BioAssay Works Iso-Gold Rapid Mouse Monoclonal Isotyping Kit

The Iso-Gold kit is a rapid-format isotyping kit that reduces the labor of the traditional ELISA kit from a day-and-a-half procedure to a five-minute assay.

determine the isotype of your monoclonal antibody
assess the purity of your purified monoclonal antibody*
verify the purity of your purchased monclonal antibody*
reduce runtime

*multiple lines apear and indicate subclass contamination

iso-gold kit

There are a number of ELISA-based isotyping kits on the market. These kits require the customer to supply their own plates, do the coating, incubation and kit development. One latex-bead-based "rapid" isotyping kit is also marketed, but the sensitivity increase of a gold-sol-based assay versus a latex-based assay gives the advantage to the gold assay.

BioAssay Works Iso-Gold™ Isotyping Kit correlation with ELISA-format isotyping assay

A study compared the monoclonal isotyping results of a widely used ELISA-format isotyping assay and a new, rapid-format BioAssay Works Iso-Gold Isotyping Kit. The results demonstrate outstanding correlation between these two assay formats.

The ELISA required controls, ELISA plate, and substrate took eight hours of hands-on time to coat the plates, block, and subsequently perform the assay on twelve different antibodies. The Iso-Gold Kit required under twenty minutes to perform the entire test on the same twelve samples. The Iso-Gold Kit pipetting time was ten minutes and all twenty assays were run simultaneously. Each was read at five minutes. Thus the total hands-on time, including scoring of the results, was under twenty minutes.

Anti-	mAb	1	2	3	4	5	6	7	8	9	10	11	12
IgGI	A	0.722	0.788	0.099	0.899	0.787	0.085	0.917	0.218	0.850	0.692	0.810	0.593
IgG2a	B	0.931	0.663	0.091	0.235	0.419	0.931	0.074	1.075	0.079	0.859	0.073	0.069
IgG2b	C	0.665	0.460	0.075	0.062	0.371	0.063	0.072	0.073	0.065	0.456	0.062	0/064
IgG3	D	0.209	0.134	0.094	0.088	0.108	0.085	0.085	0.088	0.083	0.165	0.084	0.089
IgM	E	0.158	0.739	0.853	0.089	0.069	0.056	0.053	0.057	0.054	0.186	0.061	0.069
IgA	F	0.302	0.409	0.116	0.106	0.181	0.060	0.085	0.095	0.089	0.117	0.085	0.076
Kappa	G	0.941	0.873	0.710	0.817	0.777	0.618	0.767	0.753	0.781	0.868	0.490	0.564
Lamda	H	0.249	0.209	0.107	0.098	0.109	0.085	0.090	0.090	0.084	0.154	0.255	0.084

As can be seen from the data, there is an excellent correlation with the ELISA Mouse-Monoclonal Antibody
Isotyping Kit (lot II 117753). In addition to the correct interpretation of the Isotype result, the level of contaminating antibody in each monoclonal can be accurately determined using the Iso-Gold™ Rapid Mouse-Monoclonal Isotyping Kit. Impurities in the form of contaminating isotypes and subclasses correlate well with the ELISA. Thus the rapid test has the added feature of determining the purity of the monoclonal being tested.

test image

Instructions and Procedures

For ascites fluid, the darker red line indicates the class or subclass antibody present. Weak red lines often appear on other isotypes indicating the presence of host serum immunoglobulins in the ascites.

Procedure for ascites fluid:

Add 0.5 ul of ascites fluid to 4 ml of Sample Diluent - vortex to mix
Add 150 ul of diluted ascites fluid to the Sample Well (S).
Wait 5 (five) minutes
Read results – the darker line is the isotype

NOTE: Do not read after ten (10) minutes

Procedure for cell culture fluid: For supernatants a dark red line indicates which isotype or class-specific antibody is present. In very few instances, weak red lines may appear on one or more additional isotype(s) indicating multiple hybridoma clones.

Procedure for supernatant fluid:

Add 5.0 ul of supernatant fluid to 0.5 ml of Sample Diluent - vortex to mix
Add 150 ul of diluted supernatant fluid to Sample Well (S)
Wait 5 (five) minutes
Read results

NOTE: Do not read after ten (10) minutes

For supernatants that contain the monoclonal antibody at less than 1 microgram per ml, dilute the sample 1:10 as follows:

Add 55 ul of supernatant to 500 ul of sample diluent - vortex to mix
Repeat Steps 2 through 4

Store kit and components at room temperature – Shelf life 18 months.

Assay Utility: Determining the class and subclass of a mononclonal antibody is useful in determining the best immunoglobulin purification method. For example, IgA and IgM are often best purified by size (gel exclusion) or on immuno affinity separation columns whereas IgG_2a and IgG_2b can be purified on protein A at a pH of 7 to 8. IgG₁ binds best to protein A at a pH of 8 to 9. In addition, each class and isotype can be digested to Fab fragments using the appropriate amount of pepsin or other enzymes for each isotype or antibody class.

Isotyping kit comparison, performed by BioAssay Works personnel

EIA Tests
Company Name	Format	# of Tests per Kit	# of wells needed per antibody	Test Time	Specifics	Sensitivity
BioAssay Works	Rapid Lateral Flow	10	N/A	5 min	no light chains	10 ng/ml
AbD serotec	Latex Lateral Flow (Roche)	10	N/A	5 - 10 min	+ Light Chains	not listed
BD	EIA	80	12	8 hr	+ Light Chains	not listed
BioRad	EIA	100	8	8 hr	+ Light Chains	not listed
ClonaCell (EY Labs)	Flow Through	20	N/A	1 min - 3 steps	does not include IgA/ no Light Chains	1 ug/ml
Invitrogen (Zymed)	EIA	100	8	8 hr	+ Light Chains	50 ng/ml
Pierce	EIA	100	8	8 hr	+ Light Chains	not listed
Roche	Latex Lateral Flow	10	N/A	5 min	+ Light Chains	50 ng/ml
Calbiochem (reagents to make EIA test)	EIA	100	6	2 days, 10 plates	Mab isotypes /no light chains
Note: All of the EIA Test companies listed only provide materials to make your own kit.

e-coli

The photo on the left shows three assays detecting E coli 0157 - the detection is from a low of 10-3rd to 10-5th power. The important fact to note is that the client sees Microwell ELISA sensitivity on a rapid format assay.

The founders of BioAssay Works have nearly 50 years research experience in the purification and development of bio-receptors in assay systems. Coupled with proprietary improvements that enable the synthesis of gold sols at concentrations 10-20 times current levels, BioAssay Works offers a powerful package of products and services that promise reliability and reduced costs for the development of rapid lateral-flow assays.

	Kit Components for 5 test pouch size, product code TISO-001: • 5 cassette pouches; 5 cassettes contain strips impregnated with anti-IgG₁; anti-IgG_2a;andanti-IgG_2b isotypes; and 5 cassettes that contain strips that are impregnated with anti-IgG₃, anti-IgM, and anti-IgA isotypes • Sample Diluent - 25 ml
	Kit Components for 10 test pouch size, product code ISOT-001: • 10 cassette pouches; ten (10) cassettes contain strips impregnated with anti-IgG₁; anti-IgG_2a;andanti-IgG_2b isotypes; and ten (10) cassettes that contain strips that are impregnated with anti-IgG₃, anti-IgM, and anti-IgA isotypes • Sample Diluent - 45 ml

Product Code	Description	QTY	Size
TISO-001	Iso-Gold rapid mouse-monoclonal isotyping kit	1	5 test pouches
ISOT-001	Iso-Gold rapid mouse-monoclonal isotyping kit	1	10 test pouches
KISO-001	Iso-Gold rapid mouse-monoclonal isotyping Kappa/Lambda kit (includes 5 Kappa and Lambda test strips)	1	10 test pouches
KSOT-001	Iso-Gold rapid mouse-monoclonal isotyping Kappa/Lambda kit (includes 10 Kappa and Lambda test strips)	1	20 test pouches

Store kit and components at room temperature – shelf life is 18 months.

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