Antibody solutions

The first two categories, clarifying and crossflow filters, have been very well developed and optimized for use in biotechnology and standard wastewater treatment applications. Equipment is easily available for these applications, whether as small 0.2 micron sterilizing filter used to terminally sterilize 100 ml of product solution, or a small 500 ml crossflow filter used to concentrate a small amount of antibody solution. Many vendors of this equipment to wastewater treatment applications have their origins in the CPI (Chemical Process Industries), and have incorporated many of the scale-up and optimization properties developed in much larger units used in large scale chemical production. As a result, these two filtration unit operations are one of the most optimized and efficient used in wastewater treatment. 

Here, we describe the design and preparation of antibody supramolecular complexes and their application to a highly sensitive detection method. The complex formation between antibodies (IgG) and multivalent antigens is investigated. When an antibody solution is mixed with divalent antigen, a linear or cyclic supramolecule forms [26-29]. With trivalent antigens, the antibody forms network structures. These supramolecular formations are utilized for the ampH-fication of detection signals on the biosensor techniques. 

The antibody solution (1.6x10 M) and substrate solutions with various concentration from 10 M to 10 M were mixed on a BSA-coated plate. The mixed solution of antibodies and substrates was allowed to stand for 1 day at room temperature, and then transported to the ELISA plates pre-coated with BSA-hapten and BSA blocking buffer. Absorbance at 405 nm for the resulting enzymatic hydrolysis product (p-nitrophenolate) by alkalinephosphatase of the second antibody was recorded on an Immuno-Mini NJ-2300 to determine the amount of antibody bound to BSA-hapten. 

Fig. 12a,b. The sensorgrams for the binding of the antibody dendrimer (a) or IgG (b) to the anionic porphyrin immobilized onto the surface of the sensor chip. Phosphate borate buffer (0.1 M, pH 9.0) was used. TCPP was immobilized via hexamethylenediamine spacer onto the sensor chip and then a solution of IgG or the dendrimer was injected to the flow cell. After 60 s from the injection of the antibody solutions, flow ceU was filled with buffer... 

In a second experiment, Cy5-labelled antiBSA antibodies were immobilised on a silanised glass slide precoated with metallic nanoislands using a polydimethylsiloxane (PDMS) flow-cell. The antibody solution was left for 1 hour to attach and then the cell was flushed with deionised water. The slide was then dried with N2. For this experiment, a portion of the slide was not coated with metallic nanoislands, in order to act as a reference. Figure 20 shows the image recorded using the fluorescence laser scanner mentioned previously. The enhancement in fluorescence emission between those areas with and without nanoislands (B and A, respectively) is again evident. For both chips, an enhancement factor of approximately 8 was recorded. There is considerable interest in the elucidation and exploitation of plasmonic effects for fluorescence-based biosensors and other applications. 

To each ml of the antibody solution, add 6 mg of 2-mercaptoethylamine hydrochloride (final concentration is 50mM). Mix to dissolve. Alternatively, to limit the degree of disulfide reduction, add a 500-fold molar excess of 2-mercaptoethylamine over the concentration of antibody present. 

Add 100 pi of the SIAB stock solution to each ml of the antibody solution. Mix gently to dissolve. 

Add P-galactosidase to the activated antibody solution at a ratio of 4 mg of enzyme per mg of antibody. 

In a darkened lab, slowly add 50-100pi of the NHS-fluorescein solution to the antibody solution, while mixing. Protect from light by wrapping the reaction vessel in aluminum foil. 

Add a quantity of the TCEP solution to the antibody solution with mixing to achieve a 2.75 M excess of the reducing agent over the amount of antibody present. 

With mixing, add a quantity of the dye solution to the antibody solution to provide the desired molar excess of dye. For instance, for an antibody dissolved in buffer at... 

With mixing, add a quantity of the sulfo-NHS-biotin solution to the protein solution to obtain a 12- to 20-fold molar excess of biotinylation reagent over the quantity of protein present. For instance, for an immunoglobulin (MW 150,000) at a concentration of 10 mg/ml, 20 pi of a sulfo-NHS-biotin solution (containing 8 X 10-4 mmol) should be added per ml of antibody solution to obtain a 12-fold molar excess. For more dilute protein solutions (i.e., 1-2 mg/ml), increased amounts of biotinylation reagent may be required (i.e., 20-fold molar excess or more) to obtain similar incorporation yields as when using more concentrated protein solutions. 

Add 100 pi of the NHS-iminobiotin solution to each ml of the antibody solution. Mix well to dissolve. Note Some turbidity may be present in the reaction due to incomplete dissolution of the NHS-iminobiotin. The solution may look cloudy or have a microparticulate suspension present. This is normal for many water-insoluble reagents when added to an aqueous solution in an organic solvent. As the reaction takes place, the NHS-iminobiotin will be driven into solution, both by coupling to the protein and by hydrolysis of the NHS ester. 

Add 0.3 mg of sulfo-NHS-SS-biotin (Thermo Fisher) to each ml of the antibody solution. To measure out small amounts of the biotinylation reagent, it may be first dissolved in water at a concentration of at least 1 mg/ml. Immediately transfer the appropriate amount to the antibody solution. This level of sulfo-NHS-SS-biotin addition represents about an 8-fold molar excess over the amount of antibody present. This should result in a molar incorporation of approximately 2-4 biotins per immunoglobulin molecule. 

Dissolve the protein or antibody to be conjugated in 0.1 M sodium phosphate, 0.15M NaCl, pH 7.4. The antibody solution should be as concentrated as possible, given the amount of antibody available for modification. This general protocol will work for protein or antibody concentrations ranging from about 0.5mg/ml to lOmg/ml, but an increase in the molar excess of either SANH or SFB may have to be done at lower antibody concentrations to provide the same modification yields obtained at higher concentrations. 

Add a quantity of the crosslinker solution of choice (SANH or SFB) to the antibody solution to obtain the desired molar excess of reagent over the antibody. Typically, antibody modification procedures are done with 10- to 20-fold molar excess, but for dilute antibody concentrations, this may have to be doubled, depending on how many hydrazine or aldehyde groups are desired to be introduced on the modified antibody. 

Add 6 mg of MEA to each ml of antibody solution. Alternatively, add DTT or TCEP to a final concentration equal to 3 mole equivalents per mole equivalent of antibody present. Mix to dissolve. 

Add 2-iminothiolane (Thermo Fisher) to this solution to give a molar excess of 20-40 X over the amount of antibody present (MW of Traut s reagent is 137.63). Addition of solid 2-iminothiolane may be done despite the fact that the compound is relatively insoluble in aqueous solution. As the reagent reacts, it will be completely drawn into solution. Alternatively, a stock solution of Traut s may be made in DMF and an aliquot added to the antibody solution (not to exceed 10 percent DMF in the final solution). 

Add 10—40 ul of the SATA stock solution per ml of lmg/ml antibody solution. This will result in a molar excess of approximately 12- to 50-fold of SATA over the antibody concentration (for an initial antibody concentration of lmg/ml). A 12-fold molar excess works well, but higher levels of SATA incorporation will potentially result in more maleimide-activated enzyme molecules able to couple to each thiolated antibody molecule. For higher concentrations of antibody in the reaction medium, proportionally increase the amount of SATA addition however do not exceed 10 percent DMF in the aqueous reaction medium. 

Mix the antibody solution with the enzyme solution at a ratio of 1 1 (v/v). Since an equal mass of antibody and enzyme is present in the final solution, this will result in a 3.75 molar excess of HRP over the amount of IgG. For conjugates consisting of greater enzyme-to-antibody ratios, proportionally increase the amount of enzyme solution as required. Typically, molar ratios of 4 1 to 15 1 (enzyme antibody) give acceptable conjugates useful in a variety of ETISA techniques. 

Mix the antibody solution from step 1 with the protein solution from step 2 in amounts necessary to obtain the desired molar ratio for conjugation. Often, the secondary molecule is reacted in approximately a 4- to 15-fold molar excess over the amount of antibody present. 

After the digestion is complete, add 3 ml of 10mM Tris-HCl, pH 8.0, to the gel suspension. Separate the gel from the antibody solution using filtration or by centrifugation. 

To reduce the pyridyl dithiol groups and create reactive sulfhydryls, dissolve DTT in water at a concentration of 17.2mg/ml and immediately add 500 pi of this solution to each ml of concentrated antibody solution. Mix to dissolve and react for 30 minutes at room temperature. 

Immediately mix the concentrated, thiolated antibody solution from part B with the SPDP-activated toxin from part A. 

Dissolve SMPT (Thermo Fisher) in DMF at a concentration of 4.8mg/ml. Add 27 pi of this solution to each ml of the antibody solution. Mix gently. The final concentration of SMPT in the reaction mixture is 0.13mg/ml, which translates into about a 4.8-fold molar excess of crosslinker over the amount of antibody present. 

Mix the reduced A-chain solution with activated antibody solution at a ratio of 2mg of antibody per mg of A chain. Sterile filter the solution through a 0.22 pm membrane, and react at room temperature under nitrogen for 18 hours. 

Mix the MBS-activated ricin with the partially reduced antibody in a molar ratio of 15 1 (or 6.24mg activated ricin per mg of reduced antibody). This represents a volume ratio (at lOmg/ml for both proteins) of 1ml ricin solution mixed with 160pi antibody solution. 

---