抗原多肽的设计、偶联策略

1、.：.；抗原多肽的设计、偶联战略抗体是生命科学研讨中不可或缺的工具之一，运用范围包括蛋白质表达检测和鉴定、蛋白质加工、蛋白质在细胞内的定位、免疫中和反响、蛋白质同源构造域研讨、蛋白质纯化以及疾病的免疫诊断和治疗。虽然抗体的制备过程不存在技术难点，但是抗原的选择以及所制备抗体的用途对能否获得一个优质高效的抗体至关重要。以下将对抗原多肽的设计、偶联战略等逐一引见.抗原设计 首先选择适宜的多肽序列，明确最终产物的用途对选择序列非常重要。假设仅仅需求消费针对蛋白质某个区域的特异抗体，比如研讨蛋白质N端的前提物，我们就需求设计N末端的多肽抗原。假设抗体的运用目的是识别修饰的氨基酸，如磷酸化的丝氨酸、苏氨

2、酸或者酪氨酸，乙酰化赖氨酸等，就必需对多肽进展相应的修饰。假设抗体最终用来识别自然形状下的蛋白质，对抗原的设计就要求更高。普通情况下抗血清可以识别用来免疫的多肽序列，但是不一定识别蛋白质的折叠构造。蛋白质的抗原决议簇普通由612个氨基酸构成，呈延续性或者非延续性序列。延续性抗原决议簇由延续的氨基酸序列构成，而非延续抗原决议簇包括一组非延续氨基酸，这些氨基酸由于蛋白质的折叠而构成在空间上相互毗邻。针对延续性抗原决议簇的抗体可以识别没有被埋藏在蛋白质内部的序列，而非延续性抗原的抗体能否识别抗原决议簇取决于用于抗体消费的多肽能否存在二级构造。氨基酸序列的亲水性、流露性、柔韧性决议了多肽的抗原性。许多

3、水融性的自然形状下的蛋白质其亲水序列暴露在外测，而疏水性氨基酸序列包埋在内部。抗体结合蛋白质外表的抗原决议簇，另外抗原决议簇柔韧性比较高。蛋白质的C末端经常暴露在外测并且有较高的柔韧性，因此经常被用来作为抗体消费的抗原。但是假设C末端是跨膜蛋白质的膜内部分，该序列能够由于疏水性太强而不适宜用来作为抗原。同C末端序列类似，蛋白质的N末端序列也经常暴露在蛋白质的外表，同样为首选抗原序列。预测蛋白特性例如亲水性、疏水性及二级构造例如螺旋，折叠，盘旋的一些算法有助于选择流露性较高，有抗原性的内部序列以用于抗体生成。常见预测性算法有如下三种，Hopp及Woods所描画的亲水性曲线给蛋白序列中的每一个氨基

4、分配一个平均亲水性值，对于一系列的相邻氨基，平均亲水性的最高点通常就位于抗原决议簇或在其附近。Kyte 及 Doolittle 所提出的另一算法略有不同，它主要是衡量蛋白序列的亲水性及疏水性趋势，该算法对于预测某蛋白的外部及内部区域非常有用。蛋白的二级构造那么可以经过CHOU/FASMAN 或 LIM 所提出的算法来预测。流露性或易接近区经常和螺旋区或延展的二级构造区相邻。并且，具有盘旋或双性螺旋特性的序列区也具有较好的抗原特性。目前有许多商用软件包都运用了这些不同的算法，例如MacVectorTM，DNAStarTM及PC-GeneTM。要想预测准确，不能只运用一种算法。结合各种不同的预测方

5、法来预测抗原性区域，可使胜利率大大提高。抗原性区域确定以后，接下来要确定多肽的长度。关于选定多肽长度有两种不同的观念。一种观念以为长的多肽2040个氨基酸比较好，由于长的多肽无疑会添加抗原基的数量。另一种观念那么以为小的多肽更有效，运用小的多肽能保证所产生抗体的位点特异性。但有一点是明确的，不论长度如何，所选多肽必需可以较容易地经过生化合成得到，并且能溶解到水溶性缓冲剂进展载体蛋白的耦联。由于受副反响的影响较大，高于二十个氨基酸的多肽通常很难进展高纯度合成，并且经常会含有缺失性序列。另一方面，太短的多肽10kd进展浓缩，以备测试；利用0.2M氨基乙酸glycine进展洗脱pH12，在pH2.0

6、时开场洗脱，在吸光度降到基线以下时搜集洗脱液。将洗脱液的pH降低0.5-1，直到已没有可检测到的抗体从柱上洗下来；搜集从纯化柱上搜集的各个部分，搜集后经过参与1/10体积1 M Tris-HClpH=8.0 或pH 8.0 的洗脱液将其中和；将纯化的多肽进展ELISA分析，以决议具有最高亲和性的洗液部分。将适当的抗体洗脱液浓缩至2-5mg/ml，混合50甘油在20oC下保管，或混合0.1% BSA在4oC下保管。参与0.01% NaN3作为防腐剂。3看起来有些浑浊的血清能否还可以运用？普通来讲，这不会是问题。运用前可以用0.2m的过滤器去除血清中的小颗粒Antigen design & ser

7、a purificationAntibodies to small peptides have become an essential tool in life science research, with applications including gene product detection and identification, protein processing studies, diagnostic tests, protein localization, active site determination, protein homology studies and protei

8、n purification. While it is quite easy to generate anti-peptide antibodies, it is important to carefully consider the ultimate use for the antibody and the sequence used to ensure success. This tech sheet will briefly explore peptide selection and design, coupling strategy, and carrier proteins whic

9、h are important factors in anti-peptide antisera generation. Serum purification will also be discussed. For more complete coverage of antigen design, please refer to the References1,2. Peptide Selection and DesignThe first step in the process is the selection of the appropriate peptide sequence. At

10、this step the ultimate use for the antibody must be considered. If the antibody is needed to probe a specific protein domain then the choice is simple. For example, if one is studying proteolytic processing of an N-terminal precursor, antibodies against the N-terminal region of interest would be rai

11、sed. Likewise if the goal is to monitor the phosphorylation state of a specific sequence, antibodies to the phosphorylated sequence can be used. If the goal is to raise antibodies that will recognize the protein in its native state, the problem becomes more complex. Anti-peptide antibodies will alwa

12、ys recognize the peptide. However, the same antibody may not recognize the sequence within the folded intact protein. Sequence epitopes in proteins generally consist of 6-12 amino acids and can be classified as continuous and discontinuous. Continuous epitopes are composed of a contiguous sequence o

13、f amino acids in a protein. Anti-peptide antibodies will bind to these types of epitopes in the native protein provided the sequence is not buried in the interior of the protein. Discontinuous epitopes consist of a group of amino acids that are not contiguous but are brought together by folding of t

14、he peptide chain or by the juxtaposition of two separate polypeptide chains. Anti-peptide antibodies may or may not recognize this class of epitope depending on whether the peptide used for antisera generation has secondary structure similar to the epitope and/or if the protein epitope has enough co

15、ntinuous sequence for the antibody to bind with a lower affinity. When examining a protein sequence for potential antigenic epitopes, it is important to choose sequences which are hydrophilic, surface-oriented, and flexible3. Most naturally occurring proteins in aqueous solutions have their hydrophi

16、lic residues on the surface and their hydrophobic residues buried in the interior. Antibodies bind to epitopes on the surface of proteins. Additionally, it has been shown that epitopes have a high degree of mobility4. Because the C-termini of proteins are often exposed and have a high degree of flex

17、ibility they are usually a good choice for generating anti-peptide antibodies directed against the intact protein. If the protein is an integral membrane protein and the C-terminus is part of the transmembrane segment, this sequence will be too hydrophobic and not a good choice. Like the C-terminus,

18、 the N-terminus is also frequently exposed and on the surface of the protein making it an ideal candidate for antibody generation. If a protein sequence is derived from the cDNA sequence, the leader sequence should not be included in the sequence selected for antibody generation. Algorithms for pred

19、icting protein characteristics such as hydrophilicity/hydrophobicity and secondary structure regions such as alpha-helix, beta-sheet and beta-turn aid selection of a potentially exposed, immunogenic internal sequence for antibody generation. Hydrophilicity plots as described by Hopp and Woods5 assig

20、n an average hydrophilicity value for each residue in the sequence. The highest point of average hydrophilicity for a series of contiguous residues is usually at or near an antigenic determinant. A slightly different algorithm described by Kyte and Doolittle6 evaluates the hydrophilic and hydrophobi

21、c tendencies of the sequence. This profile is useful for predicting exterior vs. interior regions of the native protein. Secondary structure can be identified by the use of algorithms developed by Chou and Fasman7 or Lim8. Surface regions or regions of high accessibility often border helical or exte

22、nded secondary structure regions. In addition, sequence regions with beta-turn or amphipthic helix character have been found to be antigenic9. Many commercial software packages such as MacVectorTM, DNAStarTM, and PC-GeneTM incorporate these algorithms. To be sucessful, none of the algorithms should

23、be used alone. Combined use of the predictive methods may result in a success rate as high as 86% in predicting antigenic determinants9,10. Once the protein region of interest has been identified, the length of the peptide must be selected. There are two differing thoughts on the topic of peptide le

24、ngth. One suggests that long peptides (20-40 amino acids in length) are optimal because it increases the number of possible epitopes. The other suggests that smaller peptides are sufficient, and their use ensures that the site-specific character of anti-peptide antibodies is retained. Clearly, any p

25、eptide selected must be chemically synthesizable and should be soluble in aqueous buffer for conjugation to the carrier protein. Peptides longer than 20 residues in length are often more difficult to synthesize with high purity because there is greater potential for side reactions, and they are like

26、ly to contain deletion sequences. On the other hand, short peptides (10 amino acids) may generate antibodies that are so specific in their recognition that they cannot recognize the native protein or do so with low affinity. The typical length for generating anti-peptide antibodies is in the range o

27、f 10-20 residues. Peptide sequences of this length minimize synthesis problems, are reasonably soluble in aqueous solution and may have some degree of secondary structure. Coupling StrategyA factor that is often over-looked when designing a synthetic peptide is the method of coupling the peptide to

28、the carrier protein. For example, N-terminal sequences should be coupled through the C-terminal amino acid and vice versa for C-terminal sequences. Internal sequences can be coupled at either end. Another consideration for internal sequences is to acetlyate or amidate the unconjugated end as the seq

29、uence in the native protein molecule would not contain a charged terminus. The most common coupling methods rely on the presence of free amino (alph-amino or Lys), sufhydryl (Cys), or carboxylic acid groups (Asp, Glu, or alpha-carboxyl). Coupling methods should be used that link the peptide to the c

30、arrier protein via the carboxy- or amino-terminal residue. The sequence chosen should not have multiple residues that may react with the chosen chemistry. If multiple reactive sites are present, try to shorten the peptide or choose the sequence so they are all localized at either the amino or the ca

31、rboxyl terminus of the peptide. For internal sequences the end furthest from the predicted epitope is normally favored as this avoids potential masking problems. The EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) or carbodiimide method is routinely used in the Sigma-Genosys labor

32、atory unless otherwise stated by the researcher. Carbodiimides can activate the side chain carboxylic groups of aspartic and glutamic acid as well as the carbooxyl terminal group to make them reactive sites for coupling with primary amines. The activated peptides are mixed with the carrier protein t

33、o produce the final conjugate. If the carrier protein is activated first, the EDC method will couple the carrier protein through the N-terminal alpha amine and possibly through the amine in the side-chain of Lysine, if present in the sequence. The m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)

34、is a heterobifunctional reagent that can be used to link peptides to carrier proteins via cysteines. The coupling takes place with the thiol group of cysteine residues. If the chosen sequence does not contain Cys it is common to place a Cys residue at the N- or C-terminus to obtain highly controlled

35、 linking of the peptide to the carrier protein. For synthesis purposes we recommend that the placement of cysteine be at the N-terminus of the peptide if possible. Glutaraldehyde is a bifunctional coupling reagent that links two compounds through their amino groups. Glutaraldehyde provides a highly

36、flexible spacer between the peptide and carrier protein for favorable presentation to the immune system. Unfortunately, glutaraldehyde is a very reactive compound and will react with Cys, Tyr and His to a limited extent. The result is a poorly defined conjugate. The glutaraldehyde method is particul

37、arly useful when a peptide contains only a single free amino group at its amino terminus. If the peptide contains more than one free amino gorup, large multimeric complexes can be formed, which are not well defined, but are highly immunogenic. Selecting the Protein CarrierConjugation to a carrier pr

38、otein is important because peptides are small molecules, that alone do not tend to be immunogenic, thus possibly eliciting a weak immune response. The carrier protein contains many epitopes that stimulate T-helper cells, which help induce the B-cell response. Many different carrier proteins can be u

39、sed for coupling to synthetic peptides. The most commonly selected carriers are keyhole limpet hemacyanin (KLH) and bovine serum albumin (BSA). The higher immunogenicity of KLH often makes it the preferred choice. Another advantage of choosing KLH over BSA is that BSA is used as a blocking agent in

40、many experimental assays. Because antisera raised against peptides conjugated to BSA will also contain antibodies to BSA, false positives may result. Although KLH is large and immunogenic, it may precipitate during cross-linking, making it difficult to handle in some cases. Ovalbumin (OVA) is anothe

41、r useful carrier protein. It is a good choice as a second carrier protein when verifying whether antibodies are specific for the peptide alone and not the carrier. Rabbit Serum Albumin (RSA) may be used when the antibody response to the carrier protein must be kept to a minimum. Rabbits immunized wi

42、th RSA conjugate are less likely to raise antibodies to the carrier, as the RSA is recognized as self. If the RSA conjugate were injected into another host, the protein would not be recognized as self. It is important to recognize that the immune system reacts to the peptide-protein carrier as a who

43、le and that there will be a portion of response directed against the conjugated peptide as well as the linker and the carrier protein1. When screening by ELISA it is advisable to use a peptide conjugate prepared using a different carrier protein. This is not necessary if performing ELISA assays wher

44、e the plates are coated directly with unconjugated peptide. Multiple Antigenic Peptides (MAPs)The MAP system represents a unique approach to anti-peptide antibody generation11. The system is based on a small immunogenically inert branched lysine core onto which multipe peptides are synthesized in pa

45、rallel. The result after synthesis is a three-dimensional molecule, which has a high molar ratio of peptide antigen to core molecule and therefore does not require the use of a carrier protein to induce an antibody response. Each core molecule may contain four identical peptides. In theory, MAP has

46、an advantage when compared to its monomeric counterpart attached to a carrier protein in that the lysine core of a MAP is small compared with the peptide antigen. Therefore, the concentration of antigen is at a maximum. The result is a highly immunogenic MAP, which exhibits significantly higher tite

47、rs when compared to its monomeric counterpart attached to a carrier protein. It should be noted that there are some synthesis concerns when making a MAP complex. The branched nature of the lysine core allows for multiple copies of the peptide to be synthesized; however, steric hindrance becomes a pr

48、oblem during the synthesis of long peptides, resulting in some arms of the dendrimer being deletion products. The high molecular weight of the complex does not lend itself to good quality control measures (mass spec and/or analytical HPLC). An indirect synthesis of the MAP can eliminate analysis pro

49、blems. In the indirect method, the peptide is first synthesized, purified then analyzed using mass spec and analytical HPLC. The peptide antigen is then coupled through a Cys to a functionalized lysine core. Choice of HostWhen attempting to raise an antibody, choose an animal that is genetically ver

50、y different from the source of immunogen. In order to achieve maximum immune response, it is important to avoid self-recognition of the immunogen by the host animal. As an example, when raising antibodies against a human protein, it is more suitable to use a rabbit or mouse host than a monkey. For h

51、ighly conserved mammalian proteins, raising antibodies in the avian (chicken) system is often a preferred alternative. Adjuvant, Immunization, & Sera CollectionSigma-Genosys routinely uses Freunds adjuvant for immunization purposes. The first injection is given in Complete Freunds adjuvant. Adjuvant

52、 is combined with the antigen to improve the immune response so that less vaccine is needed to produce more antibodies. The adjuvant allows a slow release of the antigen which allows for continual stimulation. Injections are routinely performed subcutaneously at multiple sites. A pre-immune bleed sh

53、ould be drawn from each host animal to produce a baseline to which the production bleeds can be compared. The drawn sera will contain a number of different types (IgG, IgM, IgA) and subclasses (Ig1, Ig2a, Ig2b, Ig3). Sodium azide (0.1%) can be added to the sera. Sodium azide is a broad-spectrum enzy

54、me inhibitor and acts as an antimicrobial agent. Sodium azide should not be added to sera when using in cell culture or in vivo studies. Antisera PurificationIf a high background is observed in assays using the antisera, various purification techniques are available. It is important to first check t

55、hat the background is non-specific and not due to the response against the peptide. This can be determined by performing a competitive peptide blocking study. Peptide blocking studies check that the response against the target protein is not a background artifact. Ammonium Sulfate PrecipitationAmmon

56、ium sulfate precipitation is a commonly used method for removing protein from solution. The method is a fairly crude, non-specific purification that removes the majority of plasma proteins and leaves the immunoglobulin fraction. When in solution, proteins form hydrogen bonds with water through their

57、 exposed polar and ionic groups. Adding small ions such as ammonium or sulfate removes water molecules from the protein, resulting in precipitation of the protein out of solution. It should be stated that ammonium sulfate precipitation will not result in highly purified antibodies. The contaminants

58、will consist of other high-molecular-weight proteins and proteins that are trapped in the large flocculent precipitates. It is recommended that ammonium sulfate precipitation be used as part of a purification scheme involving further purification steps. Protein A/GProtein A or Protein G purification

59、 removes the IgG fraction based on the specificity of these proteins for the Fc portion of the IgG. Protein A is produced from Staphylococcus aureus. It has the capacity to bind at least two molecules of IgG. The binding is specific to the Fc portion and does not affect the antigen binding sites. Protein G is isolated from Group G streptococci and binds the Fc region of the IgG in a similar manner to Protein A. Protein A a