Griffin:Antibody Basics - Revision history

Korey Griffin: /* Fab fragment */

Korey Griffin — Tue, 31 May 2011 21:59:16 GMT

Fab fragment

←Older revision		Revision as of 21:59, 31 May 2011
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	The 'F' stands for fragment and the 'ab' stands for antigen binding. This is the region where antigen binding takes place. The Fab portion of an antibody is made up of the N-terminal domains of both the heavy and light chains. This domain of the antibody is called the variable (V) domain (VH on the heavy chain, VL on the light chain) because there is such high variability between antibodies in this area. This allows recognition of thousands of different antigens.		The 'F' stands for fragment and the 'ab' stands for antigen binding. This is the region where antigen binding takes place. The Fab portion of an antibody is made up of the N-terminal domains of both the heavy and light chains. This domain of the antibody is called the variable (V) domain (VH on the heavy chain, VL on the light chain) because there is such high variability between antibodies in this area. This allows recognition of thousands of different antigens.
		+
		+	====Kappa/Lambda Ratios====
		+
		+	In all antibodies there are 2 light chain types, kappa and lambda.
		+
		+	*MICE 20:1
		+	*HUMAN 2:1
		+	*BOVINE 1:20

	===F(ab')2 fragment===		===F(ab')2 fragment===

Korey Griffin: /* Rodent (mouse, rat, hamster) */

Korey Griffin — Tue, 05 Apr 2011 00:10:20 GMT

Rodent (mouse, rat, hamster)

←Older revision		Revision as of 00:10, 5 April 2011
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	Small mammal that requires less antigen than the rabbit or goat. Suitable for spleenectomy and B cell fusion to produce monoclonal Ig.		Small mammal that requires less antigen than the rabbit or goat. Suitable for spleenectomy and B cell fusion to produce monoclonal Ig.
		+
		+
		+	mice spleen is isolated and fused to myeloma to produce immortal Ig producing B-Cells secreting Ig into the culture supernatant. Each subcloned 'single' hybridoma B cell produces a 'mono' single clone Ig that recognizes only 1 epitope region. Large antigens are suitable to immunize mice, however the monoclonal Ig will only target a single epitope within the antigenic region, as small as 3 amino acids.

	===Chicken===		===Chicken===

Korey Griffin: /* Direct ELISA */

Korey Griffin — Wed, 16 Jun 2010 19:19:47 GMT

Direct ELISA

←Older revision		Revision as of 19:19, 16 June 2010
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	cDNA for the gene of interest is a useful tool for antibody validation. Typically, a CHO, Cos, or 293T cell may be transfected with the cDNA in order to measure antibody specificity. A western blot can be performed to compare the nontransfected lysate with the transfectant.		cDNA for the gene of interest is a useful tool for antibody validation. Typically, a CHO, Cos, or 293T cell may be transfected with the cDNA in order to measure antibody specificity. A western blot can be performed to compare the nontransfected lysate with the transfectant.

-	===~~Direct~~ ELISA===	+	===ELISA===

	Microwell plates are coated with a sample containing the target antigen OR the immunizing peptide, and the binding of labeled antibody is quantitated by a colorimetric, chemiluminescent, or fluorescent end-point. This technique validates recognition or binding between antigen and antibody.		Microwell plates are coated with a sample containing the target antigen OR the immunizing peptide, and the binding of labeled antibody is quantitated by a colorimetric, chemiluminescent, or fluorescent end-point. This technique validates recognition or binding between antigen and antibody.
		+
		+	*Direct vs. Indirect Enzyme-linked immunosorbent assay (ELISA): Indicates whether the ELISA uses secondary antibody or not (secondary used = indirect).
		+	*Sandwich vs. Conventional: Indicates what the well is coated with (and what the overall setup of ELISA assay is). In conventional ELISA, sample is coated on well, and an antibody is used for detection. In Sandwich ELISA, "capture" primary antibody is coated on well, then the well is incubated with the sample, and then a "detection" primary antibody is used for detection of antigen bound to "capture" antibody. Both sandwich and conventional ELISAs can be either direct or indirect.
		+	*Both sandwich and conventional ELISAs can be either direct or indirect. Some sources (e.g. Wikipedia) call Sandwich ELISA a "direct ELISA". This stems from the fact that Indirect Sandwich ELISA assays are rare.

	===Peptide neutralization===		===Peptide neutralization===

Korey Griffin at 21:16, 7 May 2010

Korey Griffin — Fri, 07 May 2010 21:16:58 GMT

←Older revision		Revision as of 21:16, 7 May 2010
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	[[Image:DirectELISA.jpg\|thumb\|right\|Immunizing peptide conjugate to thr 96 well plate authenticates Serum titer for the immunogen]]		[[Image:DirectELISA.jpg\|thumb\|right\|Immunizing peptide conjugate to thr 96 well plate authenticates Serum titer for the immunogen]]

-		+	Validation of primary antibody specificity is an ongoing topic of the utmost importance to scientists who choose to utilize immunoglobulin-based approaches. The mass of histology data, the often small sample replication number, combined with common lack of sufficient (molecular biological) controls are a compelling reason for reproducibility and disclosure of all applied compounds such as antibodies. However, the pure peptide sequence alone simply does not reveal or indicate specificity or reproducibility. Adequate control experiments are essential.
-	Validation of primary antibody specificity is an ongoing topic of the utmost importance to scientists who choose to utilize immunoglobulin-based approaches. The mass of histology data, the often ~~quite~~ small n= number, combined with common lack of sufficient (molecular biological) controls are a compelling reason for reproducibility and disclosure of all applied compounds such as antibodies. However, the pure peptide sequence alone simply does not reveal or indicate specificity or reproducibility. Adequate control experiments are essential.	+

	===cDNA===		===cDNA===

Korey Griffin: /* Antibody */

Korey Griffin — Wed, 10 Mar 2010 02:08:45 GMT

Antibody

←Older revision		Revision as of 02:08, 10 March 2010
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	The 'F' stands for fragment and the 'ab' stands for antigen binding. This is the region where antigen binding takes place. The Fab portion of an antibody is made up of the N-terminal domains of both the heavy and light chains. This domain of the antibody is called the variable (V) domain (VH on the heavy chain, VL on the light chain) because there is such high variability between antibodies in this area. This allows recognition of thousands of different antigens.		The 'F' stands for fragment and the 'ab' stands for antigen binding. This is the region where antigen binding takes place. The Fab portion of an antibody is made up of the N-terminal domains of both the heavy and light chains. This domain of the antibody is called the variable (V) domain (VH on the heavy chain, VL on the light chain) because there is such high variability between antibodies in this area. This allows recognition of thousands of different antigens.
		+
		+	===F(ab')2 fragment===
		+
		+	Pepsin cleaves a whole immunoglobulin to yield one F(ab')2 fragment and this is two antigen binding fragments linked by cysteine bonds. The 'prime ( ' )' is included in F(ab')2 since this fragment contains more amino acids than the F(ab) alone.
		+
		+	F(ab')2 fragment secondary antibodies should be used when trying to avoid non-specific secondary antibody to Fc receptors on cell surfaces.

	==Primary antibody production==		==Primary antibody production==

Korey Griffin: /* Common animal types */

Korey Griffin — Wed, 10 Mar 2010 02:02:12 GMT

Common animal types

←Older revision		Revision as of 02:02, 10 March 2010
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	A unique non-mammal source for research antibodies, polyclonal IgY from yolk sack is considered more humane than drawing blood. However success has been limited.		A unique non-mammal source for research antibodies, polyclonal IgY from yolk sack is considered more humane than drawing blood. However success has been limited.
-		+
		+	Chickens produce IgY. Heavy and light chains for IgY, the heavy chain is ~65 kD and the light chain is ~27 kD for a total weight of 185 kd.
		+
	==Monoclonal Antibody purification==		==Monoclonal Antibody purification==

Korey Griffin: /* Adjuvant */

Korey Griffin — Wed, 10 Mar 2010 02:00:40 GMT

Adjuvant

←Older revision		Revision as of 02:00, 10 March 2010
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	===Adjuvant===		===Adjuvant===
		+
		+	Freund's Adjuvant is used for boost injections. Freund's Adjuvants may be used to produce water-in-oil emulsions of immunogens. Antigens in water-in-oil emulsions stimulate high and long-lasting antibody responses which can be attributed to the slow release of antigen.

	The term “adjuvant” originates from the Latin word adjuvare, which means to help or aid. An immunologic adjuvant is defined as any substance that acts to accelerate, prolong, or enhance antigen-specific immune responses when used in combination with specific vaccine antigens.		The term “adjuvant” originates from the Latin word adjuvare, which means to help or aid. An immunologic adjuvant is defined as any substance that acts to accelerate, prolong, or enhance antigen-specific immune responses when used in combination with specific vaccine antigens.

Korey Griffin: /* Carrier proteins */

Korey Griffin — Wed, 10 Mar 2010 01:59:58 GMT

Carrier proteins

←Older revision		Revision as of 01:59, 10 March 2010
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	*Bbovine serum albumin (BSA; 67kDa)		*Bbovine serum albumin (BSA; 67kDa)
	*Keyhole limpet hemocyanin (KLH; 4.5e5-1.3e7 Da).		*Keyhole limpet hemocyanin (KLH; 4.5e5-1.3e7 Da).
-	*~~MBS:~~ M-maleimidobenzoyl-N-hydroxysuccinimide ester Blue Carrier Protein	+	*M-maleimidobenzoyl-N-hydroxysuccinimide ester Blue Carrier Protein (MBS)
	*Ovalbumin (OVA)		*Ovalbumin (OVA)
	*Rabbit serum albumin (RSA)		*Rabbit serum albumin (RSA)
-	*Bovine thyroglobulin (THY).	+	*Bovine thyroglobulin (THY)

	Antigen carrier complex is subsequently mixed in with the Complete Freund's Adjuvant for a longer response time.		Antigen carrier complex is subsequently mixed in with the Complete Freund's Adjuvant for a longer response time.

Korey Griffin: /* Carrier proteins */

Korey Griffin — Wed, 10 Mar 2010 01:59:18 GMT

Carrier proteins

←Older revision		Revision as of 01:59, 10 March 2010
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	*Keyhole limpet hemocyanin (KLH; 4.5e5-1.3e7 Da).		*Keyhole limpet hemocyanin (KLH; 4.5e5-1.3e7 Da).
	*MBS: M-maleimidobenzoyl-N-hydroxysuccinimide ester Blue Carrier Protein		*MBS: M-maleimidobenzoyl-N-hydroxysuccinimide ester Blue Carrier Protein
		+	*Ovalbumin (OVA)
		+	*Rabbit serum albumin (RSA)
		+	*Bovine thyroglobulin (THY).

-	~~Also ovalbumin (OVA), rabbit serum albumin (RSA), or bovine thyroglobulin (THY).~~	+	Antigen carrier complex is subsequently mixed in with the Complete Freund's Adjuvant for a longer response time.
-		+
-		+
-	~~This antigen~~ carrier complex ~~get~~ subsequently mixed in with the Complete Freund's Adjuvant for a longer response time.	+

	===Adjuvant===		===Adjuvant===

Korey Griffin: /* Carrier Protein */

Korey Griffin — Wed, 10 Mar 2010 01:58:26 GMT

Carrier Protein

←Older revision		Revision as of 01:58, 10 March 2010
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	*Carrier proteins provide a large number of functional moieties (amino, carboxyl, and sulfhydrul groups) that serve as "anchor points" for peptide conjugation.		*Carrier proteins provide a large number of functional moieties (amino, carboxyl, and sulfhydrul groups) that serve as "anchor points" for peptide conjugation.
-	*~~The most common carrier proteins are bovine~~ serum albumin (BSA; 67kDa) ~~and keyhole~~ limpet hemocyanin (KLH; 4.~~5x105~~-1.~~3x107Da~~). ~~Common examples also include~~ ovalbumin (OVA), rabbit serum albumin (RSA), or bovine thyroglobulin (THY).	+
		+	====Carrier proteins====
		+
		+	*Bbovine serum albumin (BSA; 67kDa)
		+	*Keyhole limpet hemocyanin (KLH; 4.5e5-1.3e7 Da).
		+	*MBS: M-maleimidobenzoyl-N-hydroxysuccinimide ester Blue Carrier Protein
		+
		+	Also ovalbumin (OVA), rabbit serum albumin (RSA), or bovine thyroglobulin (THY).
		+
		+
		+	This antigen carrier complex get subsequently mixed in with the Complete Freund's Adjuvant for a longer response time.

	===Adjuvant===		===Adjuvant===