Use Of Antipeptide Antibodies Research Articles

ANTI-PEPTIDE ANTIBODIES IN THE ERA OF MONOCLONALS: APPLICATION IN DIAGNOSTICS AND RESEARCH

The areas of application of antipeptide antibodies in which the use of monoclonal antibodies is impossible or unpromising are considered. Examples of successful use of antipeptide antibodies in the diagnostics of tumor tissue damage, studies of metabolic modification and conformational transitions of proteins are given.

Replacing Immunoassays With Tryptic Digestion-Peptide Immunoaffinity Enrichment And Lc–MS/MS

For decades, immunoassays have provided the framework for protein biomarker studies in clinical medicine and in therapeutic monitoring for drug development. At the same time, investigators have uncovered many issues that make immunoassays unreliable in many human serum and plasma samples. LC-MS/MS after tryptic digestion of proteins is potentially an attractive solution, but the sensitivity of the method is not sufficient to measure many important low-abundance proteins directly. The use of antipeptide antibodies to immunoenrich peptides of interest can improve the sensitivity of the approach, greatly simplify the matrix enabling shortened chromatographic runs, and facilitate the multiplexed quantification of analytes, which could reduce the costs of quantitative protein measurements in complex specimens. We provide an overview of the method and the steps needed to develop an assay. In addition, we review the efforts to make this method generally more applicable.

Peptide‐Specific Antibody as a Tool to Evaluate TRPM3 Ion Channel Function

Calcium channel blockers can be used to selectively inhibit calcium influx across a membrane, making them potentially useful as therapeutic tools. Existing transient receptor potential (TRP) channel blockers are relatively non-specific, but have previously been used to characterise individual members of the TRP family. The activation of TRP channels can be more specifically inhibited through the use of anti-peptide antibodies targeted to particular regions of the channel structure (Xu & Beech, 2001. Circ. Res. 88:84–87). The polyclonal antibody TM3E3 was designed to specifically bind to a peptide corresponding to the 3rd extracellular loop of TRPM3, a member of the melastatin-like TRP family. The location and sequence of the peptide were determined using hydrophilicity analysis of the channel sequence (Xu et al., 2005. Nat. Biotechnol. 23:1289–1293). Here we use Ca2+-imaging to study the specificity of TM3E3 for human TRPM3 expressed in HEK-293 cells. Extracellular application of the antibody has a functional effect, inhibiting the sphingosine-induced activation of TRPM3. It does not inhibit activation of the closely related channel TRPM2 or the canonical family member TRPC5. TM3E3 will serve as a useful tool for further characterisation of endogenous TRPM3 in a variety of cells and tissues, and more importantly the results strengthen the idea that E3 targeting may be used to generate specific ion channel blockers for a variety of channels, with therapeutic potential. This work is supported by the BBSRC, the Welcome Trust and the BHF. Many thanks to C. Harteneck for providing the TRPM3 clone.

Early effects of salinity on water transport in Arabidopsis roots. Molecular and cellular features of aquaporin expression.

Aquaporins facilitate the uptake of soil water and mediate the regulation of root hydraulic conductivity (Lp(r)) in response to a large variety of environmental stresses. Here, we use Arabidopsis (Arabidopsis thaliana) plants to dissect the effects of salt on both Lp(r) and aquaporin expression and investigate possible molecular and cellular mechanisms of aquaporin regulation in plant roots under stress. Treatment of plants by 100 mm NaCl was perceived as an osmotic stimulus and induced a rapid (half-time, 45 min) and significant (70%) decrease in Lp(r), which was maintained for at least 24 h. Macroarray experiments with gene-specific tags were performed to investigate the expression of all 35 genes of the Arabidopsis aquaporin family. Transcripts from 20 individual aquaporin genes, most of which encoded members of the plasma membrane intrinsic protein (PIP) and tonoplast intrinsic protein (TIP) subfamilies, were detected in nontreated roots. All PIP and TIP aquaporin transcripts with a strong expression signal showed a 60% to 75% decrease in their abundance between 2 and 4 h following exposure to salt. The use of antipeptide antibodies that cross-reacted with isoforms of specific aquaporin subclasses revealed that the abundance of PIP1s decreased by 40% as early as 30 min after salt exposure, whereas PIP2 and TIP1 homologs showed a 20% to 40% decrease in abundance after 6 h of treatment. Expression in transgenic plants of aquaporins fused to the green fluorescent protein revealed that the subcellular localization of TIP2;1 and PIP1 and PIP2 homologs was unchanged after 45 min of exposure to salt, whereas a TIP1;1-green fluorescent protein fusion was relocalized into intracellular spherical structures tentatively identified as intravacuolar invaginations. The appearance of intracellular structures containing PIP1 and PIP2 homologs was occasionally observed after 2 h of salt treatment. In conclusion, this work shows that exposure of roots to salt induces changes in aquaporin expression at multiple levels. These changes include a coordinated transcriptional down-regulation and subcellular relocalization of both PIPs and TIPs. These mechanisms may act in concert to regulate root water transport, mostly in the long term (> or =6 h).

Production of antibodies against degradative neoepitopes in aggrecan.

The use of synthetic peptides to generate rabbit polyclonal anticatabolic neoepitope antibodies that can be used to study the presence of defined proteolytic cleavage sites in aggrecan is described. Principles of peptide design and methods for preparation and characterization of ovalbumin conjugates are presented along with approaches for the characterization and affinity purification of the resulting antisera. Limitations associated with the use of antipeptide antibodies to study authentic protein neoepitopes are discussed.

Assessment of P450 induction in the marmoset monkey using targeted anti-peptide antibodies

The identity and expression of hepatic P450 enzymes in marmosets was investigated using a panel of anti-peptide antibodies originally targeted against human P450 enzymes. In immunoblotting, of 12 antibodies examined, 10 bound specifically to bands in marmoset liver microsomal fraction corresponding to P450 enzymes. It is proposed that these represent marmoset CYP1A1, CYP1A2, CYP2A, CYP2B, CYP2C forms (CYP2C-1 and CYP2C-2), CYP2D19, CYP3A21 and another CYP3A form (CYP3A-m). The antibodies, together with an anti-marmoset CYP2E1 antibody, were used to investigate the expression of 10 P450 enzymes in marmosets treated with P450-inducing chemicals. Treatment with phenobarbitone caused CYP2B, CYP2C-2 and CYP3A21 levels to increase, rifampicin caused increases in CYP2B and CYP2C-1 and a decrease in CYP3A21 levels, whereas dioxin caused CYP1A1 and CYP1A2 levels to increase and CYP2E1 levels to decrease. Clofibric acid did not induce any P450. P450 enzyme activities were assessed using 8 different substrates and increases were found after treatment with phenobarbitone, rifampicin, and dioxin. However, due to species differences in substrate selectivity, it proved difficult to ascribe these changes to individual P450 enzymes. Thus, the use of anti-peptide antibodies provides a more informative way of assessing the levels of specific P450 enzymes than enzyme activity measurements.

Molecular cloning and characterization of HE1, a major secretory protein of the human epididymis.

The cDNA sequence of HE1, a novel human epididymal gene product isolated by differential screening, predicts an abundant, small secretory glycopeptide. HE1 is encoded by a single copy gene and seems to be well conserved among mammals. The predicted HE1 peptide is identical to that of EPI-1, the chimpanzee homologous gene product, and to ESP14.6, the macaque homologous product. Its tentative N-terminus also shows similarity to that of a major porcine epididymal sperm-associated glycopeptide. Northern blot analysis of various bovine and rat tissues indicated that their HE1-homologous gene products showed highly increased expression in the epididymis, suggesting that the tissue distribution observed in humans is also conserved. With use of antipeptide antibodies, HE1-related antigen was shown by immunohistochemical methods to be present within the epithelium and lumen of the human corpus epididymidis. High amounts were also present in the lumen of the cauda epididymidis and deferent duct. Moreover, HE1 seemed to be associated with epididymal spermatozoa, and association with ejaculated spermatozoa remained doubtful. With Western blotting, an antiserum raised against the synthetic HE1 epitope specifically cross-reacted with proteins of human seminal plasma in the range of 25-27 kDa. An antiserum raised against chimpanzee EPI-1 recognized human seminal plasma antigens of the same apparent molecular size.

GPIIIa(90-102) and GPIIIa(631-653) epitopes as markers of conformational changes occurring during the activation of the glycoprotein IIb/IIIa complex.

To evaluate the range of conformational changes in the GPIIIa subunit upon the interaction of GPIIb/IIIa complex with fibrinogen, we employed two polyclonal antipeptide antibodies against synthetic peptides spanning chain segments (90-102) and (631-653) of GPIIIa. The first peptide fragment derives from the region preceding the putative binding site for the RGD sequence, while the second one is located in the vicinity of membrane lipid bilayer. The resting platelets bound about 13,700 and 11,500 molecules of anti-[GPIIIa(90-102)] and anti-[GPIIIa(631-653)] antibodies, respectively. The anti-[GPIIIa(90-102)] antibodies preferentially bound to the active form of GPIIb/IIIa complex. This binding was significantly reduced after dissociation of the complex, indicating that the recognized epitopes became partially hidden in the GPIIIa subunit. In contrast to anti-[GPIIIa(90-102)], the anti-[GPIIIa(631-653)] antibodies reacted optimally with the EDTA-treated GPIIb/IIIa. Upon stimulation of platelets with ADP, the number of binding sites anti-[GPIIIa(631-653)] was reduced 2.8-fold. The binding affinity of anti-[GPIIIa(90-102)] antibodies to the resting and ADP-stimulated platelets differed 4.7-fold, indicating that the stimulation of the platelets with ADP resulted in better exposure of this epitope. This binding was inhibited almost totally by fibrinogen in a dose-dependent fashion. Similarly, fibrinogen inhibited binding of the anti-[GPIIIa(631-653)] antibodies to the platelets. In the reversed system, binding of fibrinogen to platelets was inhibited only to 60% by the anti-[GPIIIa(90-102)] antibodies. Such inhibition of fibrinogen binding was sufficient to block aggregation of ADP-stimulated platelets by the anti-[GPIIIa(90-102)] antibodies in platelet-rich plasma. In contrast, the anti-[GPIIIa(631-653)] potentiated fibrinogen binding by 20%, confirming that there were additional interacting sites for fibrinogen in other regions of the GPIIb/IIIa complex. The presented data indicate that the activation of the fibrinogen receptor is associated with extensive conformational changes in the GPIIIa subunit, detectable by use of antipeptide antibodies recognizing not only the epitopes located next to the putative ligand-binding site but also the epitopes in the vicinity of the platelet membrane lipid bilayer.

Detection of cells producing murine interferon‐α using antipeptide antibodies

The purpose of this study was to produce antibodies which could be used to investigate the expression of murine (Mu)IFN-alpha. Rabbits were immunized with a peptide, corresponding to the 15 COOH-terminal amino acids of MuIFN-alpha-1, conjugated to keyhole limpet haemocyanin (KLH), and the resulting antipeptide antibodies were identified by indirect ELISA. Antipeptide antibodies were purified from rabbit immune sera by immunoadsorption to peptide immobilized on nitrocellulose and any remaining antibodies to KLH removed by immunoadsorption to KLH-Sepharose. The characterization of the antipeptide antibodies by ELISA, immunoprecipitation, affinity chromatography and immunofluorescence demonstrated that the antibodies recognize the peptide immunogen and the native IFN-alpha molecule. Using these antibodies for immunofluorescence staining and flow cytometric analyses of stained cells, we have shown that unstimulated murine spleen cells produce IFN-alpha. This finding is in agreement with the recent demonstration of constitutive IFN-alpha production by unstimulated human leucocytes and has important implications for the functions of interferons. The production, characterization and use of antipeptide antibodies as described herein may also have broader application for studies of the expression of other cytokines.

Galactocerebrosidase from human urine: Purification and partial characterization

Galactocerebrosidase (GALC, EC 3.2.1.46) was purified from human urine by a series of hydrophobic affinity column chromatography steps. The activity was enriched 176000-fold from concentrated urine by only four columns, including octyl Sepharose, hydroxylapatite, butyl Sepharose and ethyl-agarose. The overall recovery was about 20% but only low amounts were obtained due to its low abundance. The estimated final specific activities of several batches were between 1 and 2 mmol/h per mg protein. The final purified fractions were essentially free of other lysosomal enzyme activities. The most pure fractions showed a series of bands between 50 and 53 kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis which were determined to have identical N-terminal amino acid sequence. In addition, gel filtration of partially purified GALC after disassociation showed one peak of activity estimated to have a molecular mass near 50 kDa. GALC was also purified from human brain and human placenta using the same methods demonstrating the usefulness of this procedure in obtaining GALC from solid human tissues. In addition to the bands migrating near 50 kDa from urine, there were also bands at 80 kDa and 30 kDa in some preparations. By N-terminal sequencing and the use of antipeptide antibodies, the 80 kDa band was demonstrated to have the same N-terminal amino acids as the 50−53 kDa bands. The 30 kDa band had a unique sequence. The relationship between the different molecular weight species remains to be determined. The purification of GALC and the securing of amino acid sequence information will aid in the cloning of the GALC gene. This enzyme is deficient in human patients with Krabbe disease and several animal species.

A High Temperature Transfer Procedure for Detection of G Protein γ Subunits by Immunoblotting

The production of antibodies against synthetic peptides derived from amino acid sequences common or unique to a particular protein(s) is an important tool in the identification of structurally related members of an ever-increasing number of protein families. The successful use of anti-peptide antibodies requires that the protein(s) of interest be properly transferred and fully denatured prior to detection by Western-type immunoblotting. In this paper, we demonstrate that conventional transfer procedures are not successful in presenting the G protein γ subunits in a suitable state for immunodetection. We describe a high temperature (70°C) transfer procedure that results in a more than 20-fold enhancement in the sensitivity of immunodetection of the various G protein γ subunits. The effect of high temperature transfer could not be duplicated by including 0.2% SDS in the buffer during transfer to nitrocellulose, or by baking or autoclaving the nitrocellalose after transfer. Thus, high temperature transfer is a powerful procedure for enhancing immunoblot detection of protein(s) that may be resistant to denaturation and/or subject to renaturation during the transfer and/or binding to nitrocellulose.

Use of anti-peptide antibodies for the design of antigen-specific immune complex assays

A simple and sensitive method is described for the detection of circulating immune complexes (ICs) in an antigen-specific manner. The method is based on the use of anti-peptide antibodies as solid-phase capture reagents to bind antigen which is complexed to serum antibodies. The bound serum antibody is detected with a labelled second antibody. The method requires that the anti-peptide antibodies bind native protein efficiently, and that the anti-peptide antibodies do not compete with antibodies raised against the native protein which are involved in IC formation. Two anti-peptide antibodies specific for the hepatitis B surface antigen (HBsAg) and the hepatitis B e antigen (HBeAg), which possessed the requisite characteristics, were chosen as models for IC assay development. The solid-phase, anti-peptide based assays efficiently detected HBsAg and HBeAg-containing ICs in preformed antigen/antibody mixtures and in the serum of chronically infected hepatitis B patients.

Specific immunodetection of G-proteins in plant cell membranes

At present, much is known concerning the biochemistry of heterotrimeric G-proteins in animal cells [l] and lower eukaryotic cells, e.g. Saccharomyces cerevisiae 121 and many examples of Ga, GP and G8 subunits have been identified by cloning and sequencing studies. The a-subunits, for which most examples have been discovered, show a number of highly conserved regions, which are concerned with GTP binding and hydrolysis and other less conserved regions which are responsible for coupling to receptor and effector molecules respectively. Synthetic peptides which correspond in sequence to these regions have been used to generate antisera which are panspecific to Ga subunits (Gac antisera) or specific to the various GQ subtypes, e.g. Gia, Gsa [3,41. In contrast, no G-proteins have yet been isolated from plant cells. However, for some time there has been strong circumstantial evidence indicating their existence and involvement in plant signal transducing pathways. Non-hydrolysable GTP analogues were shown to modulate inositol phospholipid turnover in Acer pseudoplatinus cells [5] and it has been suggested that auxins initiate G-protein coupled events in plant cells [6]. In addition, microsomal membrane and highly resolved plasmalemma preparations from various species including Arabidopsis thaliana [7] and Cucurbita pep0 [8] have been shown to bind GTPVS with high affinity (Kb= 10-SM) and to possess polypeptides of M= 31-37 kDa that crossreacted with Gac antisera. Finally, genes for a Ga subunit and a GP subunit homologs have been cloned and sequenced in A . thaliana [9] and Chlamydamonas rheinhardtii [lo] respectively. In the present communication we report the specific immunodetection of Ga subunits in plant cell membrane preparations and comment on the use of antipeptide antibodies. A number of antisera and affinity purified antibodies directed against the Gac, Gia, Goa and GP sequences [3,4] were used to probe immunoblots of microsomal fraction and plasmalemma membrane proteins prepared from higher plants. Typical positive data from these studies are shown in Fig.1. Microsomal membranes from A. thaliana . and Pisum sativum were shown to possess polypeptides of Mr 37 kDa (tracks 1,3) which were labelled with anti-Gia whilst a 41 kDa protein in Zed mays microsomal membranes was labelled with antiGac antiserum (track 5). Preabsorption of the antisera with the relevant synthetic peptides (tracks 2,4,6) was shown to abolish the immune detection. In addition, the anti-Gac serum was also shown to detect the a-subunit of an authentic G-protein, bovine transducin (Gta tracks 7,8). In addition to the immunological data presented, specific high affinity binding of the GTP analogue, GTPXS was also found in purified plasmalemma from the above species (not shown). The ability of the synthetic peptides to block the immune detection is important since all of the antibodies used displayed crossreactivity of various strengths and against polypeptides of Mr's between 17 and 90 kDa (data not shown). Some of the proteins detected were indeed in the Mr range expected for Ga subunits (37-50 kDa) and GP subunits (35-36 kDa). However, with the exception of the polypeptides indicated in Fig.1, in these cases the immune detection could not be abolished and the crossreactivity must indicate a non-specific labelling or the presence of antibodies that are directed towards epitopes other than those represented by the complete synthetic peptide sequence. 34

Use of antipeptide antibodies to probe the catalytic activity of p37 v-mos

Several site-directed antipeptide antibodies were generated to probe the kinase function of p37 v-mos. Anti- mos(3755) antibodies allowed autophosphorylation of p37 v-mos, as well as transphosphorylation of exogenously added purified bovine vimentin. Rabbit antipeptide antibodies against v- mos residues 158–170, 194–206, 260–271, and 363–374 and a mouse monoclonal antibody against residues 344–359 completely inhibited p37 v-mos protein kinase activity in vitro. p37 v-mos autophosphorylation and vimentin transphosphorylation were affected similarly. These results suggest important roles for insert and the carboxy-terminal domains in the catalytic activity of p37 v-mos.

Identification of a cleavage site directing the immunochemical detection of molecular abnormalities in type IIA von Willebrand factor.

Proteolytic cleavage of the von Willebrand factor subunit may be important for processing and/or function of the molecule and is altered in certain subtypes of von Willebrand disease. It results in the generation of two main fragments with apparent molecular masses of 140 kDa and 176 kDa from the 225-kDa subunit. We have now obtained chemical evidence to locate the protease-sensitive bond between residues Tyr-842 and Met-843, a site that appears to reflect the specificity of calcium-dependent neutral proteases (calpains). Antibodies were raised against four synthetic peptides that represented sequences immediately preceding or following or including the cleavage site. One antibody (against the fragment from Ala-837 through Asp-851) reacted only with the intact subunit, and its epitope included the cleavage site. All others reacted specifically with either the 140-kDa or the 176-kDa fragment, demonstrating their origin from a single cleavage. In samples of purified von Willebrand factor from four of five patients with type IIA von Willebrand disease, the anti-peptide antibodies showed markedly decreased reactivity with either the 140-kDa or the 176-kDa fragment, suggesting the existence of distinct molecular abnormalities clustered around the cleavage site. Thus, in the majority of type IIA patients, a common pathogenetic mechanism may lead to the disappearance of the larger multimers as a consequence of structural changes that may expose a sensitive bond to the action of specific proteases. These studies demonstrate the use of anti-peptide antibodies directed at a relevant structural domain for the immunochemical differentiation of normal and mutant molecules.

Adenovirus type 12 tumour antigen synthesis differs during infection of permissive and non-permissive cells

Synthesis of the adenovirus type 12 E1A and E1B tumour antigens was compared in productively infected human (KB) cells and in abortively infected baby hamster kidney (BHK) cells. By the use of anti-peptide antibodies, the E1A tumour antigens were easily detectable in infected KB extracts as early as 6 h post-infection, but were not detectable in infected BHK extracts until 12 h post-infection. The level of E1A tumour antigens detected in BHK extracts was 10 to 15% of that detected in KB extracts. The level of the E1B 163R (19K) tumour antigen was also lower in BHK extracts: 5 to 10% of that detected in KB extracts. Stability of the E1A tumour antigens was not significantly different in the two infected cell species, indicating that the lower E1A level during abortive infection was due to a lower rate of synthesis of these proteins. These data suggest that early protein synthesis is not the same in abortively infected cells as it is in productively infected cells.

Localization and characterization of three different β‐adrenergic receptors expressed in Escherichia coli

After fusion with the N-proximal portion of the outer membrane protein LamB, three beta-adrenergic receptors, the human beta 1- and beta 2- and turkey beta 1-adrenergic receptor, were expressed in Escherichia coli with retention of their own specific pharmacological properties. Molecular characterization and localization of the three receptors in bacteria and comparison of the behaviour of each hybrid protein are reported. The bacteria were lysed and fractionated on a sucrose gradient. Saturable [125I]iodocyanopindolol binding activity was found associated mainly with the inner membrane fraction, suggesting that the receptor is correctly folded in this membrane. Binding activity was also found in the outer membrane fraction but varied according to the receptor type. Photoaffinity labeling experiments revealed that the receptors exhibit binding activity only after proteolytic removal of the LamB moiety from the fusion protein. The three hybrid proteins, detected in immunoblots by anti-peptide antibodies, were found mainly in the outer membrane fraction. Each of them exhibited different susceptibility to intrinsic bacterial proteolytic enzymes; sites of proteolytic cleavage were localized by the use of anti-peptide antibodies. The functional expression in E. coli of three beta-adrenergic receptors with similar structure but different amino acid sequences suggests that this expression system may be a general feature among similar receptors of the family of G-protein-coupled receptors. The level of expressed binding activity of a given receptor will be within the control of proteolytic degradation processes, depending on the primary sequence of the receptor. Constructions of new hybrid proteins, in combination with expression in protease mutants of E. coli, should help in controlling such processes.

Detection of T cells in paraffin wax embedded tissue using antibodies against a peptide sequence from the CD3 antigen.

Rabbit polyclonal antibodies were raised against a proline rich, peptide sequence, comprising 13 amino acids, in the cytoplasmic domain of the CD3 epsilon chain. Immunoprecipitation experiments showed that this antibody preparation recognised the CD3 antigen on human T lymphoblasts. The antibody stained normal T cells strongly in tissue sections which had been fixed in formalin or Bouin's solution and embedded in paraffin wax. Its reactivity with T cell lymphoma, when evaluated on a series of 96 previously phenotyped cases, closely agreed with the results obtained on cryostat sections. These results indicate that the specific detection of T cells in routinely processed tissue biopsy specimens is now technically feasible on a wide scale in diagnostic laboratories using CD3 peptide antibodies, and they also suggest that in future the use of anti-peptide antibodies may detect other lineage specific antigenic markers in paraffin wax sections.

Use of anti-peptide antibodies to study the transmembranous topography and structure-activity relationships of (Ca2+-Mg2+)-ATPase

Polyclonal antibodies were raised against peptides corresponding to segments of (Ca2+-Mg2+)-ATPase from fasttwitch adult rabbit skeletal muscle by the following methods. Peptides corresponding to residues 1 1 1, 504-5 1 1 and 985-994 were synthesized by the method of Merrifield (1986) or Sheppard (Atherton et af., 1981). For peptide 1-11, a cysteine residue was added to the C-terminus to facilitate coupling to the carrier protein and the same modification was made to the N-termini of peptides 504-51 1 and 985-994. These last two peptides correspond to the T, trypsin cleavage site and the C-terminus of the (Ca2'Mg2')-ATPase, respectively (Brandl et af., 1986). All three peptides were coupled to keyhole limpet haemocyanin with m-maleimidobenzoyl-N-hydroxysuccinamide ester by the method of Green et af. (1982). Peptides 567-582 and 38 1-400 (corresponding to segments of the protein on the nucleotide-binding and phosphorylation domains, respectively) were used without coupling to a carrier. New Zealand White rabbits were immunized by intradermal injection with 500 pg of peptide or peptide linked to carrier in Freund's complete adjuvant. Animals were boosted with a repeat intramuscular injection 6-8 weeks later and the serum was collected 5-10 days later. Except for peptide 504-5 1 1, specific antibodies were obtained, which in e.1.i.s.a. (Hudson & Hay, 1983) were capable of recognizing the peptides against which they were raised with minimal cross-reactivity with the other peptides. Anti-peptide antibodies against peptide 504-5 1 1 not only recognized the peptide against which they were raised but also peptide 381-400. This was observed on two occasions with serum from separate rabbits. No reciprocal cross-reactivity was observed with antibodies against peptide 38 1-400. An examination of the sequence of these peptides reveals no obvious similarities. All anti-peptide antibodies, except those against peptide 567-582, strongly recognized both the ATPase and sarcoplasmic reticulum (SR) in e s.a., suggesting that not only are these segments of the ATPase on the cytoplasmic face of the SR vesicles, which with the exception of the C-terminus segment, has already been substantiated by other techniques (Klip et af., 1980; Tong, 1980; Allen et al., 1980; Brandl et al., 1986), but they are also exposed on the surface of the cytoplasmic domains. The reason for the poor recognition of the ATPase by anti-peptide 567-582 antibodies is unclear. This anti-peptide antibody was also unable to recognize denatured ATPase, making it unlikely that the lack of recognition was due to the epitope being inaccessible. To further investigate the transmembranous location of the C-terminus, binding of the anti-peptide antibodies to

Quantitation of a guanine nucleotide binding regulatory protein by an enzyme-linked immunosorbent competition assay

We have developed a new method using a competitive enzyme-linked immunosorbent assay, ELISA, for the determination of levels of the stimulatory guanine nucleotide binding protein, G s, in membrane extracts. The method is based on the use of antipeptide antibodies generated in rabbits directed against amino acids 28–42 in the α-subunit, α s, of G s. The peptide is utilized as the stationary phase in the ELISA and anti- α s antibody bound to the microtiter plate is assessed by a peroxidase-coupled anti-rabbit immunoglobulin G antibody that yields detectable color development at 490 nm. G s purified from rabbit liver is utilized as the standard to assess the ability of G s present in cholate extracts of membrane samples to compete with bound peptides for primary antibody. This assay provides a direct means to quantify changes in levels of native G s in membranes and cells.