About Us

Contact Us

Distributors

FAQ

Links

Order

Products Overview

Products New

Services

Site Map

Tradeshows

Product Code	Description	QTY	Size
LPKIB01-B009 Black	Latex-in-a-Box conjugation kit	1 Kit	1x9 ml of 1% Black latex particles plus reagents
LPRIB01-B009 Red	Latex-in-a-Box conjugation kit	1 Kit	1x9 ml of 1% Red latex particles plus reagents
LPLIB01-B009 Blue	Latex-in-a-Box conjugation kit	1 Kit	1x9 ml of 1% Blue latex particles plus reagents

Lateral-flow chromatographic and flow-through tests offer fast detection of critical components for use in point-of-care testing. The key to these tests is the ability to covalently attach antibodies to intensely colored, nanoscale particles. Latex polystyrene particles that bind ligands through a carboxyl bond or via passive absorption (hydrophobically) have proved highly successful for this application.

For optimal binding of the antibody or protein while retaining a high degree of specific activity, the pH of the latex suspension must be adjusted to slightly above the iso-electric point of the coating antibody or protein. This is done through a series of pH titrations with the provided buffers. Varying amounts of buffers A and B, and varying amounts of buffers C and D, are added to the latex particles to create a pH 5-11 range. Next, antibody or protein is added, and after 30 minutes the reaction is stopped.

After a wash step, the particles are ready for testing. This convenient Latex-in-a-Box™ kit allows you to quickly (typically in less than 50 minutes) determine the pI, and optimal coating range for your antibody or soluble protein.

For in vitro research only - not for diagnostic or therapeutic use.

Kit Components:

1. 1% Black, Blue, or Red 0.3 micron latex particles - (9 mL)
2. Buffer Solution A - 1.0 mL (cap with black dot)
3. Buffer Solution B - 1.0 mL (cap with green dot)
4. Buffer Solution C - 1.0 mL(cap with blue dot)
5. Buffer Solution D - 1.0 mL (cap with red dot)
6. BSA Blocking Stabilizer Solution - 2.0 mL (clear cap)
7. Conjugate Drying Buffer – 2 mL (cap with purple dot)

Materials required but not provided:

1. Clean glass or polystyrene test tubes (12 x 75 mm)
2. Pipettes and tips

Sample Preparation
The antibodies or proteins used with this kit must be at a concentration of 1 mg/mL or greater and should be in a 0.5 X PBS buffer solution. If they are not in a 0.5 X PBS buffer solution, then dialyze the antibody or protein against 0.5 X PBS. Proteins at a concentration of 2 mg/ml or greater should be in 1 X PBS.

Generic Passive Coating Procedure:
Note: Use aseptic technique when handling the latex particles.
Helpful Hint: If you know the isoelectric of your antibody, choose the buffer conditions from the chart at / around the isoelectric point. E.g., if the pI of your antibody is 7.3, choose buffer conditions for pHs 7.3; 7.8 and 8.2

• Shake or swirl latex particles to resuspend any settled latex particles. Place 0.5 mL latex particles into ten (10) clean individual test tubes.
• Label each tube with the pH value (or, 1 through 10) from the provided pH charts.
• Add Buffer Solutions as specified in pH charts below to each test tube. Shake to mix.
• Place each tube on a low-speed vortexer and add antibody solution - mix thoroughly (about 2 to 3 seconds)
       For the 300 nm latex particles, 250 µl of a 1 mg /mL solution of antibody or protein is optimal.
       Note: The saturation point for 300 nm latex particles is about 500 µg of antibody per mL of latex particles.
• Allow the reaction to continue for a total of 30 minutes.
• Add 50 µl of BSA Blocking Stabilizer Solution to each test tube
• Add 2 mL of PBS
• Wash particle solution by spinning in a centrifuge at 10,000 xG for 10 minutes
• Vacuum the supernatant fluid and vortex the pellet
• Add 2 mL of PBS
• Repeat the wash procedure
• Vacuum off the supernatant fluid and vortex pellet
• Resuspend particle solution in 0.5 mL of Storage Buffer, (PBS, 0.1% BSA , 0.05%proclin 950)

Note: cross-linking of the latex often causes an aggregation of latex particles. In order to disperse the latex particles, use a bath sonicator to break down any aggregates. Usually, sonication for 10 to 30 seconds is sufficient.

Note: Do no heat latex solution to greater than 50° C.

Note: See section below on “Stability of Latex particles Conjugates"

Covalent Coupling of Protein to Latex Beads
Note: For MES Buffer formulation, see “MES Wash & Coupling Buffer Formulation” below

• Add 1.0 mL of 0.1 Molar MES buffer to 1.0 mL of latex particles
• Wash the 2.0 mL particle solution by spinning in a centrifuge at 10,000 XG for 10 minutes
• Vacuum off the supernatant fluid and vortex the pellet
• Repeat wash step
• Vacuum off the supernatant fluid and vortex the pellet
• Resuspend the latex particle solution in 0.25 mL MES buffer
• Dissolve 10 mg EDC or CMC in 0.25 mL MES buffer
• Add freshly prepared EDC to the particle solution and mix gently for 10 minutes at room temperature
• After incubation, wash the particles 2 x with 2 mL 0.1 Molar MES buffer
• Resuspend the activated particles in 0.25 mL MES buffer
• Add proteins (e.g., 250 µg protein dissolved in MES buffer) to the activated particles and mix the suspension gently for two hours at room temperature
• Wash the particles 3 x with 2 ml PBS
• Resuspend the particles in Blocking/Storage buffer (PBS, 0.1% BSA , 0.05% Proclin 950)

Note: cross-linking of the latex often causes an aggregation of latex particles. In order to disperse the latex particles, use a bath sonicator to break down any aggregates. Usually, sonication for 10 to 30 seconds is sufficient. Note: Do no heat latex solution to greater than 50 C

MES Wash & Coupling Buffer Formulation

0.1 M 2-(N-Morpholino) ethanesulfonic acid MES, pH 5.0

Water soluble carbodiimide:

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or
1-cyclonhexyl-3(2-morphonlinoethyl) carbodiimide metho-ptoluensulfonate (CMC)

Blocking & Storage Buffer

PBS, 01% BSA , 0.05% proclin 950

Stability of Latex Particles Conjugates

Latex particles completely coated with protein take on the properties of the coating proteins and become very stable in solutions of high ionic strength.

However, cross linking of the latex often causes an aggregation of latex particles. In order to disperse the latex particles, use a bath sonicator to break down any aggregates. Usually, sonication for 10 to 30 seconds is sufficient. Note: Do no heat latex solution to greater than 50°C.

In order to effectively dry down the latex particles, add 0.1 mL of Latex Particles Drying Buffer for every 1.0 ml of conjugate. Mix thoroughly. Apply gradually and evenly to either glass fiber or polyester ribbon

Place polyester ribbon in a vented 37° C oven or incubator for four (4) hours to dry thoroughly. Glass fiber ribbon should be left in the incubator overnight.

Alternative Drying Procedure for Polyester Ribbon

If a vented incubator is not available, use a hairdryer set to deliver 30°C - 37°C heat at a ten (10) inch distance from the ribbon surface. Usually, three (3) or four (4) minutes in a wave-motion will suffice to thoroughly dry the ribbon.

Store all dried ribbons in a tightly sealed glass container containing ample desiccants (granular or pouch-form).

This generic procedure may be modified or scaled as needed. When developing a new assay, it is important to determine the optimal amount of ligand to add to the latex particles. Once the tubes have been assayed, it is useful to further optimize binding by both decreasing or increasing the amount of antibody added to each tube. Often, a 20% increase or decrease in antibody or protein added is sufficient to yield an optimal coating procedure. A few cases require a 40% or more increase or decrease in coating antibody or protein.

Discussion

A sensitive lateral-flow assay requires that all of the antibody or protein that is added to the latex particles be irreversibly bound to the beads. Any free antibody or protein serves to short-circuit the assay. This behavior ultimately sets the sensitivity limits of an assay.

Latex particles remain in solution because they repel each other due to a significant negative charge.

Application of Latex Particles Conjugates

The latex particles conjugate is excellent for use in a variety of latex particle amplified assay procedures. This includes BioAssay Works' patented, ultra-sensitive C-FLAT technology. Researchers interested in evaluating this technology may contact BioAssay Works for a research-use license with no fee.

pH Charts for Optimal Coating
at a pH of 5-11 (per 0.5 ml of latex particles)

Tube Number	pH	Buffer A	Buffer B
1	5.4	9 µl	1 µl
2	6.6	8 µl	2 µl
3	7.3	6 µl	4 µl
4	7.8	4 µl	6 µl
5	8.2	2 µl	8 µl

Tube Number	pH	Buffer C	Buffer D
6	8.4	10µl	0 µl
7	8.8	8 µl	2 µl
8	9.2	6 µl	4 µl
9	9.6	4 µl	6 µl
10	10.1	2 µl	8 µl

BioAssay Works®, LLC. | 10075 Tyler Place, Suite 18 • Ijamsville, MD, 21754 | sales@bioassayworks.com
phone 301.874.8888 | fax 301.874.8889