NZ215967A - Composition of peptides of known weight for determining weight of unknown peptide - Google Patents

Description

21 5967 Priority Date(s): ..

Ccvp'ete Specification Filed: - C&1KAS/J.2- : i - - CO L .. 3..QMAY. 198ft... • - . „ ; :: No.: Date: COMPLETE SPECIFICATION "NOVEL MOLECULAR WEIGHT STANDARDS AND METHODS OF USE THEREOF" ?/We, COMMONWEALTH SERUM LABORATORIES COMMISSION, a body corporate established under the Commonwealth Serum Laboratories Act 1961, of 45 Poplar Road Parkville, in the State 'of Victoria, Commonwealth of Australia, hereby declare the invention for which K/ we pray that a patent may be granted to 143S&/us, and the method by which it is to be performed, to be particularly described in and by the following statement:- (followed by page -la-) 2 1 59 1CK "NOVEL MOLECULAR WEIGHT STANDARDS AND METHODS OF USE THEREOF" This invention relates to compositions for use in the determination of molecular weights of proteins, and to methods utilising such compositions.

The technique of Western transfer and protein blotting of electrophoretically separated protein mixtures is now widely applied in biological sciences. The technique employs electrical current to effect the transfer of proteins resolved on polyacrylamide (or other polymeric) gels onto the surface of treated semi-rigid or rigid membrane or paper support which strongly bind the proteins. The technique was discussed by S.Gershoni and G.Paladie (Anal.Bioch. (1983) 131, 1-15). Methods to classify the transferred proteins according to their molecular weight require that suitable protein standards of known molecular weight are also simultaneously transferred from the same, adjacent or non-contiguous regions of the separation gel onto the same, adjacent or non-contiguous regions of the treated semi-rigid or rigid paper or membrane support. The region of paper or membrane support containing electrophoretically transferred molecular weight markers is removed and stained for protein. Molecular weight i * 215967 markers suitable for the above methods are commercially available from several companies.

The present invention provides new molecular weight markers which offer the following advantages over the known protein molecular weight markers: (i) they can be detected on all supports. Protein staining methods cannot be used on all supports (e.g. cationized nylon membranes) because staining reagents also stain the support thus preventing identification of molecular weight markers. (ii) detection of molecular weight markers and sample protein is in general simultaneous and only one procedure is required. A separate staining procedure is required when other protein molecular weight markers are used. (iii) detection of markers does not require the removal of lanes containing the markers from the rest of the support. Supports containing other protein molecular weight markers must be removed for the separate detection procedure - these procedures can affect the dimensions of the support making it difficult to align immunodetected components with the molecular weight markers. 14 A series of C labelled protein molecular weight markers are available from Amersham (Buckinghamshire, England). These markers can be detected simultaneously with immunodetected components but they are expensive and can only be used when the 1 i T^irT Sti-.' 21 5967 3 immunodetection procedure utilises radioactive labels (e.g. radiolabelled antibodies, or Protein A).

According to a first aspect of this invention, there is provided a composition for use in the determination of the molecular weight(s) of protein(s), which comprises at least one protein or polypeptide of known molecular weight selected from the group consisting of the immunoglobulins and fragments and polymers thereof (hereinafter referred to as Ig Components).

Whilst the present invention extends to the use of all classes of immunoglobulins, including for example immunoglobulin A and immunoglobulin M, presently preferred compositions in accordance with this invention are compositions comprising at least one protein or polypeptide of known molecular weight(s) selected from the group consisting of immunoglobulin G and fragments and polymers thereof (hereinafter referred to as IgG Components).

Preferably, the composition of this aspect of the invention will comprise two or more Ig Components, preferably in equal amounts, and typically at least four or five such components. In such preferred compositions, the Ig Components are selected so as to have calibrated molecular weights falling at intervals, preferably at approximately regular intervals, over a range of molecular weights. By way of example, IgG Components may be selected to cover the range of approximately 200 - 25 KDa, preferably with regular molecular weight intervals between the components. The 2 159 4 Ig Components may be derived from different animal species as described in greater detail below.

In this aspect, the invention thus provides a composition comprising proteins of defined molecular weight derived, from different Ig classes, preferably from immunoglobulin G (gammaglobulin), which can be used to provide molecular weight markers on SDS-polyacrylamide gels and on Western Blots. When used to provide molecular weight markers on Western Blots the composition of the invention allows simultaneous detection of antigen(s) and molecular weight standards thus providing a measure of the efficiency of the transfer and detection procedure when markers of the same or cross-reacting species to the antigen(s) species are used.

The range of molecular weight components in the compositions of this invention may be obtained by fragmentation of an immunoglobulin molecule such as the IgG molecule by known methods into the various combinations of heavy and light chain components and other components by partial or full reductive alkylation, enzymatic digestion, chemical cleavage with polymerisation of the various fragments where necessary.

In another aspect, this invention provides a method for the detection and/or determination of the molecular weight(s) of one or more sample proteins, which method comprises the steps of: a. simultaneously subjecting the sample protein(s) and a composition of the present invention as described « 2159 above to electrophoretic resolution on a polymeric gel; and b. detecting and comparing the resolved sample protein(s) and Ig Components in order to detect and/or determine the molecular weight(s) of the sample protein(s).

The step of electrophoretic resolution may be conducted under one- or two-dimensional conditions. Preferably, a sodium dodecyl sulphate-polyacrylamide gel is used for this resolution.

Detection of the resolved sample protein(s) and Ig Components may be performed directly in the polymeric gel. Alternatively, an intermediate transfer step may be performed whereby the resolved materials are transferred onto a separate support, with the step of detection and comparison being performed on the transferred materials, for example by electrophoretic transfer onto a semi-rigid or rigid support following the technique of Western transfer and protein blotting described above.

Because of the favourable characteristics of the immunoglobulin fragments, and particularly IgG ~\ fragments, they can be electrophoretically transferred onto semi-rigid or rigid polymeric films where they can be detected using a variety of reagents including protein A, enzyme-linked anti-immunoglobulins (intact, H- or L- chain), and radio-labelled analogues. By using immunoglobulins and fragments thereof from various and j appropriate species, including human, mouse, goat, bovine, rabbit etc., this method thus allows reliable 21596? 6 molecular weight determinations and also quantification of transfer and immunodetection efficiency. By way of example, human immunoglobulin fragments can be used in detection systems where anti-human immunoglobulins are employed as second antibodies.

Starting materials for the preparation of the preferred IgG Components of the composition of this invention include purified gammaglobulin from a variety of animal species (e.g. human, rabbit, rat, mouse, goat etc.) derived directly from blood or monoclonal sources. Preferably the starting material consists only of the major subtype of IgG where applicable.

Dissociation of gammaglobulin chains into combinations of heavy (H) and light (L) polypeptides can be achieved by incubating the gammaglobulin with a reducing agent (e.g. dithiothreitol, mercaptoethanol) over a range of concentrations and subsequently alkylating the reduced proteins with a suitable alkylating reagent such as iodoacetamide or iodoacetic acid. This produces stable mixtures of IgG L and H chain monomers, dimers, trimers and tetramers of the combinations H2L, , HL, H and L with varying abundance which can be used directly for molecular weight calibration. Alternatively, the components in these mixtures can be isolated by a variety of chromatographic methods including gel filtration (size exclusion) ion exchange chromatography and affinity chromatography such as with Protein A as the ligand. For reasons of detection efficiency, the preferred reagent is obtained by appropriate reconstitution of these combinations to form a product with equal amounts of these components. Alternatively, following 2 1 5967 7 isolation, these components may be treated with various cross-linking reagents, for example with formaldehyde or glutaraldehyde, the bisoxirans, or with bifunctional reagents including para-azidophenacyl bromide, the N-succinimidyl esters (e.g. N-succinimidyl (4-iodoacetyl) aminobenzoate; succinimidyl 4-(N-maleimidomethyl) cyclohexane-^l-carboxylate) and the C6-8 alkandiimidates (e.g. dimethyladipimidate; dimethyl pimelimidate, etc.), over a range of concentrations to produce oligomeric IgG fragments. By reconstituting these mixtures a product may be obtained with equal mass of each of these different oligomers.

Molecular weight markers can also be generated from this immunoglobulin G starting material by enzymatic digestion. In this procedure IgG is digested with various proteolytic enzymes (e.g. papain, pepsin, plasmin) to yield the desired fragments. IgG fragments of different molecular weight are then isolated. The isolation can be achieved by a variety of methods including gel filtration, ion exchange chromatrography and affinity chromatography such as Protein A affinity chromatography. Following isolation the fragments may be treated with a crosslinking reagent (e.g. glutaraldehyde, para-azidophenacyl bromide, etc.) over a range of concentrations to produce mixtures of IgG fragments with varying degrees of polymerization. Once again, by reconstituting these mixtures a product with equal amounts of each molecular weight component is generated.

Alternatively, IgG fragments may be generated by other methods of chemical fragmentation (e.g. using cyanogen bromide), then processed and crosslinked as 21 59 8 described for the alkylated and enzymatically derived fragments.

IgG fragments obtained by one or any of the above methods may be mixed to obtain a product with the desired mixture of fragments.

As described above, the preferred gaitunaglobulin derived molecular weight markers of this invention can be used on SDS-polyacrylamide gels as standards. Similarly, following transfer, they function as standards on "Western Blots". The products can be used on all SDS-PAGE systems (e.g. Reisfeld, R.A., Lewis, U.J. and Williams, D.E. Nature (1962) 195 281-283; Laemmeli, U.K., Nature (1970) 227 680-685; Weber, K. and Osborne, M., J.Biol.Chem. (1969) 244 4406-4412) which require molecular weight markers.

The immunoglobulin derived molecular weight markers in accordance with this invention can be chosen from any species of Ig when used as molecular weight standards on SDS-PAGE gels. For use as molecular weight standards on Western Blots, the species of Ig used is important. In these procedures, sample protein(s) and Ig Components as molecular weight standards are electrophoresed on an SDS polyacrylamide gel and electrotransferred to a charged or reactive support (e.g. nitrocellulose or nylon membranes or diazophenylthio paper) to produce a "Western Blot". Reactive or charged sites on the support that are not occupied are blocked with a blocking buffer usually containing a detergent (e.g. Triton X-100, Tween 20) and/or protein (e.g. gelatin, albumin), then the support is incubated with buffer containing antibody (the 2 1 5967 9 primary antibody) directed against the required antigen. The Ig Components used as molecular weight standards should be of the same species as the primary antibody. Bound primary antibody and the Ig Components can be detected by incubating the blot with a variety of reagents including labelled or enzyme conjugated Protein A, or a labelled or enzyme conjugated secondary antibody directed against the species Ig of the primary antibody. Thus simultaneous detection of antigen and molecular weight markers is achieved. Such use of immunoglobulin derived molecular weight markers also provides a measurement of the sensitivity of the immunodetection and transfer procedures.

The following Examples further illustrate the compositions and methods of the present invention, in association with the accompanying Figures, in which: Figure 1 is a calibration curve for IgG Molecular Weight Standards against conventional molecular weight standards (M, H and R refer to approx. 5yg loads of inununo standard from mouse, human and rabbit IgG respectively, and these are bordered by 5yg loads of low and high molecular weight standards respectively of a commercial reagent (Pharmacia); and Figure 2 illustrates the use of the IgG molecular weight standards in a Western blotting procedure. A description of the protocols of the various tracts is to be found in Example 4.

EXAMPLE 1 Human gammaglobulin from Cohn Fraction II paste was reduced with dithiothreitol according to the following procedure: 2159 IgG at 2.5 mg/ml was incubated with 0.5raM and 5mM dithiothreitol. The reactions were allowed to proceed for 30 minutes at room temperature and stopped by addition of iodoacetamide to give a final concentration of iodoacetamide of lOmM. Alkylation was allowed to proceed for 10 minutes at room temperature. Solutions were then dialysed at 4°C in 50mM Tris/Cl, 50mM NaCl pH 8.0. Untreated gammaglobulin and reduced and alkylated gammaglobulin were then subjected to SDS-PAGE according to the method of Laemmeli on a 10% gel. 2ug of protein was loaded per sample. The gel was then stained for protein by the silver staining method of Wray. (Wray W., Boulikas, T., Wray, V.P. & Hancock, R. Analytical Biochem. (1981) 118 197-203).

The silver stain revealed that all possible breakdown products from the reduction and alkylation of gammaglobulin were generated using both concentrations of dithiothreitol, and corresponded to H2L2, H2L, H2, HL, H and L chains. Plots of the logarithm of molecular weight versus relative mobility demonstrated linear dependencies for the various IgG Components, which included components of molecular weight of 150, 125, 100, 75, 50 and 25 KDa respectively.

EXAMPLE 2 Gammaglobulin fragments were prepared and electrophoresed as described in Example 1 and electrotransferred to nitrocellulose according to the method of Towbin (Towbin, H., Staehelin, T., Gordon, J. Proc.Natl.Acad.Sci.USA (1979) 76 4350-4354) . Electrotransfer was carried out for 3 hours at 70 volts 2 159 l r> 11 at 2°C. Following transfer the nitrocellulose membrane (NCM) was incubated at 20mM Tris/Cl pH 7.5, 0.05% Triton ^ X-100, 5% gelatin, 150mM NaCl buffer (Buffer A) for 60 minutes at room temperature then incubated in 5ml of Buffer A containing 2 x 10 counts per minute per mL of labelled Protein A. The NCM was then given three ten minute washes in Buffer A, was thoroughly rinsed with 125 water and then dried. Bound I -Protein A was then detected by autoradiography.

Five discreet bands were detected and corresponded to 112^2' H2L' **2' an(^ H Co,nPonents gammaglobulin.

EXAMPLE 3 The gamma globulin fragments were obtained from IgG from three species (human, mouse and rabbit) as follows:- Human IgG was isolated from myeloma serum by Protein A Sepharose and consisted mainly of igG^. Aliquots of protein concentration 3.75 mg/mL were made ImM and 4mM with respect to dithiothreitol and incubated at room temperature for 30 minutes. The reduction reaction was stopped by the addition of iodoacetamide (final concentration 8mM) and after ten minutes at room temperature, the solutions were dialysed against 50mM Tris/HCl, 50mM NaCl pH 8.0. Following preliminary analysis on 5-16% polyacrylamide gels in the presence of V SDS and visualization of the proteins by the silver staining method of Wray (Wray, S., Boulikas, T., Wray, V.P. and Hancock, R., Analytical Biochem. (1981) 118 197-203) , the two solutions were blended to produce a reagent with approximately equal quantities of all / possible combinations of light and heavy chains. c 2159 v_..- 12 Mouse monoclonal IgG was Isolated from ascites fluid by Protein A Sepharose. Aliquots of protein concentration 0.2 mg/mL were made 0.25, 1 and 4mM with respect to dithiothreitol and incubated at room temperature for 30 minutes. The reduction reaction was stopped by the addition of iodoacetamide (final concentration 8mM) and after ten minutes at room temperature, the solutions were dialysed against 50mM Tris/HCl, 50mM NaCl pH 8.0. Following preliminary analysis on 5-16% polyacrylamide gels in the presence of SDS and visualization of the proteins by the silver staining method of Wray, the three solutions were blended to produce a reagent with approximately equal quantities of all possible combinations of light and heavy chains.

Rabbit IgG was isolated from serum by Protein A Sepharose and divided into three aliquots. One aliquot was made 16mM with respect to dithiothreitol and incubated at room temperature for 30 minutes.

Alkylation was effected by making the solution 32mM with respect to iodoacetamide and leaving at room temperature for ten minutes. The solution was then dialysed against 50mM Tris/HCl, 50mM NaCl pH 8.0. The second aliquot was incubated with papain (Worthington 28u/mg) in the presence of lOmM cysteine at 37° for 1 1/2 hours. The reaction was stopped by the addition of iodoacetamide and the solution dialysed against 50mM Tris/HCl, 50mM NaCl pH 8.0. The Fc fragments of the digested IqG were isolated on Protein A Sepharose and the product dialysed against 50mM phosphate 145mM NaCl pH 7.0. This material was reduced by making the solution 14mM with respect to dithiothreitol and incubating at room temperature for 30 minutes. Alkylation was effected by making the solution 30mM with respect to iodoacetamide followed by dialysis * © 2 J 59 13 against the pH 8.0 tris buffer. The third aliquot was fe not treated further. All three aliquots were examined I by SDS-PAGE and silver stained as for the human and mouse IgG. From this data, a blend was made to produce a mixture containing native and fragmented IgG.

Aliquots of the blends of the components from the .-7* three species, prepared as described above, were run on i a 5-16% polyacrylamide gel, in parallel with commercial I ' high and low molecular weight standards (Pharmacia). f The proteins were visualized by silver staining and the result is illustrated in Fig.l. i A calibration curve was constructed of versus r log molecular weights of the standards and, from this curve, it was found that the IgG fragments had the following molecular weights:-;■ Mouse 130 , 122, 108, 81 , 46 and 22 Human 135, 127, 113, 86, 47 and 22 Rabbit 122, 43, 25 EXAMPLE 4 Two 5-16% polyacrylamide gels were prepared with slots to accommodate single samples at each side and with the rest of the gel dedicated to a single sample. 5pg of the mouse immuno standard (described in Example 3) in SDS buffer was added to the two single side tracks on one gel and 5ug of the human immuno standard was added to these tracts on the other gel. 80pg (in 200jj1) of an extract of B.Pertussis (Whooping Cough) bacteria was loaded on to the middle, wide track of each gel. The gels were run at 36m.amp constant current for approximately 2 1/2 hours. The separated proteins were then transferred to nitrocellulose membranes by a 21 14 constant voltage of 70 volts for 16 hours. The nitrocellulose membranes were cut into strips and transferred to BioRad incubation trays. The strips containing B.Pertussis antigens were probed with various mouse and human antibodies. All strips (including immuno standards) were finally probed with either Horse Radish Peroxidase (HRP)-conjugated goat anti-mouse IgG . or with HRP-conjugated goat anti-human IgG, depending on i ' the species from which the primary antibody and the f. immuno standards had been derived.

J Fig.2 shows the results obtained. Tracks 1-4 correspond to B.Pertussis extract probed with (1) mouse Vj hyperimmune (vaccine) serum, (2) mouse polyclonal serum raised against a preparation of filamentous haemagglutin (FHA) purified from B.Pertussis extract, (3) mouse monoclonal anti-FHA and (4) mouse polyclonal L» anti-pertussigen (major B.Pertussis toxin). The two ! bridging tracks correspond to the mouse immuno standard.

| Tracks 5-7 correspond to B.Pertussis extract probed with | three different human sera at 1/100 dilution; from (5), a patient suspected of having Whooping Cough, (6) a healthy vaccinated individual and (7) a pool of sera from patients known to have low anti-FHA and anti-pertussigen titres by ELISA tests. These are bridged by the human immuno standard.

It will be seen from the mouse panel that the IgG molecular weight (immuno) standards provide a useful range of molecular weight species to determine the molecular weights of the antigen recognised by the various sera. From the human panel, it is obvious that the overall immunodetection is rather weak, illustrating the usefulness of the standards in checking the O 215967 e effectiveness of the electrophoresis, transfer and immunodetection stages. <?■ v. o

Claims (14)

215967 16 WHAT UE CLAIM IS

1. A composition for use in the determination of the molecular weight(s) of protein(s), which comprises at least one protein or polypeptide of known molecular weight selected from the group of Ig Components consisting of the immunoglobulins and fragments and polymers thereof.

2. A composition according to claim 1, wherein said at least one protein or polypeptide of known molecular weight is selected from the group of IgG Components consisting of Immunoglobulin G and fragments and polymers thereof.

3. A composition according to claim 1 or claim 2, which comprises at least two said proteins or polypeptides of known molecular weight.

4. A composition according to claim 3, which comprises at least four said proteins or polypeptides of known molecular weight.

5. A composition according to claim 3, wherein said proteins or polypeptides of known molecular weight are present in approximately equal amounts in said composition.

6. A composition according to claim 3, wherein said proteins or polypeptides of known molecular weight are selected so as to have molecular weights falling at regular intervals over a range of molecular weights. 2 1 59 17

7. A composition according to claim 2, wherein said proteins or polypeptides of known molecular weight comprise the HL, H and/or L chains of immunoglobulin G. ■.-s

8. A method for detection and/or determination of the molecular weight(s) of one or more sample proteins, which method comprises the steps of: a. simultaneously subjecting the sample protein(s) and a composition as defined in claim 1 to electrophoretic resolution on a polymeric gel; and b. detecting and comparing the resolved sample protein(s) and Ig Components in order to detect and/or determine the molecular weight(s) of the sample protein(s).

9. A method according to claim 8, wherein said polymeric gel is a sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE).

10. A method according to claim 8, wherein the detection of the resolved sample protein(s) and Ig Components is performed directly in said polymeric gel.

11. A method according to claim 8, wherein prior to said detection step, the resolved sample protein(s) and Ig Components are transferred to a separate support, and the detection step is performed on the transferred materials. O

12. A method according to claim 11, wherein the resolved sample protein(s) and Ig Components are transferred by electrophoretic transfer onto a semi-rigid or rigid, charged or reactive support. 215967 H f : y

13. A composition as claimed in any one of claims 1-7 substantially as hereinbefore described with reference to any example thereof and/or any of the accompanying drawings.

14. A method as claimed in any one of claims 8-12 when performed substantially as hereinbefore described with reference to any example thereof and/or any of the accompanying drawings. u.\) .1) i , Or djfPC\ A. J. PARK & SON per agents FOR THE APPLICANTS