely sensitive, and specific, if costly, and it is especially useful in diagnosing and treating autoimmune diseases such as Hashimoto's thyroiditis and Systemic Lupus Erythematosus.

	===Enzyme-Linked ImmunoSorbent Assay (~~[http://www.diagnosticautomation.com/elisa.htm~~ ELISA])===		===Enzyme-Linked ImmunoSorbent Assay (ELISA)===
	A sample of antigen is applied to the surface of a well, and the spaces between the sample are filled with proteins that do not bind antibody. A large amount of antibody, linked to a certain enzyme that can create a color change, is added to the sample under conditions that allow only Ab/Ag binding; ''there must be enough Ab added to react with every possible Ag molecule in the sample--if you have more Ag than Ab, the extra Ag will '''not''' be detected by the test''. The well is then washed, and the color-changing reaction is activated. The level of color change can be measured by spectrometry, and the concentration of antigen can be determined via comparison of color change with a standard curve.		A sample of antigen is applied to the surface of a well, and the spaces between the sample are filled with proteins that do not bind antibody. A large amount of antibody, linked to a certain enzyme that can create a color change, is added to the sample under conditions that allow only Ab/Ag binding; ''there must be enough Ab added to react with every possible Ag molecule in the sample--if you have more Ag than Ab, the extra Ag will '''not''' be detected by the test''. The well is then washed, and the color-changing reaction is activated. The level of color change can be measured by spectrometry, and the concentration of antigen can be determined via comparison of color change with a standard curve.
	*'''Sandwich (capture) ~~[http://www.diagnosticautomation.com/elisa.htm~~ ELISA]'''--for Antigens of very low concentration, such as cytokines, binding to the well plate can be difficult. This method uses an Ab specific for one of the epitopes on the Ag, attaching these bottom antibodies to the plate well, where they bind the added small concentration of Ag. A second type of antibody, one that binds to a different epitope on the Ag, is added to the well. Thus, the Ag is sandwiched between a bottom "holding" antibody and an upper, "detector" antibody that is linked to a color-changing enzyme.		*'''Sandwich (capture) ELISA'''--for Antigens of very low concentration, such as cytokines, binding to the well plate can be difficult. This method uses an Ab specific for one of the epitopes on the Ag, attaching these bottom antibodies to the plate well, where they bind the added small concentration of Ag. A second type of antibody, one that binds to a different epitope on the Ag, is added to the well. Thus, the Ag is sandwiched between a bottom "holding" antibody and an upper, "detector" antibody that is linked to a color-changing enzyme.

	===Competitive Inhibition Assay===		===Competitive Inhibition Assay===

wikademia>Thommiddleton: /* Enzyme-Linked ImmunoSorbent Assay (ELISA) */

2009-04-23T18:11:20Z

Enzyme-Linked ImmunoSorbent Assay (ELISA)

← Older revision		Revision as of 18:11, 23 April 2009
Line 40:		Line 40:
	The technique is both extremely sensitive, and specific, if costly, and it is especially useful in diagnosing and treating autoimmune diseases such as Hashimoto's thyroiditis and Systemic Lupus Erythematosus.		The technique is both extremely sensitive, and specific, if costly, and it is especially useful in diagnosing and treating autoimmune diseases such as Hashimoto's thyroiditis and Systemic Lupus Erythematosus.

	===Enzyme-Linked ImmunoSorbent Assay (ELISA)===		===Enzyme-Linked ImmunoSorbent Assay ([http://www.diagnosticautomation.com/elisa.htm ELISA])===
	A sample of antigen is applied to the surface of a well, and the spaces between the sample are filled with proteins that do not bind antibody. A large amount of antibody, linked to a certain enzyme that can create a color change, is added to the sample under conditions that allow only Ab/Ag binding; ''there must be enough Ab added to react with every possible Ag molecule in the sample--if you have more Ag than Ab, the extra Ag will '''not''' be detected by the test''. The well is then washed, and the color-changing reaction is activated. The level of color change can be measured by spectrometry, and the concentration of antigen can be determined via comparison of color change with a standard curve.		A sample of antigen is applied to the surface of a well, and the spaces between the sample are filled with proteins that do not bind antibody. A large amount of antibody, linked to a certain enzyme that can create a color change, is added to the sample under conditions that allow only Ab/Ag binding; ''there must be enough Ab added to react with every possible Ag molecule in the sample--if you have more Ag than Ab, the extra Ag will '''not''' be detected by the test''. The well is then washed, and the color-changing reaction is activated. The level of color change can be measured by spectrometry, and the concentration of antigen can be determined via comparison of color change with a standard curve.
	*'''Sandwich (capture) ELISA'''--for Antigens of very low concentration, such as cytokines, binding to the well plate can be difficult. This method uses an Ab specific for one of the epitopes on the Ag, attaching these bottom antibodies to the plate well, where they bind the added small concentration of Ag. A second type of antibody, one that binds to a different epitope on the Ag, is added to the well. Thus, the Ag is sandwiched between a bottom "holding" antibody and an upper, "detector" antibody that is linked to a color-changing enzyme.		*'''Sandwich (capture) [http://www.diagnosticautomation.com/elisa.htm ELISA]'''--for Antigens of very low concentration, such as cytokines, binding to the well plate can be difficult. This method uses an Ab specific for one of the epitopes on the Ag, attaching these bottom antibodies to the plate well, where they bind the added small concentration of Ag. A second type of antibody, one that binds to a different epitope on the Ag, is added to the well. Thus, the Ag is sandwiched between a bottom "holding" antibody and an upper, "detector" antibody that is linked to a color-changing enzyme.

	===Competitive Inhibition Assay===		===Competitive Inhibition Assay===

wikademia>Dallas1278 at 00:13, 21 July 2008

2008-07-21T00:13:01Z

← Older revision		Revision as of 00:13, 21 July 2008
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	[[Category:Immunology]]		[[Category:Immunology]]
			{{alphabetical\|I}}

64.131.153.31: /* Knock-Out Mice */

2007-11-28T05:10:51Z

Knock-Out Mice

← Older revision		Revision as of 05:10, 28 November 2007
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	===Knock-Out Mice===		===Knock-Out Mice===
	Genetic factors that produce certain cytokines (or certain cell types themselves) can be studied ~~throught~~ the use of knock-out mice. In these mice, a certain gene is inactivated and the animal is allowed to grow. The resulting phenotype is compared against other knock-out mice, in an attempt to piece together the genetic basis of different cellular functions and phenotypes. The process of gene knockout has become fine-tuned in recent years. Basically, a gene library is searched for a candidate gene to be knocked-out. A full mouse genome with the candidate gene knocked-out is developed and grown in a preliminary stem cell, and it is injected into a mouse stem cell via electroporation. Besides making the candidate gene inoperable, the altered genome will usually have two factors:		Genetic factors that produce certain cytokines (or certain cell types themselves) can be studied through the use of knock-out mice. In these mice, a certain gene is inactivated and the animal is allowed to grow. The resulting phenotype is compared against other knock-out mice, in an attempt to piece together the genetic basis of different cellular functions and phenotypes. The process of gene knockout has become fine-tuned in recent years. Basically, a gene library is searched for a candidate gene to be knocked-out. A full mouse genome with the candidate gene knocked-out is developed and grown in a preliminary stem cell, and it is injected into a mouse stem cell via electroporation. Besides making the candidate gene inoperable, the altered genome will usually have two factors:
	*A gene to make the cultured preliminary stem cell resistant to a certain antibiotic. Because not all of the stem cells will incorporate the altered genome, an antibiotic is applied to the treated cells, and only those that survive have therefore been injected with the altered genome (including both the antibiotic resistance gene and the knocked-out gene sequence)		*A gene to make the cultured preliminary stem cell resistant to a certain antibiotic. Because not all of the stem cells will incorporate the altered genome, an antibiotic is applied to the treated cells, and only those that survive have therefore been injected with the altered genome (including both the antibiotic resistance gene and the knocked-out gene sequence)
	*A gene to make the phenotype of a fully-altered knockout mouse obvious. For example, the altered mouse genome might include a gene to make the knockout mice pure white in color. The stem cells, before being treated, are collected from a black mouse. Thus, when the black fur mother mouse is injected with the treated stem cells, she will give birth to baby mice with varied coat colors, from black to white and grey in between. The nearly-white progeny are then cross-bred with each other, and the process is repeated until mice with pure white coats are created. These mice are most likely to have both the pure white genotype and the knocked-out gene. Thus, linked phenotypes are a marker for linked genotypes.		*A gene to make the phenotype of a fully-altered knockout mouse obvious. For example, the altered mouse genome might include a gene to make the knockout mice pure white in color. The stem cells, before being treated, are collected from a black mouse. Thus, when the black fur mother mouse is injected with the treated stem cells, she will give birth to baby mice with varied coat colors, from black to white and grey in between. The nearly-white progeny are then cross-bred with each other, and the process is repeated until mice with pure white coats are created. These mice are most likely to have both the pure white genotype and the knocked-out gene. Thus, linked phenotypes are a marker for linked genotypes.

wikademia>Jguk at 20:56, 5 July 2006

2006-07-05T20:56:15Z

← Older revision		Revision as of 20:56, 5 July 2006
Line 64:		Line 64:
	[[Immunology\|Back to Immunology]] \|\|		[[Immunology\|Back to Immunology]] \|\|
	[[Immunology_-_Vaccines\|Previous Chapter: Vaccines]]		[[Immunology_-_Vaccines\|Previous Chapter: Vaccines]]

			[[Category:Immunology]]

wikademia>Daniel.lenhoff at 04:54, 20 December 2005

2005-12-20T04:54:57Z

@@ Line 22: / Line 22: @@
 ===Hematopoietic Stem Cell Concentration===
 Irradiation of a mouse can wipe out the HSCs in the bone marrow, destroying the mouse's ability to produce red blood cells and leukocytes.  These cells can be replaced with the bone marrow cells of an immunologically identical mouse, although in the injected replacement cells very few cells are actually HSCs.  In order to concentrate the amount of actual HSCs in a sample, mice can be irradiated and replacement bone marrow cells are injected with fluorescent immunoglobulins that bind to mature RBCs and WBCs.  A flow cytometry method can now be used to weed out these labelled cells, and the process can be repeated until only those that are most likely undifferentiated remain.  The HSCs can be found in a subset of cells that contain the CD34 receptor on their surface; the CD34+ subpopulation is almost totally HSC with few differentiated cells.
 ----

wikademia>Daniel.lenhoff: /* Stem Cells */

2005-12-19T08:58:30Z

Stem Cells

← Older revision		Revision as of 08:58, 19 December 2005
Line 1:		Line 1:
	==Experimental Methods in Immunology==		==Experimental Methods in Immunology==
	===Stem Cells===		===Stem Cells===
	The growth of stem cells in the bone marrow is the basis for cellular immunity. In vitro mimicry of this growth can be achieved by filling a semisolid medium with stromal cells. By adding different growth factors and cytokines, as well as stem cells of various differentiation level, the influence of chemical mediators of hematopoiesis can be understood.		The growth of stem cells in the bone marrow is the basis for cellular immunity. In vitro mimicry of this growth can be achieved by filling a semisolid medium with stromal cells. By adding different growth factors and cytokines, as well as stem cells of various differentiation level, the influence of chemical mediators of hematopoiesis can be understood.
			HSCs can be taken from a donor and injected into a person who has a defective or absent hematopoietic system. As little as 10% of the donor's bone marrow is removed and injected into the recipient, and the HSCs will find their way to the bone without direction, replenishing the hematopoietic system of the recipient.
			There are several types of stem cell grafts:
			*autologous--donor is the recipient themself; the recipient can freeze HSCs prior to chemotherapy or radiation therapy for cancer; additionally, genetically engineered autologous HSCs can be injected back into the patient
			*syngeneic--donor is genetically identical to recipient (identical twins)
			*allogeneic--donor is genetically different from recipient
			**this can lead to graft-vs.-host disease (GVHD)
			**GVHD is based on MHC/HLA types, and so a bone marrow "match" occurs when an allogeneic member of the population is found to have relatively similar MHC/HLA types relative to the recipient

	===Knock-Out Mice===		===Knock-Out Mice===
	Genetic factors that produce certain cytokines (or certain cell types themselves) can be studied throught the use of knock-out mice. In these mice, a certain gene is inactivated and the animal is allowed to grow. The resulting phenotype is compared against other knock-out mice, in an attempt to piece together the genetic basis of different cellular functions and phenotypes. The process of gene knockout has become fine-tuned in recent years. Basically, a gene library is searched for a candidate gene to be knocked-out. A full mouse genome with the candidate gene knocked-out is developed and grown in a preliminary stem cell, and it is injected into a mouse stem cell via electroporation. Besides making the candidate gene inoperable, the altered genome will usually have two factors:		Genetic factors that produce certain cytokines (or certain cell types themselves) can be studied throught the use of knock-out mice. In these mice, a certain gene is inactivated and the animal is allowed to grow. The resulting phenotype is compared against other knock-out mice, in an attempt to piece together the genetic basis of different cellular functions and phenotypes. The process of gene knockout has become fine-tuned in recent years. Basically, a gene library is searched for a candidate gene to be knocked-out. A full mouse genome with the candidate gene knocked-out is developed and grown in a preliminary stem cell, and it is injected into a mouse stem cell via electroporation. Besides making the candidate gene inoperable, the altered genome will usually have two factors:

wikademia>Daniel.lenhoff at 08:38, 19 December 2005

2005-12-19T08:38:53Z

@@ Line 1: / Line 1: @@
 ----
 [[Immunology|Back to Immunology]] ||
 [[Immunology_-_Vaccines|Previous Chapter: Vaccines]]