Elisa For Detection And Quantification Of Staphylococcal Protein Biology Essay

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Protein A is one of a number of adhesion factors and toxins produced by S aureus and is known to react with many eukaryotic targets especially those that will have an immunological effect within the cell.

It is also widely known for its uses in the purification of rhMAb's (recombinant human monoclonal antibodies) such as IgG in the downstream processing of such products. (Ey et al., 1978) For the production of monoclonal antibodies Protein A resin accounts for approximately 10% of the cost of downstream processing while virus filtration can account for 40% (Gottschalk et al., 2006). It is used in the chromatographic process to remove the sample from solution as Staphylococcal Protein A has a high affinity to the Fc fraction of immunoglobulins through interaction with the heavy chain, especially IgG due to Protein A's multiplicity of binding sites (shown in "Table 1"). It was shown that each of the binding sites on the Staphylococcal Protein A (E, D, A, B and C) has a high affinity towards the Fc fragment of the immunoglobulin IgG. Jansson et al showed that each of the fragments of Protein A were expressed individually and through the use of E.coli and their specificity and binding capabilities studied in detail through the use of real-time bio specific analysis (Birger Jansson et al., 1997).

Protein A affinity chromatography is one of the most commonly used methods of monoclonal antibody purification due to various advantages it has over other affinity compounds. The affinity chromatography process is an easy, fast and extremely specific process for the purification of the target. Because of its specificity an affinity-purification step can be introduced at an early stage in the purification process to effectively reduce the number of successive unit operations in the purification process. The affinity between SpA and IgG was one of the first studied for the use in an affinity chromatography process and has led to the widespread use of SpA, or ligands derived from it, in protein purification (Sophia Hober et al., 2006)

There are alternatives to the use of SpA in the purification process such as the use of Streptococcal Protein G (SpG) for the purification of serum and IgG subclass III due to the low affinity of SpA to this subclass (Table 1). Aside from these cases the use of SpG is limited due to the much greater stability of SpA. Methods have also been applied via protein engineering to help stabilise the SpA and SpG with relation to CIP (cleaning in place) and help maintain their affinity over numerous uses (S.Gulich et al., 2002). Fully synthetic ligands (pseudobiospecific ligands) have also been developed to completely remove the necessity for the use of potentially toxic SpA, with high selectivity for antibody-like molecules and enhanced stability but have still not become as widely used due to high cost of production (Ana C.A. Roque et al., 2007)

Though advances are being made, the long term utilization of SpA as antibody-purification supports ensures they are still widely used and are being produced on a large scale via recombinant production. Protein A is the ligand of choice with regard to purification and has been optimised over the years for high capacity, high throughput and reusability so it can be used effectively and in a cost efficient manner in large scale monoclonal antibody production (Duncan Low et al., 2006)

Protein A Binding

The multiple binding sites of SpA is the main reason it is advantageous in affinity chromatography for the purification of monoclonal antibodies, however this binding strength can lead to difficulties when retrieving the product from the column and lead to leaching of the SpA due to the conditions required for the retrieval of the monoclonal antibody.

Using fragmentation methods followed by the implementation of amino acid sequence analysis showed that SpA has four unique Fc binding sites (A, B, C and D) and a C terminal region which is used for binding the SpA to the cell wall of S aureus when it is in situ (Tomas Moks et al., 1986).

Fig 2. Structure of an immunoglobulin molecule. (Tizard IR 2001)

A study carried out on the binding capabilities of SpA show that it is only capable of binding two immunoglobulin molecules at a time. Following further studies it was shown that due to its low chemical and proteolytic stability, Protein A resins cannot withstand post chromatographic cleaning procedures in harsh conditions. Also over extended usage and exposure to sera, the eluted antibodies can become heavily contaminated by whole ligand from the SpA or fragments of the binding sites that are produced from ligand leakage due to column degradation, and additional purification steps can be required due to the possible toxicity of these contaminants in the product (Gagnon., 1996)

Protein A Toxicity

Protein A has been shown to be linked with toxicity in humans and animals when present in therapeutic monoclonal antibody preparations (Bloom JW et al., 1989). Staphylococcal Protein A (SpA) has been shown to induce inflammatory responses in human corneal epithelial cells by exposing them to varying concentrations of SpA in vitro.

The results showed that lower levels of SpA (5-10µg/ml) had little or no effect on the cells however when concentrations were risen to 50µg/ml there was a marked response by the cells which led them to produce high levels of TNF-α and IL-8 which were subsequently detected in the culture media of the cells and are known to induce inflammation. Using these methods they also successfully showed SpA's ability to act as an activator for TNFR1 (TNF receptor-1) which initiates a cascade of events that can attribute to the development of staphylococcal pneumonia (Ashok Kumar et al 2007).

Accordingly immunoglobulin products where their intended use is of a clinical nature must be shown to be free of any quantifiable trace of SpA impurities. Hence the development of a method for the detection and quantification of Protein A post chromatographic process to levels as low as parts per million (ppm) is essential to ensure the safety of the product.

Fig 3. Protein A levels in varying monoclonal antibody batches over three years (Rodolfo Valdez Veliz et al., 2003)

ELISA (Enzyme-linked immunosorbent assay)

The ELISA method was developed originally because of the dangers associated with its predecessor radioimmunoassay which used radioactively labelled antibodies or antigens to detect the presence of an analyte in a sample first described in 1960 (Rosalyn S. Yalow et al., 1960). To circumvent the use of these dangerous methods a safer method was sought and developed in 1971 with the use of enzymes linked to the antibody used as the method of detection (Engvall E., 1971).

There are now many types of ELISA and the type that is used is dependent on a varying number of factors that relate to the analyte such as, the number of binding sites for the antibodies, the presence of high levels of contamination in the sample and also the sensitivity required in the testing process.

"Indirect" ELISA (Fig 4 (A))

The analyte is added to the wells of the micro titre plate and allowed to adhere to the plastic through basic charge interactions.

Non-reacting protein is then added to the well to block areas on the well that have remained unbound to prevent interference during testing

The primary antibody is subsequently added to the wells of the plate where it will bind to the analyte.

A secondary antibody is then added to the reaction which will have an enzyme attached (e.g. HRP - Horseradish peroxidase), where it will have negligible effect on the binding capability of the antibody.

A substrate for the enzyme is then added which will lead to a reaction which can be quantified.

The higher the level of analyte in the sample the greater the reaction which allows quantification.

One of the major dis-advantages with regard the use of the indirect ELISA is that the method of analyte immobilization is non-specific and hence, if there are other proteins present in the sample (serum sample) they will competitively bind to the micro titre plate preventing the analyte from binding.

Fig 4. (A) In indirect ELISA, the production of colour indicates the amount of an antibody to a specific antigen. (B) In sandwich ELISA, the production of colour indicates the quantity of antigen. (R.A. Goldsby et al., 2000)

Sandwich ELISA (Fig 4 (B))

A known quantity of capture antibody is added to the well of the micro titre plate and allowed to adhere through incubation

Non-reacting protein is then added to the well to block areas on the well that have remained unbound to prevent interference during testing

The sample containing the analyte is then added to the plate and incubated

Unbound sample is then washed away with a washing step

A specific antibody for the analyte is then added to the well which is conjugated to an enzyme where the enzyme will have little effect on the binding of the antibody to the analyte

Again the plate is washed to remove the unbound antibody enzyme complex from the well

A substrate is then added which when catalysed by the enzyme will release a measureable effect which allows for quantification of the analyte.

Use of the capture antibody in this case allows for greater specificity with regard to the analyte being tested. It also allows for the sample being tested to be part of a complex mixture as would be the case in testing the eluent from the chromatographic process which would contain the monoclonal antibody that is being produced and also the test analyte which would be the SpA. In this case the capture antibody would be specific to the SpA and selectively bind this though considerable consideration must be taken to choose the capture antibody. (Norman E Crook et al., 1979)

Competitive ELISA

The well of the micro titre plate is coated with unlabelled primary antibody and incubated to allow attachment.

Non-reacting protein is then added to the well to block areas on the well that have remained unbound to prevent interference during testing

Unknown samples and standards are then added to the well and incubated so the analyte will bind to the primary antibody

Immunogen that is conjugated to an enzyme is then added to the wells which will bind to any location where the analyte didn't.

The substrate is then added and the reaction can be observed.

The more analyte in the sample the less available space for the conjugated immunogen to bind and a lesser reaction will be observed which is inversely proportional to the amount of analyte in the sample.

The competitive ELISA approach is used when two "matched pair" antibodies cannot be found for your analyte thus not allowing the other types of assay to be carried out. Another advantage of the competitive ELISA is the ability to use crude or impure samples and still have specific binding and quantification of the analyte without effect by other proteins that may be present in the sample. (DM Kennedy., 1988)

Current Methods

Due to the onset of clinical applications for monoclonal antibodies there are now numerous products being developed based on this technology for various healthcare applications (D Glover., 1999). There is now large scale use of SpA due to its high specificity for immunoglobulins in the purification of rhMAbs from ascetic fluid or the supernatant from cell culture due to the high yields obtained, and the purity levels than can be obtained from the chromatographic process (Ey et al., 1978). However due to the release of free SpA or SpA-Immunoglobulin complex and its toxicity, there have been large numbers of test methods developed for the detection and quantification of the SpA with varying levels of reliability.

These varying levels of reliability are due to the complex nature of the development of a quantitative test for SpA. This is due to the recognition of the Fc fragment in most of the immunoglobulins by SpA (Table 1). These tests also have to be carried out in the presence of high levels of monoclonal antibodies which can lead to interference of the test results due to unwanted binding or competition. (Rodolfo Valdez Veliz et al., 2003).

Commercial Kits

There are a number of commercial kits available for the detection of SpA or rProtein A (recombinant Protein A) which is also used in chromatographic processes. These tests vary in price sensitivity and also in the methods that they use.

Protein A ELISA (Fig 5)

This is a commercially available kit that uses a 96 well micro titre plate and works on the principle of coating the plate with affinity-purified chicken anti-SpA. Then the sample is added to the well where it will bind to the micro well and it will then be detected by biotinylated chicken anti-SpA antibody. A streptavidin horseradish peroxidase (HRP)

conjugate is then bound to the biotin conjugate and finally the substrate for the HRP is added to each well giving a colour change in each well which is representative of the amount of SpA present in the sample.

This test works upon the principle that it can remove contamination caused by the monoclonal antibodies that will be present in the sample. In samples containing

mammalian IgG, immunologically active epitopes of SpA can be blocked due to the nonspecific binding that can occur between IgG and SpA. To overcome this, the test is carried out at a low pH (Berglund A. et al., 1987) since this causes IgG and SpA to dissociate and allow the SpA to bind. To further remove contamination the samples are boiled in a water bath. This eliminates interference caused by excess IgG by heat-inactivation while the SpA will remain unaffected (Rune Nilsson., 1990). However some binding can still occur so flexibility is allowed with pH dependant on the specific test being carried out. By using chicken anti-SpA the specificity of the test is greatly increased as chicken IgY is one of the few immunoglobulins that do not bind SpA in the Fc region (A. Larsson et al., 1992).

This test has a functional sensitivity of 0.15 ng/mL (0.15 ppm) and comes at a cost of €425 with the ability to test 44 samples along with the standards giving a total of 96 micro titre wells.

Fig 5. Protein A ELISA Kit from Immunsystem AB (www.proteinaelisa.com)

MSD Protein A Kit

This is a variant on the common method for the detection and quantification of Protein A that is available commercially. Meso Scale Discovery are a company who use proprietary MULTI-ARRAY® and MULTI-SPOT® microplates with electrodes integrated into the bottom of the plate. Their test is not a typical ELISA kit as it is not an enzyme linked immunoassay. Alternatively their assay uses Electrochemiluminescence for the detection of the SpA in the sample. The electrodes in their multi well micro titre plates are made from carbon so biological reagents can be attached to the carbon simply by passive adsorption and can still retain high levels of biological activity even after adsorption.

As with other types of ELISA the test uses an anti-protein A antibody as a capture molecule which is coated to the base of the micro titre well. In place of a tag that will incur an enzymatic reaction there is an electrochemiluminescent label (SULFO-TAGTM ) which emits light when electrochemically stimulated. The detection process is initiated at electrodes located in the bottom of MSD's micro titre plates. Only labels near the electrode are excited and detected, enabling non-washed assays which remove many wash steps from a normal ELISA protocol (Fig 6)

Fig 6. MSD Protein A assay format

This particular assay has a range from 50pg/ml to 10ng/ml if the sample matrix is free of IgG. A method of buffering the effect of any bound IgG is also supplied in the form of gamma globulin to extend the assay range from 500pg/ml to 250ng/ml. This process along with the dissociation protocols of many of the common Protein A detection methods can allow the assay to be much more sensitive.

The fact that this assay uses proprietary equipment and software can raise the price of implementation of this type of assay in a laboratory. However the financial loss can be attributed to the gain in dynamic range of the assay with a sensitivity of 50pg/ml in comparison to the sensitivity of the Protein A ELISA from Immunsystem AB which has a poorer sensitivity of 150pg/ml along with the removal of the cumbersome wash steps from the procedure. (http://www.meso-scale.com)

Recent Research

Though much of the work in this field was done in the late 80's to early 90's there have been some studies done recently that have tried to develop on the methods that are available and try to combine the developments that have been achieved.

Since 2000 there have been studies carried out in this area due to the current rise in the numbers of rhMAb products that are under development and the widespread use of Protein A as a capture mechanism in many affinity chromatography columns now used. In 2003 Rodolfo Valdes Veliz et al., developed a sandwich immunoassay for the detection of Protein A for its use in immunoaffinity chromatography of recombinant hepatitis B virus surface antigen (rHBsAg) which is used as a vaccine against the virus. They developed a specific sandwich ELISA for this purpose which used polyclonal sheep antibodies specific against SpA. They achieved a quantification limit of 0.39ng/ml for this assay (Rodolfo Valdez Veliz et al., 2003). This assay however does not account for the possible interference from binding of the product and the SpA in the sample being tested which may lead to interference in the results obtained and not give a total quantification of the SpA in the sample, only giving the level of unbound SpA.

The most recent study carried out by Judith Zhu-Shimoni et al in 2009 accounts for this interference and was done with the purpose of developing a method for the quantification of SpA in bioprocess samples that removed this interference, which may be observed when there are large quantities of IgG in the sample being tested. They were developing on a method where Protein A/IgG samples were successfully dissociated using a defined combination of chelators, detergents and heat, breaking the bonds that form the complex while not disturbing the Protein A structure to an extent that it would affect its binding capability (Steindl et al., 2000)

Up to now the presence of IgG in the sample had to be accounted for and a separate assay validated in each case with regard to the various products that would be produced in a factory. The objective was to develop an assay that completely removed the need for this revalidation. They worked on the basis of removing the possibility of interference by introducing Protein A/IgG Complex Dissociation Buffer which enabled the dissociation of the Protein A/IgG bond and removed interference that this may cause. (Fig 7)

Fig 7. Dissociation of Protein A/IgG complex to allow for interference free quantification of Protein A in sample. (Judith Zhu-Shimoni et al., 2009)

They effectively showed that the presence of varying levels in the sample can interfere with the quantification of the analyte and even different forms of rhMab's can affect the assay in different ways and the addition of this dissociation step can effectively remove this interference. (Fig 8)

Fig 8. Effect of rhMabs on Protein A standard curves with ranges from 0.39 to 50 ng/ml (Judith Zhu-Shimoni et al., 2009)

While exploring this multiplex assay, which can be used for a wide range of IgG products, they also explored the effect of heating on the dissociation process and the use of two different methods of heating. Initially slower heating times were used to a temperature of 96oC using a PCR thermocycler, ranging from 15mins to 3hrs. Results showed that times less than 1hr showed insufficient dissociation of the Protein A/IgG samples, while heating for 3hrs of the sample without interfering rhMab showed a decrease in recovery which infers denaturation of the samples leading to decreased recovery (Fig 9). Due to the capacity of the thermocycler very few samples could be tested simultaneously which lead to variances between cycles.

Fig 9. Effect of heat on dissociation of Protein A/IgG complex in presence or absence of rhMab using PCR thermocycler (Judith Zhu-Shimoni et al., 2009)

Microwave assisted heating times were then tested using a microwave capable of holding 96 well micro titre plates which greatly increased the throughput of samples. In this instance heating times and temperatures were varied to find the optimal range for dissociation of the sample complex. Using full factorial Design of Experiments (DOE) approach the optimal conditions for the process were 80oC for 10mins allowing both forms of the rhMab tested to dissociate from the Protein A giving optimal quantification of Protein A (Fig 9).

Fig 10. Effect of heat on dissociation of Protein A/IgG complex in presence or absence of rhMab after microwave-assisted heating. (Judith Zhu-Shimoni et al., 2009)

Removal of interference caused by the product being present in the sample allows for the assay to be used and was then described as a Multiproduct (MP-PA) assay. They tested this efficient against assays that do not account for interference and are known as Product-specific (PS-PA) assays. The MP-PA assay showed a comparable difference with greater sensitivity and range with regard to the PS-PA assay that was previously used. (Table 2)

MP-PA

PS-PA

Recovery

Standard

96-110%

94-104%

Control Spike

88-106%

97-102%

Real Sample Spike

80-121%

62-70%

Precison

Intra-assay

<9%

<4%

Inter-assay

<15%

<9%

Sensitivity (ng/ml)

Limit Of Detection

0.043

0.081

Working Range (ng/ml)

Upper limit

12.5

50

Lower Limit

0.098

0.391

Table 2. Assay Performance for both Multiproduct and Product-specific assays run in triplicate over different days (Judith Zhu-Shimoni et al., 2009)

Alternative Methods

Because of the knowledge and specificity of the ELISA method of detection and quantification of analytes in samples there have been very few other methods that have been developed to detect Protein A. Before the use of the ELISA method for the quantification of Protein A there were methods present which allowed for the detection but not the quantitation of the amount of SpA in a sample. One of the original methods was the single-tube mixed agglutination test. This was a rapid mixed agglutination test where sheep erythrocytes which had been sensitized to SpA were mixed with the sample and the presence of agglutination meant a positive result for the presence of SpA (Peter E. Maxim et al., 1976). This test however is of little use as it does not allow the amount of SpA present to be quantified. It could however be used as a preliminary test though results would not be of the standard required within industry.

In 1999 Ihab Abdel-Hamid et al., described a novel method for the determination of SpA whereby an amperometric immunosensor is used which consists of carbon molecules that have been dispersed and conjugated with anti-protein A antibodies. Its uses the amperometric immunosensor combined with a sandwich assay scheme using peroxidase-labelled anti-IgG antibodies for the quantitation of the SpA in the sample giving a detection limit in the nanomolar range. This system allows for the automation of the detection of Protein A and also allows for much faster determination with an assay time as low as 22mins. The system can also be arranged as a portable device to allow for point to point detection throughout the manufacturing process giving a more accurate estimation of which point in the system is causing the leaching of the SpA into the product (Ihab Abdel-Hamid et al., 1999)

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