Elisa For Detection And Quantification Of Staphylococcal Protein Biology Essay

Protein A is one of a figure of adhesion factors and toxins produced by S aureus and is known to respond with many eucaryotic marks particularly those that will hold an immunological consequence within the cell.

It is besides widely known for its utilizations in the purification of rhMAb ‘s ( recombinant human monoclonal antibodies ) such as IgG in the downstream processing of such merchandises. ( Ey et al. , 1978 ) For the production of monoclonal antibodies Protein A rosin histories for about 10 % of the cost of downstream processing while virus filtration can account for 40 % ( Gottschalk et al. , 2006 ) . It is used in the chromatographic procedure to take the sample from solution as Staphylococcal Protein A has a high affinity to the Fc fraction of Igs through interaction with the heavy concatenation, particularly IgG due to Protein A ‘s multiplicity of adhering 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 Ig IgG. Jansson et Al showed that each of the fragments of Protein A were expressed separately and through the usage of E.coli and their specificity and adhering capablenesss studied in item through the usage of real-time bio specific analysis ( Birger Jansson et al. , 1997 ) .

Protein A affinity chromatography is one of the most normally used methods of monoclonal antibody purification due to assorted advantages it has over other affinity compounds. The affinity chromatography procedure is an easy, fast and highly specific procedure for the purification of the mark. Because of its specificity an affinity-purification measure can be introduced at an early phase in the purification procedure to efficaciously cut down the figure of consecutive unit operations in the purification procedure. The affinity between SpA and IgG was one of the first studied for the usage in an affinity chromatography procedure and has led to the widespread usage of SpA, or ligands derived from it, in protein purification ( Sophia Hober et al. , 2006 )

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There are options to the usage of SpA in the purification procedure such as the usage 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 instances the usage of SpG is limited due to the much greater stableness of SpA. Methods have besides been applied via protein technology to assist brace the SpA and SpG with relation to CIP ( cleaning in topographic point ) and assist keep their affinity over legion utilizations ( S.Gulich et al. , 2002 ) . Fully man-made ligands ( pseudobiospecific ligands ) have besides been developed to wholly take the necessity for the usage of potentially toxic SpA, with high selectivity for antibody-like molecules and enhanced stableness but have still non become as widely used due to high cost of production ( Ana C.A. Roque et al. , 2007 )

Though progresss are being made, the long term use of SpA as antibody-purification supports ensures they are still widely used and are being produced on a big graduated table via recombinant production. Protein A is the ligand of pick with respect to purification and has been optimised over the old ages for high capacity, high throughput and reusability so it can be used efficaciously and in a cost efficient mode in big scale monoclonal antibody production ( Duncan Low et al. , 2006 )

Protein A Binding

The multiple binding sites of SpA is the chief ground it is advantageous in affinity chromatography for the purification of monoclonal antibodies, nevertheless this binding strength can take to troubles when recovering the merchandise from the column and lead to leaching of the SpA due to the conditions required for the retrieval of the monoclonal antibody.

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

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

A survey carried out on the binding capablenesss of SpA show that it is merely capable of adhering two immunoglobulin molecules at a clip. Following farther surveies it was shown that due to its low chemical and proteolytic stableness, Protein A rosins can non defy station chromatographic cleansing processs in rough conditions. Besides over extended use and exposure to sera, the eluted antibodies can go to a great extent contaminated by whole ligand from the SpA or fragments of the binding sites that are produced from ligand escape due to column debasement, and extra purification stairss can be required due to the possible toxicity of these contaminations in the merchandise ( Gagnon. , 1996 )

Protein A Toxicity

Protein A has been shown to be linked with toxicity in worlds and animate beings when nowadays in curative monoclonal antibody readyings ( Bloom JW et al. , 1989 ) . Staphylococcal Protein A ( SpA ) has been shown to bring on inflammatory responses in human corneal epithelial cells by exposing them to changing concentrations of SpA in vitro.

The consequences showed that lower degrees of SpA ( 5-10Aµg/ml ) had small or no consequence on the cells nevertheless when concentrations were risen to 50Aµg/ml there was a pronounced response by the cells which led them to bring forth high degrees of TNF-I± and IL-8 which were later detected in the civilization media of the cells and are known to bring on redness. Using these methods they besides successfully showed SpA ‘s ability to move as an activator for TNFR1 ( TNF receptor-1 ) which initiates a cascade of events that can impute to the development of staphylococcal pneumonia ( Ashok Kumar et Al 2007 ) .

Consequently immunoglobulin merchandises where their intended usage is of a clinical nature must be shown to be free of any quantifiable hint of SpA drosss. Hence the development of a method for the sensing and quantification of Protein A station chromatographic procedure to degrees every bit low as parts per million ( ppm ) is indispensable to guarantee the safety of the merchandise.

Fig 3. Protein A degrees in changing monoclonal antibody batches over three old ages ( Rodolfo Valdez Veliz et al. , 2003 )

ELISA ( Enzyme-linked immunosorbent check )

The ELISA method was developed originally because of the dangers associated with its predecessor radioimmunoassay which used radioactively labeled antibodies or antigens to observe the presence of an analyte in a sample foremost described in 1960 ( Rosalyn S. Yalow et al. , 1960 ) . To besiege the usage of these unsafe methods a safer method was sought and developed in 1971 with the usage of enzymes linked to the antibody used as the method of sensing ( Engvall E. , 1971 ) .

There are now many types of ELISA and the type that is used is dependent on a variable figure of factors that relate to the analyte such as, the figure of adhering sites for the antibodies, the presence of high degrees of taint in the sample and besides the sensitiveness required in the testing procedure.

“ Indirect ” ELISA ( Fig 4 ( A ) )

The analyte is added to the Wellss of the micro titer home base and allowed to adhere to the plastic through basic charge interactions.

Non-reacting protein is so added to the well to barricade countries on the well that have remained unbound to forestall intervention during proving

The primary antibody is later added to the Wellss of the home base where it will adhere to the analyte.

A secondary antibody is so added to the reaction which will hold an enzyme attached ( e.g. HRP – Horseradish peroxidase ) , where it will hold negligible consequence on the binding capableness of the antibody.

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

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

One of the major dis-advantages with respect the usage of the indirect ELISA is that the method of analyte immobilisation is non-specific and therefore, if there are other proteins present in the sample ( serum sample ) they will competitively adhere to the micro titer home base forestalling the analyte from adhering.

Fig 4. ( A ) In indirect ELISA, the production of coloring material indicates the sum of an antibody to a specific antigen. ( B ) In sandwich ELISA, the production of coloring material indicates the measure of antigen. ( R.A. Goldsby et al. , 2000 )

Sandwich ELISA ( Fig 4 ( B ) )

A known measure of gaining control antibody is added to the well of the micro titer home base and allowed to adhere through incubation

Non-reacting protein is so added to the well to barricade countries on the well that have remained unbound to forestall intervention during proving

The sample incorporating the analyte is so added to the home base and incubated

Unbound sample is so washed away with a washing measure

A specific antibody for the analyte is so added to the well which is conjugated to an enzyme where the enzyme will hold small consequence on the binding of the antibody to the analyte

Again the home base is washed to take the unbound antibody enzyme composite from the well

A substrate is so added which when catalysed by the enzyme will let go of a measureable consequence which allows for quantification of the analyte.

Use of the gaining control antibody in this instance allows for greater specificity with respect to the analyte being tested. It besides allows for the sample being tested to be portion of a complex mixture as would be the instance in proving the eluent from the chromatographic procedure which would incorporate the monoclonal antibody that is being produced and besides the trial analyte which would be the SpA. In this instance the gaining control antibody would be specific to the SpA and selectively adhere this though considerable consideration must be taken to take the gaining control antibody. ( Norman E Crook et al. , 1979 )

Competitive ELISA

The well of the micro titer home base is coated with unlabeled primary antibody and incubated to let fond regard.

Non-reacting protein is so added to the well to barricade countries on the well that have remained unbound to forestall intervention during proving

Unknown samples and criterions are so added to the well and incubated so the analyte will adhere to the primary antibody

Immunogen that is conjugated to an enzyme is so added to the Wellss which will adhere to any location where the analyte did n’t.

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

The more analyte in the sample the less available infinite for the conjugated immunizing agent to adhere and a lesser reaction will be observed which is reciprocally relative to the sum of analyte in the sample.

The competitory ELISA attack is used when two “ matched brace ” antibodies can non be found for your analyte therefore non leting the other types of check to be carried out. Another advantage of the competitory ELISA is the ability to utilize petroleum or impure samples and still hold specific binding and quantification of the analyte without consequence by other proteins that may be present in the sample. ( DM Kennedy. , 1988 )

Current Methods

Due to the oncoming of clinical applications for monoclonal antibodies there are now legion merchandises being developed based on this engineering for assorted health care applications ( D Glover. , 1999 ) . There is now big graduated table usage of SpA due to its high specificity for Igs in the purification of rhMAbs from ascetic fluid or the supernatant from cell civilization due to the high outputs obtained, and the pureness degrees than can be obtained from the chromatographic procedure ( Ey et al. , 1978 ) . However due to the release of free SpA or SpA-Immunoglobulin complex and its toxicity, there have been big Numberss of trial methods developed for the sensing and quantification of the SpA with changing degrees of dependability.

These changing degrees of dependability are due to the complex nature of the development of a quantitative trial for SpA. This is due to the acknowledgment of the Fc fragment in most of the Igs by SpA ( Table 1 ) . These trials besides have to be carried out in the presence of high degrees of monoclonal antibodies which can take to intervention of the trial consequences due to unwanted binding or competition. ( Rodolfo Valdez Veliz et al. , 2003 ) .

Commercial Kits

There are a figure of commercial kits available for the sensing of SpA or rProtein A ( recombinant Protein A ) which is besides used in chromatographic procedures. These trials vary in monetary value sensitiveness and besides in the methods that they use.

Protein A ELISA ( Fig 5 )

This is a commercially available kit that uses a 96 good micro titer home base and works on the rule of surfacing the home base with affinity-purified lily-livered anti-SpA. Then the sample is added to the well where it will adhere to the micro good and it will so be detected by biotinylated lily-livered anti-SpA antibody. A streptavidin horseradish peroxidase ( HRP )

conjugate is so bound to the vitamin H conjugate and eventually the substrate for the HRP is added to each good giving a coloring material alteration in each well which is representative of the sum of SpA nowadays in the sample.

This trial works upon the rule that it can take taint caused by the monoclonal antibodies that will be present in the sample. In samples incorporating

mammalian IgG, immunologically active antigenic determinants of SpA can be blocked due to the nonspecific binding that can happen between IgG and SpA. To get the better of this, the trial is carried out at a low pH ( Berglund A. et al. , 1987 ) since this causes IgG and SpA to disassociate and let the SpA to adhere. To farther take taint the samples are boiled in a H2O bath. This eliminates intervention caused by extra IgG by heat-inactivation while the SpA will stay unaffected ( Rune Nilsson. , 1990 ) . However some binding can still happen so flexibleness is allowed with pH dependent on the specific trial being carried out. By utilizing lily-livered anti-SpA the specificity of the trial is greatly increased as poulet IgY is one of the few Igs that do non adhere SpA in the Fc part ( A. Larsson et al. , 1992 ) .

This trial has a functional sensitiveness of 0.15 ng/mL ( 0.15 ppm ) and comes at a cost of a‚¬425 with the ability to prove 44 samples along with the criterions giving a sum of 96 micro titre Wellss.

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

MSD Protein A Kit

This is a discrepancy on the common method for the sensing and quantification of Protein A that is available commercially. Meso Scale Discovery are a company who use proprietary MULTI-ARRAYA® and MULTI-SPOTA® microplates with electrodes integrated into the underside of the home base. Their trial is non a typical ELISA kit as it is non an enzyme linked immunochemical assay. Alternatively their check uses Electrochemiluminescence for the sensing of the SpA in the sample. The electrodes in their multi good micro titre home bases are made from C so biological reagents can be attached to the C merely by inactive surface assimilation and can still retain high degrees of biological activity even after surface assimilation.

As with other types of ELISA the trial uses an anti-protein A antibody as a gaining control molecule which is coated to the base of the micro titer good. In topographic point of a ticket that will incur an enzymatic reaction there is an electrochemiluminescent label ( SULFO-TAGTM ) which emits visible radiation when electrochemically stimulated. The sensing procedure is initiated at electrodes located in the underside of MSD ‘s micro titer home bases. Merely labels near the electrode are excited and detected, enabling non-washed checks which remove many wash stairss from a normal ELISA protocol ( Fig 6 )

Fig 6. MSD Protein A check format

This peculiar check has a scope from 50pg/ml to 10ng/ml if the sample matrix is free of IgG. A method of buffering the consequence of any edge IgG is besides supplied in the signifier of gamma globulin to widen the check scope from 500pg/ml to 250ng/ml. This procedure along with the dissociation protocols of many of the common Protein A sensing methods can let the check to be much more sensitive.

The fact that this assay uses proprietary equipment and package can raise the monetary value of execution of this type of check in a research lab. However the fiscal loss can be attributed to the addition in dynamic scope of the check with a sensitiveness of 50pg/ml in comparing to the sensitiveness of the Protein A ELISA from Immunsystem AB which has a poorer sensitiveness of 150pg/ml along with the remotion of the cumbersome wash stairss from the process. ( 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 surveies done late that have tried to develop on the methods that are available and seek to unite the developments that have been achieved.

Since 2000 there have been surveies carried out in this country due to the current rise in the Numberss of rhMAb merchandises that are under development and the widespread usage of Protein A as a gaining control mechanism in many affinity chromatography columns now used. In 2003 Rodolfo Valdes Veliz et al. , developed a sandwich immunochemical assay for the sensing of Protein A for its usage in immunoaffinity chromatography of recombinant hepatitis B virus surface antigen ( rHBsAg ) which is used as a vaccinum against the virus. They developed a specific sandwich ELISA for this intent which used polyclonal sheep antibodies specific against SpA. They achieved a quantification bound of 0.39ng/ml for this check ( Rodolfo Valdez Veliz et al. , 2003 ) . This assay nevertheless does non account for the possible intervention from binding of the merchandise and the SpA in the sample being tested which may take to interference in the consequences obtained and non give a entire quantification of the SpA in the sample, merely giving the degree of unbound SpA.

The most recent survey carried out by Judith Zhu-Shimoni et Al in 2009 histories for this intervention and was done with the intent of developing a method for the quantification of SpA in bioprocess samples that removed this intervention, which may be observed when there are big measures of IgG in the sample being tested. They were developing on a method where Protein A/IgG samples were successfully dissociated utilizing a defined combination of chelators, detergents and heat, interrupting the bonds that form the composite while non upseting the Protein A construction to an extent that it would impact its binding capableness ( Steindl et al. , 2000 )

Up to now the presence of IgG in the sample had to be accounted for and a separate check validated in each instance with respect to the assorted merchandises that would be produced in a mill. The aim was to develop an check that wholly removed the demand for this revalidation. They worked on the footing of taking the possibility of intervention by presenting Protein A/IgG Complex Dissociation Buffer which enabled the dissociation of the Protein A/IgG bond and removed intervention that this may do. ( Fig 7 )

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

They efficaciously showed that the presence of changing degrees in the sample can interfere with the quantification of the analyte and even different signifiers of rhMab ‘s can impact the check in different ways and the add-on of this dissociation measure can efficaciously take this intervention. ( Fig 8 )

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

While researching this manifold check, which can be used for a broad scope of IgG merchandises, they besides explored the consequence of heating on the dissociation procedure and the usage of two different methods of heating. Initially slower heating times were used to a temperature of 96oC utilizing a PCR thermocycler, runing from 15mins to 3hrs. Consequences showed that times less than 1hr showed deficient dissociation of the Protein A/IgG samples, while heating for 3hrs of the sample without interfering rhMab showed a lessening in recovery which infers denaturation of the samples taking to decreased recovery ( Fig 9 ) . Due to the capacity of the thermocycler really few samples could be tested at the same time which lead to discrepancies between rhythms.

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

Microwave assisted heating times were so tested utilizing a microwave capable of keeping 96 good micro titer home bases which greatly increased the throughput of samples. In this case heating times and temperatures were varied to happen the optimum scope for dissociation of the sample composite. Using full factorial Design of Experiments ( DOE ) approach the optimum conditions for the procedure were 80oC for 10mins leting both signifiers of the rhMab tested to disassociate from the Protein A giving optimum quantification of Protein A ( Fig 9 ) .

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

Removal of intervention caused by the merchandise being present in the sample allows for the check to be used and was so described as a Multiproduct ( MP-PA ) assay. They tested this efficient against checks that do non account for intervention and are known as Product-specific ( PS-PA ) assays. The MP-PA check showed a comparable difference with greater sensitiveness and scope with respect to the PS-PA check that was antecedently 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

& lt ; 9 %

& lt ; 4 %

Inter-assay

& lt ; 15 %

& lt ; 9 %

Sensitivity ( ng/ml )

Limit Of Detection

0.043

0.081

Working Range ( ng/ml )

Upper bound

12.5

50

Lower Limit

0.098

0.391

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

Alternate Methods

Because of the cognition and specificity of the ELISA method of sensing and quantification of analytes in samples at that place have been really few other methods that have been developed to observe Protein A. Before the usage of the ELISA method for the quantification of Protein A there were methods present which allowed for the sensing but non the quantitation of the sum of SpA in a sample. One of the original methods was the single-tube assorted agglutination trial. This was a rapid assorted agglutination trial where sheep erythrocytes which had been sensitized to SpA were assorted with the sample and the presence of agglutination meant a positive consequence for the presence of SpA ( Peter E. Maxim et al. , 1976 ) . This trial nevertheless is of small usage as it does non let the sum of SpA nowadays to be quantified. It could nevertheless be used as a preliminary trial though consequences would non be of the criterion required within industry.

In 1999 Ihab Abdel-Hamid et al. , described a fresh method for the finding of SpA whereby an amperometric immunosensor is used which consists of C molecules that have been dispersed and conjugated with anti-protein A antibodies. Its uses the amperometric immunosensor combined with a sandwich check strategy utilizing peroxidase-labelled anti-IgG antibodies for the quantitation of the SpA in the sample giving a sensing bound in the nanomolar scope. This system allows for the mechanization of the sensing of Protein A and besides allows for much faster finding with an assay clip every bit low as 22mins. The system can besides be arranged as a portable device to let for point to indicate sensing throughout the fabrication procedure giving a more accurate appraisal of which point in the system is doing the leaching of the SpA into the merchandise ( Ihab Abdel-Hamid et al. , 1999 )