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Keywords:

  • protein complexes;
  • solid-phase complex preparation;
  • immune complexes

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. REFERENCES

Protein–protein conjugation is usually achieved by solution phase methods requiring concentrated protein solution and post-synthetic purification steps. In this report we describe a novel continuous-flow solid-phase approach enabling the assembly of protein complexes minimizing the amount of material needed and allowing the repeated use of the same solid phase. The method exploits an immunoaffinity matrix as solid support; the matrix reversibly binds the first of the complex components while the other components are sequentially introduced, thus allowing the complex to grow while immobilized. The tethering technique employed relies on the use of the very mild synthetic conditions and fast association rates allowed by the avidin–biotin system. At the end of the assembly, the immobilized complexes can be removed from the solid support and recovered by lowering the pH of the medium. Under the conditions used for the sequential complexation and recovery, the solid phase was not damaged or irreversibly modified and could be reused without loss of binding capacity. The method was specifically designed to prepare protein complexes to be used in immunometric methods of analysis, where the immunoreactivity of each component needs to be preserved. The approach was successfully exploited for the preparation of two different immunoaffinity reagents with immunoreactivity mimicking native squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) and alphafetoprotein-immunoglobulin M (AFP-IgM) immune complexes, which were characterized by dedicated sandwich enzyme-linked immunosorbent assay (ELISA) and immunoblot. Besides the specific application described in the paper, the method is sufficiently general to be used for the preparation of a broad range of protein assemblies. Copyright © 2010 John Wiley & Sons, Ltd.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. REFERENCES

Chemical conjugation is the simplest approach to protein tagging or to the implementation of different biochemical properties in the same protein complex. Though rather simplistic, this approach has allowed the development of the large range of conjugates routinely used by virtually any immunometric methods of analysis. Conjugation usually relies on the formation of covalent bonds between the different partners by exploiting homo- or hetero-bifunctional reagents reacting with aminoacid side chains, usually lysine, cysteine or carboxylic acids conveniently found on the protein surface. Alternatively, a reactive group, such as an aldehyde, can be introduced by oxidation of the sugar moieties at the protein glycosylation sites and then made to react with the free lysine side chains of another protein species forming an imine or a secondary amine if a suitable reducing agent is present. These conjugation procedures are well described in the literature and are routinely used in the practice (Wong, 1991); other approaches, such as the click-chemistry and the Staudinger ligation, are increasingly being pursued (Brennan et al., 2006; Gauthier and Klok, 2008; Best, 2009). Despite their widespread use, standard conjugation protocols present major drawbacks related to the need of concentrated protein solutions, usually in the range of 1–10 mg/ml or more, (Wong, 1991) to ensure an efficient conjugation and the need of post-synthetic purification steps to remove large polymeric species or unreacted materials. This feature limits the applicability of the approach to cheap proteins stable in concentrated solutions and readily available in large amounts. Conversely, the conjugation of proteins available in small amounts or prone to the formation of aggregates in concentrated solutions would be difficult to achieve by this method. An approach which may, in principle, overcome some of these limitations is the development of efficient solid-phase protocols allowing the preservation of protein structure and functional activity reducing the need of post-synthetic purification processes. Some reports in the literature have described the effort to devise solid-phase approaches to protein conjugation and functionalization indicating a growing attention towards these alternative protocols. So far, all of these methods have displayed both advantages and limitations related to the specific chemical approach being used. Among the most interesting methods, Russell et al. (2002; 2004) proposed a batch-mode solid-phase protein conjugation procedure, exploiting the thiol-maleimide chemistry. However, this batch-wise method exploits the same chemistry for both conjugation and protein immobilization and does not allow the repeated use of the solid phase. Another approach to the implementation of solid-phase protocols in protein conjugation has been reported by Hu and coworkers (Hu et al., 2003; Hu and Su, 2003); the authors used an ion exchange matrix to temporarily immobilize bovine haemoglobin aiming to obtain dimers of the protein; in this case glutaraldehyde was used as linker. The same group has also reported a method for haemoglobin pegylation requiring immobilization of the protein on ion exchange columns (Suo et al., 2009). Here, we present a different solid-phase approach for the preparation of protein complexes where a special attention is paid to preserve the immune reactivity of the single components. The method is entirely based on the use of strong and specific non-covalent interactions between different protein building blocks in addition to the biotin–avidin technology. The development of the reported method arose from the need to obtain immunoreactive molecules similar to natural antigen-immunoglobulin M (IgM) immune complexes. The design of the approach was guided by practical considerations related to the limitations of the costs associated to the purification of the final product.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. REFERENCES

Materials

Bovine serum albumin (BSA), inorganic and organic materials used for buffers preparation were obtained from standard suppliers, the reagent grade water was obtained by using the Elix 3 and the milli-Q Synthesis A10 systems (Millipore). All the buffers used were freshly prepared and filtered through 0.45 µm nalgene filters (Millipore) prior to use. Protein purifications by affinity chromatography were run on the ÄKTA Purifier system (Amersham Bioscience). Analytical gel filtration chromatography were run using either a Biosep-SEC-S4000 column (Phenomenex) assembled on a waters 600 HPLC system equipped with an in-line degasser and dual wavelength detector. sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblot analysis were performed using the vertical electrophoresis device Mini PROTEAN 3 cell (Bio-Rad) in conjunction with the power supply EPS 301 (Amersham Bioscience). The molecular marker PageRulerTM Plus prestained protein ladder were obtained from Fermentas. The immunoblot analysis were carried out using the Trans-Blot® Transfer Medium Pure Nitrocellulose Membrane (0.45 µm) from Bio-Rad.

Centrifugal filtrations were performed using either the Amicon® Ultra 10 000 MWCO or the microcon filtration devices (Millipore). Enzyme-linked immunosorbent assay (ELISA) assays were run using high binding 96 wells plates (Greiner Bio-One). The washing procedures required in ELISA assays were automatically performed by using the Biotrak II Plate Washer (Amersham Biosciences). The ELISA assays required the use of the chromogenic substrate ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) (Sigma Aldrich), the colour development was monitored at 405 nm using the Viktor3 TM 1420 Multilabel Counter plate reader (Perkin Elmer) or the Biotrak II Plate Reader (Amersham Biosciences). Rabbit polyclonal anti-squamous cell carcinoma antigen (anti-SCCA) antibody is a product of Xeptagen SpA, goat polyclonal anti-human IgM complexed to horseradish peroxidase (HRP) was obtained from Sigma-Aldrich. The total protein assay was performed in a microwell format according to the Bradford method against bovine serum albumin or human immunoglobulin G (IgG) used as standards, optical densities of the solutions were read at 620 nm using the Biotrak II Plate Reader (Amersham Biosciences).

Purification of IgM from human serum

Purification of human IgM from human serum was accomplished by affinity chromatography on goat-anti-human IgM (µ-chain specific) agarose (Sigma-Aldrich A9935). The affinity matrix was loaded onto a glass column (0.5 cm ID, 2.0 ml bed volume) and packed at a flow rate of 1.5 ml/min using PBS as mobile phase. The column was conditioned at a flow rate of 0.5 ml/min with PBS pH 7.2 using at least five column volumes of buffer prior to sample loading. A sample of diluted human serum obtained diluting 3.0 ml of serum to 12 ml with PBS pH 7.2 were loaded onto the column at a flow rate of 0.5 ml/min; the chromatographic run was monitored at 220 and 280 nm. The unretained material (unbound fraction) was collected and the column was washed with reagent grade water at 1.5 ml/min flow rate until the conductivity of the flowtrought dropped to zero. The bound fraction was then eluted using glycine/HCl buffer 0.1 M at pH 2.5, the flow rate used during elution was 1.5 ml/min. The collected solution was neutralized adding a saturated solution of NaHCO3, 100 µl of saturated bicarbonate solution were used for each millilitre of collected solution. In a typical experiment, the bound fractions from three different chromatographic runs were pooled together and the buffer was exchanged by centrifugal filtration by using the Amicon® Ultra 10 000 MWCO centrifugal filtration devices (Millipore). To completely remove any traces of glycine, the concentrate was treated with PBS (10 × 5 ml) and eventually reduced to about 0.5 ml; the protein concentration was determined by the Bradford method. The solution was divided into 200 µl aliquots and stored at −20°C until further use.

Human IgM biotinylation

The concentration of the IgM solution resulting from the purification process was adjusted to 1260 µg/ml adding PBS; a solution of bicarbonate buffer 0.1 M pH 8.5 containing NaCl 0.5 M was added to the solution in a 1:1.5 v/v ratio. The mixture was treated with a freshly prepared solution of N-hydroxysuccinimide activated biotin (SIGMA B3295-50MG) 1.0 mg/ml in DMSO in a 1:12 v/v ratio, the solution was stirred for 4 h and afterwards dialysed overnight against 1 L of PBS. The dialysis process was repeated two more times against 1 L of PBS. The protein solution was collected and assayed for total protein content according to the Bradford method.

Biotinylation of SCCA

Squamous Cell Carcinoma Antigen was obtained from cell lysate as previously described. (De Falco et al., 2001) 500 µl of SCCA solution in morpholinoethansulphonic acid (MES) was subjected to centrifugal filtration in order to change the milieu to phosphate buffered saline (PBS) using the microcon centrifugal filtration devices as recommended by the manufacturer. The resulting solution was diluted to 600 µl with PBS. An equal volume of bicarbonate buffer 0.1 M pH 8.5 containing NaCl 0.5 M was added, to the solution were added 60 µl of a solution of NHS activated biotin (SIGMA B3295-50MG) 1.0 mg/ml in DMSO, and then the solution was stirred for 4 h and afterwards dialysed overnight against 1 L of PBS. The protein solution was collected and assayed for total protein content according to the Bradford method.

Biotinylation of alphafetoprotein (AFP)

500 µl of AFP solution in PBS was subjected to centrifugal filtration in order to remove any free amines present in the medium using the microcon centrifugal filtration devices as recommended by the manufacturer. The resulting solution was diluted to 600 µl with PBS. An equal volume of bicarbonate buffer 0.1 M pH 8.5 containing NaCl 0.5 M was added, to the solution were added 60 µl of a solution of N-hydroxysuccinimide activated biotin (SIGMA B3295-50MG) 1.0 mg/ml in DMSO, and then the solution was stirred for 4 h and afterwards dialysed overnight against 1 L of PBS pH 7.2. The protein solution was collected and assayed for total protein content according to the Bradford method.

ELISA assays for the estimation of the complex reactivity

SCCA-IgM complex

High binding 96-wells microplates were sensitized with polyclonal rabbit anti-SCCA at the concentration of 10 µg/ml, 100 µl/well. The coating procedure was performed overnight at 4°C in a humid closed chamber. The solution was removed and the plates washed with PBS containing 0.05% Tween20 (3 × 300 µl/well). The wells were filled with 300 µl of PBS containing 3% bovine serum albumin to saturate the residual reactive sites of the plastic plate; the plate was allowed to stand at room temperature for 2 h. The samples were loaded on the plate by performing a two-fold serial dilution starting from the concentration of 100 µg/ml, PBS containing 1% BSA and 0.05% Tween20 was used as dilution buffer. The final volume was 100 µl/well, the samples were incubated for 1 h at room temperature. The solutions were removed by suction and the plate was washed with PBS containing 0.05% Tween20 (6 × 300 µl/well). A solution of goat-anti-human IgM complexed to HRP at the 1:1000 dilution (100 µl/well) was added to the wells and incubated for 1 h at room temperature. The solution of secondary antibody was removed by suction and the plate washed with a PBS containing 0.05% Tween20 (6 × 300 µl). A solution containing ABTS 0.2 mg/ml and 5 mM hydrogen peroxide in phosphate-citrate buffer pH 5.0 was added to each well (150 µl/well), the enzymatic reaction was allowed to proceed for 20 min at 37°C in the dark. Absorbance was read at 405 nm.

AFP-IgM complex

High binding 96-wells microplates were sensitized with polyclonal rabbit anti-AFP (DAKO A008) at the concentration of 10 µg/ml, 100 µl/well. The coating procedure was performed overnight at 4°C in a humid closed chamber. The solution was removed and the plates washed with PBS containing 0.05% Tween20 (3 × 300 µl/well). The wells were filled with 300 µl of PBS containing 3% bovine serum albumin to saturate the residual reactive sites of the plastic plate, the plate was allowed to stand at room temperature for 2 h. The samples were loaded on the plate by performing a two-fold serial dilution starting from the concentration of 100 µg/ml, PBS containing 1% BSA and 0.05% Tween20 was used as dilution buffer. The final volume was 100 µl/well, the samples were incubated for 1 h at room temperature. The solutions were removed by suction and the plate was washed with PBS containing 0.05% Tween20 (6 × 300 µl/well). A solution of goat-anti-human IgM complexed to HRP at the 1:1000 (100 µl/well) was added to the wells and incubated for 1 h at room temperature. The solution of secondary antibody was removed by suction and the plate washed with a PBS containing 0.05% Tween20 (6 × 300 µl). A solution containing ABTS 0.2 mg/ml and 5 mM hydrogen peroxide in phosphate-citrate buffer pH 5.0 was added to each well (150 µl/well), the enzymatic reaction was allowed to proceed for 20 min at 37°C in the dark. Absorbance was read at 405 nm.

Solid-phase assembly of the SCCA-IgM complex

The affinity matrix featuring covalently linked goat antibodies raised against human IgM was packed in a glass column (0.5 cm ID, 5 cm height) and conditioned at 0.5 ml/min flow rate using PBS as mobile phase. The solution of biotinylated IgM (250 µg/ml) was injected in 250 µl aliquots (6 × 250 µl), the process was monitored at 280 nm and the unbound material was collected. After the addition of the IgM solution was complete, the column was briefly washed with PBS. A solution of avidin (600 µg/ml) was injected in small aliquots (4 × 250 µl), monitoring the process at 280 nm. After the addition of avidin, the column was washed with PBS. A solution of biotinylated SCCA (200 µg/ml) was added in small portions (4 × 250 µl) monitoring the injection process at 280 nm, at the end of the addition, the column was briefly washed with PBS and afterwards a solution of N-hydroxysuccinimide activated biotin previously reacted with lysine (biotin concentration 100 µg/ml) was added (3 × 250 µl), the immobilized complex was washed with PBS pH 7.2. The complex was recovered by changing the mobile phase to 0.1 glycine buffer pH 2.5. The complex eluted as a relatively narrow peak, the eluted solution was neutralized by the addition of a saturated sodium bicarbonate solution.

Solid-phase assembly of the AFP-IgM complex

The affinity matrix featuring covalently linked goat antibodies raised against human IgM was packed in a glass column (0.5 cm ID, 5 cm height) and conditioned at 0.5 ml/min flow rate using PBS as mobile phase. The IgM solution (250 µg/ml) was injected in 250 µl aliquots (5 × 250 µl), the process was monitored at 280 nm and the unbound material was collected. After the addition of the IgM solution was complete, the column was briefly washed with PBS. A solution of avidin (600 µg/ml) was injected in small aliquots (4 × 250 µl), monitoring the process at 280 nm. After the addition of avidin, the column was washed with PBS. A solution of biotinylated AFP (100 µg/ml) was added in small portions (2 × 250 µl) monitoring the effluent at 280 nm, at the end of the addition, the column was briefly washed with PBS and afterwards a solution of N-hydroxysuccinimide activated biotin previously reacted with lysine (biotin concentration 100 µg/ml) was added (3 × 250 µl) and the immobilized complex was washed with PBS. The complex was recovered by changing the mobile phase to 0.1 citrate buffer pH 2.5. The complex eluted as a relatively narrow peak and the eluted solution was neutralized by the addition of a saturated sodium bicarbonate solution.

SDS–PAGE and immunoblot of SCCA-IgM complex

SDS–PAGE of SCCA-IgM complex was performed according to the Laemmli procedure on a discontinuous gel system consisting of a 10% resolving gel and 4% stacking gel. The protein samples (5 µg) were treated with 1:6 (v/v) of sample buffer containing 0.5 M Tris-Cl pH 6.8, 0.35 M SDS, glycerol 30% (v/v), 0.175 mM Bromophenol Blue and 6 mM DTT. 16 µl of a solution of SCCA-IgM complex were treated with 1:3.2 (v/v) of a modified sample buffer containing 0.5 M Tris-Cl pH 6.8, 0.83 M SDS, glycerol 30% (v/v), 0.29 mM Bromophenol Blue and 10 mM DTT. Prior to the loading in the gel, samples were heated for 5 min at 100°C and briefly centrifuged. The separation was carried out at the constant voltage of 250 V until the tracking dye exited the gel bottom. The total run time was 45 min. After electrophoresis stacking gel was discarded and the transfer of polyacrylamide gel resolved proteins to the nitrocellulose membrane was carried out as described by Towbin et al. (1979) at the constant voltage of 100 V; the total running time was 1 h. After the transfer, the nitrocellulose membrane was dried overnight at the room temperature. The dried membrane was immersed for 1 h in 10 ml of blocking buffer (PBS containing 0.05% Tween20 and 5% skimmed milk). The membrane was incubated with a solution 10 µg/ml of oligoclonal rabbit IgG anti-SCCA (Xeptagen SpA) for 1 h at the room temperature and then washed for 15 min four times with PBS containing 0.05% Tween20. A solution of goat-anti-rabbit IgG complexed to the HRP at the 1:1000 (10 ml) was added to the membrane as secondary antibody and incubated for 1 h at the room temperature. The membrane was washed for 15 min four times with PBS containing 0.05% Tween20 and then immersed in 6 ml of a chemiluminescent solution (Immobilon Western substrate HRP, Millipore) for 1 min. After the detection of the reactive bands, the membrane was washed, blocked as described above and was incubated for 1 h at the room temperature with a solution of goat-anti-human IgM complexed to the HRP at the 1:1000 (10 ml). After the antibody incubation, the membrane was washed for 15 min four times with PBS containing 0.05% Tween20 and then treated with the chemiluminescent solution as described above. The reactive bands were scanned using the Image Station 440 CF (Kodak) and analysed with the Kodak 1D Software.

Gel filtration chromatography

A preliminary mass analysis of the oligomeric complex was carried out by gel-filtration, using a BioSep SEC-s4000 column (Phenomenex), the column was calibrated by using a set of proteins of known molecular weight, spanning the range 900–17 KDa (human IgM, human IgG, BSA, horse myoglobin). A total volume of 200 µl of complex solution (50 µg/ml) was injected at 1.0 ml/min flow rate using PBS pH 7.2 as mobile phase. The obtained chromatogram (Figure 5) displays a single peak eluting at 6.6 min. The column effluent was collected in 500 µl fractions, and their immunoreactivity was assayed by dedicated ELISA. A significant immune reactivity was detected in only those fractions eluting at 6.6 min (Kav 0.08), thus confirming the nature of the peak.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. REFERENCES

Principle and rationale of the method

A solid-phase approach to the preparation of protein complexes by exploiting solely non-covalent interactions is conceivable but it would be effective only if the non-covalent interactions holding together the assembled complex are stronger than those exploited to immobilize the first building block to the solid support. In fact, only in these conditions a certain degree of selectivity allowing the recovery of the intact complex could be attained. From this starting point, we selected the antigen–antibody interaction to immobilize the first component of the complex on a solid support and the avidin–biotin interaction to assemble the complex; the differences in the association constants for the two processes should be sufficient to achieve a reasonable degree of selectivity. Of course, this approach requires the use of biotinylated protein building blocks and the use of avidin as ‘linker’. The tremendous advantage of using avidin as ‘linker’ is its high specificity and its high binding constant towards biotinylated molecules which in turn allows the use of diluted solutions of biotinylated species. The outlined method requires to use a solid phase consisting of an immunoaffinity matrix featuring covalently linked antibodies against the first of the complex components. The first biotinylated protein building block, once immobilized on the solid support by the antigen–antibody interactions, would serve as a handle to build the assembled species by sequential addition of avidin and other different biotinylated molecules. Of course because of the heterogeneity in protein biotinylation, a certain degree of heterogeneity could also be expected in the assembling process, leading to different reactivities of different protein complexes. This is indeed the case for all the conjugation processes not relying on protein engineering which, instead, ensure the precise identification of the reaction sites and an unequivocal definition of the complex architecture.

The working principle of the method for the assembling of just two different biotinylated protein building blocks (BB1; BB2) is graphically depicted in Figure 1. In Step 1 the first biotinylated building block, BB1, is captured by the antibody specific to it covalently bound to the solid support; the species not recognized by the antibody are washed away. In Step 2, an excess of avidin is added to the resin-bound BB1 forming a complex with the available biotin moieties on its surface. The other binding sites of avidin far apart from the BB1 surface will remain free and will be thus available for further binding with other biotinylated species. During this process, some of the available biotin moieties on BB1 will remain unreacted either because of steric hindrance due to avidin molecules bound to nearby biotins, or because of their limited accessibility on the side of the BB1 molecule facing the antibody bound to the bead surface. The addition of a different biotinylated building block, BB2, in Step 3 allows the formation of the immobilized complex by reaction with the free biotin binding sites on the avidin molecules bound to the immobilized BB1. In Step 4, the non-reacted biotin binding sites on avidin still available for binding but not reacted to BB2 must be saturated in order to prevent oligomerization after cleavage of the complex from the solid support; this can easily be achieved by adding an excess of free biotin providing an efficient capping methodology. At the end of the assembling process, the complex can be removed from the solid support by changing the pH, thus breaking the antibody–protein interaction responsible for its immobilization (Step 5). The pH change does not affect the biotin–avidin interaction allowing the recovery of the intact complex. After cleavage the column could be re-used without loss of binding capacity. From a general point of view, what molecule should be used as first building block and what should be used as the second is largely a matter of choice and may vary according to the intended use of the complex. As mentioned above, the mandatory requisites for the method were to be cost effective and require no purification of the final product. Clearly these considerations strongly restrict the number of possible approaches to those optimizing the use of the most expensive species. Therefore, aiming to obtain molecular complexes with an immune reactivity similar to that of natural immune complexes formed by tumour antigens and IgM, the use of the antigen was required to be as limited as possible. These considerations prompted us to devise the presented method, which required the immobilization of the IgM as the first building block and proceeding to the formation of the complex by addition of avidin and antigen. This approach has the advantage of using as the first building block a cheap species, which does not interfere in the assay in case of incomplete assembling of the complex.

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Figure 1. Schematic representation of the continuous-flow synthetic process. In Step 1, the first biotinylated protein building block is immobilized on the beads carrying its specific antibody, all the other protein species present are washed away. In Step 2, an excess of avidin is added in order to react with the accessible biotin moieties. Some of the biotins will not react because of steric hindrance or limited accessibility. In Step 3, the second biotinylated building block is added reacting with the free biotin binding sites on avidin. To provide an efficient capping methodology, in Step 4 an excess of free biotin is added to saturate any remaining binding sites on the avidin molecules. In Step 5, a pH change of the medium favours the ‘cleavage’ of the complex from the resin weakening the interaction between the BB1 and the antibody specific to it. The resin can be washed and reused for another synthesis.

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On the contrary, in case of incomplete reaction, the immobilization of the antigen as the first building block would have led to some free antigen in the same solution containing the complex, which could compete for the latter in the immunoassay, thus lowering the observed reactivity. In this case a post-synthetic purification step would have been necessary. As a concluding remark, it is worth stressing that the solid-phase complex preparation described above allows a certain degree of control on the topology of the complex, maintaining a high surface to volume ratio. An alternative solution phase method using the same building blocks is conceivable but in this case the topology of the complex would be much difficult to control. In closer details, a solution phase method would produce a polymeric species with many molecules buried inside the complex and thus inaccessible for recognition by the antibodies used in ELISA assays. In this case only those molecules on the surface would be available for recognition, while the rest could just serve as scaffold. This approach is clearly less efficient than the solid-phase method in terms of use of materials.

Solid-phase assembly of the SCCA/IgM and AFP/IgM complexes

The proof of principle of the method outlined above was obtained by preparing two different complexes containing IgM and tumour associated antigens, the latter represents an example of relatively expensive proteins usually available in limited amounts, while the former represents an example of a protein prone to form aggregates. In closed details, the first complex prepared comprised human IgM (BB1) and the Squamous Cell Carcinoma Antigen (SCCA) (BB2), while the second complex comprised human IgM (BB1) and alpha-fetoprotein (AFP) (BB2). The reason for the preparation of these complexes resides in the important role played by SCCA-IgM and AFP-IgM immune complexes in the diagnosis of hepatocellular carcinoma and in the urgent industrial need of synthetic complexes displaying immunoreactivity mimicking that of the natural cognate immune complexes. The choice of using IgM instead of the tumour antigen as the first building block arose from a practical consideration. In case of incomplete assembly of the complex, the free antigen released upon cleavage would have interfered in the ELISA assay. In this case, a post-synthetic purification step would have been necessary. The experimental design required the use of an anti-IgM antibody covalently linked to a solid phase to temporarily immobilize the biotinylated IgM; the resin was packed in a standard affinity column mounted on a standard FPLC system. The column was conditioned in PBS at a flow rate of 0.5 ml/min, the addition of the biotinylated protein was achieved by repeatedly injecting 250 µl of diluted protein solution, the injection process and the flow-through of unretained material were monitored at 280 nm, the resulting ‘chromatographic’ trace is displayed in Figure 2 (preparation of SCCA-IgM complex displayed in panel A, preparation of AFP-IgM complex displayed in panel B).

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Figure 2. Chromatographic traces recorded during the continuous-flow synthesis of the SCCA-IgM (Panel A) and AFP-IgM complex (Panel B). The chromatograms were recorded at 280 nm, dashed lines represents the injections of the various species.

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The immobilized IgM were then reacted with an excess of avidin thus saturating the accessible biotin moieties on the IgM surface (Figure 1, Step 2). The reaction occurred by injecting into the column a solution of avidin in PBS; at this stage, the avidin molecules display some of their free binding sites available for binding with other biotinylated species. The addition of the biotinylated SCCA or biotinylated AFP results in the formation of the complex. The saturation of the remaining binding sites of avidin eventually present could be achieved by injecting into the column an excess of biotin. This step is necessary in order to avoid the polymerization of the complexes once they are removed from the solid phase. By using this procedure, multiple conjugation cannot be ruled out. The release of the complexes was achieved by changing the pH of the mobile phase to 2.5, thus weakening the antibody–IgM interaction without affecting the avidin–biotin binding. In a typical experiment, the recovery of the complex in terms of total protein content was about 70% of the total protein loaded on the column. The procedures used for the biotinylation of the building blocks were optimized in order to reduce the number of biotin moieties introduced in each building block. This was necessary in order to limit the number of avidin molecules involved in the formation of the complex. A harsher biotinylation procedure generally resulted in a diminished reactivity of the complex, probably because of the steric hindrance caused by the presence of a large number of avidin molecules.

Characterization of the complexes

The collected protein complexes were analysed to ascertain the existence of connectivity between IgM and SCCA or IgM and AFP within the same molecular species as a direct indication of the successful assembling. To this aim dedicated ELISA assays were developed by using anti-SCCA or anti-AFP antibodies as capturing phases and the use of an anti-IgM antibody complexed to HRP as tracing species; in these experiments ABTS was used as chromogenic substrate in the presence of hydrogen peroxide. The results of the ELISA analysis, reported in Figure 3, indicate that SCCA and IgM were present within the first complex and AFP and IgM were present in the second. In both cases a mixture of uncomplexed SCCA and IgM or AFP and IgM did not display significant reactivity (Figure 3).

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Figure 3. Immunoreactivity of the SCCA-IgM (squares, solid squares first synthesis, open squares second synthesis, the concentration of the complex was adjusted to 100 µg/ml in both cases) and AFP-IgM (circles, open circles first synthesis, solid circles second synthesis, the concentration of the complexes was adjusted at 100 µg/ml in both cases) complexes. Two mixtures comprising an equimolar amounts of biotinylated IgM and SCCA (diamonds) and equimolar amounts of biotinylated IgM and AFP with a total protein concentration of 100 µg/ml but in absence of avidin (cross) were used as negative control.

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The repeatability of the synthetic method was tested for the preparation of both the SCCA-IgM and AFP-IgM complexes. The observed immune reactivity of the different preparations was very similar (Figure 3).

In the case of SCCA-IgM mimetic complex, the presence of both SCCA and IgM within the same species was confirmed by Western blot (Figure 4). About 1 µg of the complex was subjected to SDS–PAGE analysis, under reducing conditions, on a 0.75 mm (10%) polyacrylamide gel slab, the bands were transferred on nitrocellulose membranes and reacted with anti-IgM and anti-SCCA polyclonal antibodies. In these conditions, the complex falls apart, and the identity of the bands was confirmed by comparison with original samples. Due to the relatively large expected molar mass of the complex, SDS–PAGE analysis could only give a confirmation of the simultaneous presence of both molecules in the complex rather than allowing a precise identification of the complex mass distribution.

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Figure 4. Negative image of the Western blot analysis of the SCCA-IgM complex. Panel A reports the detection of SCCA by incubation of the nitrocellulose membrane with rabbit anti-SCCA polyclonal antibody and subsequent reaction with goat-anti-rabbit IgG complexed to horseradish peroxidase. Paned B reports the detection of IgM heavy chains after incubation of the nitrocellulose membrane with goat-anti-human IgM (µ-chain specific) antibody complexed to horseradish peroxidase. Prior to incubation with the anti-IgM antibody, the membrane was soaked in 0.5 M NaOH for 5 min to strip the antibodies used for the SCCA detection, little SCCA reactivity from the authentic sample used as reference is however still detectable.

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Gel filtration analysis of the complexes

A sample of the artificial SCCA-IgM complex was analysed by gel-filtration chromatography (Figure 5), the complex eluted with a retention time of 6.6 min. The effluent was collected in 500 µl fractions and each fraction was analysed by ELISA in order to confirm the nature of the compound. The immune reactivity of the complex was detected in the fractions eluting at a retention volume close of about 6.6 ml, the calculated Kav for the eluted species was 0.08, no reactivity was detected in other fractions. From the calibration curve of the column, the molecular mass of the complex could be estimated to be in the range of 1–2 MDa. However, since the resolution of the ordinary stationary phases for gel filtration chromatography is low in this range of molecular masses, these values should be considered as lower bounds.

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Figure 5. Gel filtration and immune reactivity analyses of the SCCA-IgM complex. Panel A reports the gel filtration profile, panel B reports the measured immune reactivity in the collected fractions. Insert displays the calibration curve of the column.

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CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. REFERENCES

In this report, we preliminarily described a novel methodology for the continuous-flow solid-phase assembling of two different biotinylated molecules. As a proof of principle, the method has been validated by preparing complexes with immune reactivity mimicking that of the natural SCCA-IgM and AFP-IgM immune complexes, but other applications of the procedure can be envisaged. The design of the approach was led by practical considerations aiming to limit the costs associated to the process. The method allowed the preparation of robust protein complexes by using a limited amount of proteins in mild conditions. The immobilization of the first building block on the resin by means of a specific antibody blocks, from the steric point of view, part of the protein surface from any further reaction, ensuring at the same time the recovery of a significant immune reactivity after the synthesis. The crude preparations of the protein complex displayed immune reactivity mimicking that of the natural immune complexes, and no purification steps were required. In both cases, the chemical connectivity between SCCA and IgM or between AFP and IgM within the same complex was firmly established by dedicated sandwich ELISA; the signals obtained in the same ELISA by using unassembled proteins were negligible. The presence of both species within the same complex was also confirmed by immunoblot analysis with specific antibodies. The synthetic strategy devised exploits ‘orthogonal’ non-covalent interactions for protein immobilization and functionalization and allows the repeated use of the same solid phase.

The implementation of this process on a larger scale suitable for industrial production is an ongoing project and will be reported in due course.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. REFERENCES
  • Best MD. 2009. Click chemistry and bioorthogonal reactions: unprecedented selectivity in the labeling of biological molecules. Biochemistry 48: 65716584.
  • Brennan JL, Hatzakis NS, Tshikhudo TR, Dirvianskyte N, Razumas V, Patkar S, Vind J, Svendsen A, Nolte RJM, Rowan AE, Brust M. 2006. Bionanoconjugation via click chemistry: the creation of functional hybrids of lipases and gold nanoparticles. Biocomplex Chem. 17: 13731375.
  • De Falco S, Ruvoletto MG, Verdoliva A, Ruvo M, Raucci A, Marino M, Senatore S, Cassani G, Alberti A, Pontisso P, Fassina G. 2001. Cloning and expression of a novel hepatitis B virus-binding protein from HepG2 cells. J. Biol. Chem. 276: 3661336623.
  • Gauthier MA, Klok HA. 2008. Peptide/protein-polymer complexes: synthetic strategies and design concepts. Chem. Commun. 25912611.
  • Hu T, Li D, Su Z. 2003. Preparation and characterization of dimeric bovine hemoglobin tetramers. J. Protein Chem. 22: 411416.
  • Hu T, Su Z. 2003. A solid phase adsorption method for preparation of bovine serum albumin-bovine hemoglobin complex. J. Biotechnol. 100: 267275.
  • Russell J, Colpitts T, Holets-McCormack S, Spring T, Stroupe S. 2004. Defined protein complexes as signaling agents in immunoassays. Clin. Chem. 50: 19211929 .
  • Russell LC, Colpitts TL, Holets-McCormack SR, Spring TG, Stroupe SD, Vogt AD, Wong ST. 2002. Solid phase assembly of defined protein complexes. Bioconjug. Chem. 13: 958965.
  • Suo X, Zheng C, Yu P, Lu X, Ma G, Su Z. 2009. Solid phase pegylation of hemoglobin. Artif. Cells Blood Substit. Immobil. Biotechnol. 37: 147155.
  • Towbin H, Staehelin T, Gordon J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76: 43504354.
  • Wong SS. 1991. Chemistry of Protein Conjugation and Cross-linking. CRC press: USA. (and references cited therein).
Abbreviations:
SCCA

Squamous Cell Carcinoma Antigen

AFP

alphafetoprotein

IgM

Immunoglobulin M

ELISA

Enzyme-linked Immunosorbent Assay

BSA

Bovine Serum Albumin

IgG

immunoglobulin G

NHS

N-hydroxysuccinimide

HRP

Horseradish Peroxidase

ABTS

2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)

SDS–PAGE

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis