**4. Application of antibodies**

**3.4. Activation of the SAA by separation from HDL under febrile temperatures and** 

Moreover, when the infection remains limited, a local APR will take care of it.

neity and its known functions, see the reviews [1, 2].

The functions of the four human isotypes, SAA1, SAA2, (SAA3 in humans is only transcribed in some cells) and SAA4 have not been fully analyzed. They have arisen through gene duplications, thus indicating important individual functions either alone or in combination. As described before, the human acute-phase A-SAA has two very similar isotypes, A-SAA1 and A-SAA2, in the APR mostly synthesized in the liver and expressed in most body cells (see below) and the constitutive C-SAA4 and some allotypes in SAA1 and SAA2. For a review of the SAA heteroge-

Another discovery was the discontinuous separation of SAA from HDL described above at different temperatures, meaning that not all SAA molecules are being separated from HDL at a single temperature except for the temperature of 42°C (**Figures 3** and **4**). In fact, these figures show that the separation of SAA is spreading out over the whole febrile temperature range starting from 38°C to 42°C and above. In addition, based on these observations in **Figure 4**, it is possible that SAA isotypes and allotypes are separated from HDL at different febrile temperatures and thereafter fulfill their different functions locally or systematically as individual SAA species as is also to be derived from **Figure 3**. Another indication for the differential release of the SAA species can be detected in **Figure 4** in the different shapes of the protein blots of the SAAs devoid of HDL, thus indicating possible SAAs with distinct isoelectric points (IP). In **Figure 4**, there are free dots before 37°C named (for convenience) "arc SAA," the least acidic SAA. The SAA species released from HDL after 37°C ("38 SAA") are probably the more acidic ones. In this sense, the dot changes also occur later on 39°C-, 40°C-released SAA, etc. Analyzing the spots for the identity of the various SAA species could show whether these indications did discover a mechanism by which the different SAA species can be released from HDL and thereby are being activated at specific temperatures alone or with other SAAs for special purposes, which need to be analyzed. These points may also be of therapeutical interest. This proposed temperature selection of SAA isotypes could specify the needed APR function for a specific purpose. The increase of the organism's temperature is being induced

Taken together [15, 17, 18, 21], it is clear that the mechanism of separation of SAA from HDL in vitro is also strictly regulated in vivo by body temperatures above 37°C. Therefore, this is a key mechanism that can be induced and activated basically by two different manifestations. The most common is the orthologic APR activation [33] of SAA. This occurs with a maximal SAA concentration of up to 1000 times within a day as a systemic "biochemical thunderstorm" with a myriad of activating and inhibiting events simultaneously, which are not understood in detail today [1, 2]. During these events, the cause of the APR will be eradicated and the APR becomes curative. With this beneficial outcome, the normal immune homeostasis returns in a foreseeable future. However, when the APR cannot overcome its initial cause, it will become a pathologic APR [33] with a "persistent biochemical thunderstorm" and lacking a self-driven cure. The consequences can be summarized in an exhaustion of the resources of the organism and decline of the metabolic activity through a multitude of clinically challenging conditions exemplified by severe viral and bacterial chronic inflammations, systemic inflammatory response syndrome (SIRS) or uncontrolled chronic infections, sepsis and septic shock [1, 2].

**consequences**

76 Infectious Process and Sepsis

### **4.1. Polyclonal and monoclonal antibodies prepared against AA and SAA**

In a collaborative study, each of the eight species-specific polyclonal AA antibodies against eight species (including humans) was immunohistochemically tested against the AA amyloids of eleven different species, including those of humans. The results showed a strong reactivity only with the homologous species and with only some cross-reaction with a related species. The reactivity was in general species specific, but a universal generic AA antibody could not be obtained in these eight polyclonal antibodies [37].

The next step was to produce murine monoclonal antibodies against AA and SAA [20]. Their value and merit have been documented by the inventors Köhler and Milstein [38]. Monoclonal antibodies are represented by one amino acid sequence and have the value of a chemical reagent. We selected 20 stable clones (see **Table 1**), which were epitope mapped [31] and immunohistochemically tested on AA amyloids in 10 different mammals, many humans and 9 different birds. Some cross-reactivity with some monoclonals was detected. Most of the 19 AA amyloids tested could be identified with the two monoclonals mc4 and mc29, showing that most of these AA amyloids have some peptides in common and these antibodies recognize the same or very similar epitopes of AA in different species. In addition, antibodies of all clones were tested for binding with 15 synthetic SAA peptides in only 4 clones the epitope could be identified. These included the known clones mc4 and mc29 (see above), and the two new ones, mc1 and mc20 (see **Table 1**). In APS, two different charge variants of SAA have been detected with these monoclonals [22].

The cause of the failing reactivity of most of the synthetic peptides with most of the monoclonals may be due to the presence of more discontinuous epitopes. This could also be deduced from the fact that SAA shows multiple short peptides that alternate between the invariable (red) and the variable (white) peptides, as shown in **Figure 5** (see also below).

Moreover, since mc21 was negative with the linear peptides, but reacted very strongly with AA amyloid in tissues, it was epitope mapped differently. It was mapped with endoproteinase Asp-N-generated peptides from a pure and partially amino acid-sequenced human AA (KIR) protein of 8.4 kDa. Of the 11 distinct peptides separated by RP-HPLC, mc21 reacted only with a single peptide, which was aa 33–42 of SAA [32]. This peptide is almost identical with the largest invariant peptide of SAA (see **Figure 5**). Two other monoclonals mc9 and mc13 did not show any reaction with these 11 distinct HPLC peaks [32] although they were reactive with AA in tissue sections. Here again, in linear SAA peptides, the discontinuous epitopes of SAA may not be preserved.


**4.2. Identification of functional SAA epitopes by the monoclonals: finding their use** 

on the α-helical coils 1–4 and the contribution of the invariable (red) and variable peptides are also visible.

Monoclonals (see **Table 1**) are being applied for classification of AA amyloid in tissue sections when a recent amyloidosis was diagnosed in a patient or in an animal. Every amyloid has to be classified for therapeutic and prognostic purposes. This is exemplified in exploiting the generic monoclonals mc4 and mc29 (in Refs. [23, 24]) by either the immunohistochemical classification (IHC) on paraffin section of animal AA amyloidosis [23, 24] or by the immunoelectron microscopic classification (EM) of human AA amyloid on ultrathin sections [25].

**Figure 5.** Structure of SAA and epitope-mapped AA monoclonal antibodies. The structure of SAA 1–104 and its fragments AA 1-76 and SL-77-104 with invariant (in red) and variant (white in between the red) peptides and epitope mapped of five monoclonal AA/SAA antibodies. There are three species-independent (mc4, mc21 and mc29), and two variant as well as species-specific (mc1 and mc20) monoclonal antibodies against AA and SAA. The positions of epitopes

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The SAA1 and SAA2 proteins are presented in **Figure 5** as a continuous string with 1–104 amino acids. SAA consists of two parts, the N-terminal AA 1–76 polypeptide, which causes AA amyloidosis in humans [11] and animals under unfavorable inflammatory conditions [23, 24], and the C-terminal SL 77–104 polypeptide, whose function is stabilizing the two double coils [39, 40]. **Figure 5** was constructed using data from the USCS Genome Browser

The structure of SAA consists of four α-helical coils, 1–4, with 1–27, 32–47, 50–69 and 73–88 aa in length, respectively, followed by a tail after the 4th coil. These four coils are arranged in two antiparallel double coils, whereby 1 joins 3 and 2 joins 4 [39]. Each α-helix and its tail contain alternating blocks of twenty invariable peptides (**Figure 5**, in red, numbered 1–20). The variable peptides that can be species specific are the white, unstained sites in between the invariable peptides. Variables are also the peptides joining the coils, which

**and detecting their function**

*4.2.1. Diagnostic application*

*4.2.2. The structure of SAA*

(GRCH38hg38) Assembly, as reported by 2.

Explanations: IHC, immunohistochemistry; EM, immunoelectron histochemistry.\* Available from Dako, Denmark.

**Table 1.** Monoclonal AA and SAA antibodies [20].

Therefore, another strategy for the epitope mapping has been worked out that is the cooperative precipitation with either the antigens AA or SAA in 1.5% agarose gel. Applied were various combinations of two different monoclonals on one antigen, respectively. A precipitation showed that the two given monoclonals react with two epitopes. This approach resulted in precipitations and the epitope could be estimated roughly in some of the antibodies (unpublished). This has been expected since all the AA/SAA antibodies were selected by reactivity with amyloid in tissues. Finally, precipitation with SAA but not with AA pointed to a monoclonal against the SL peptide (see **Figure 5**, aa 77–104). Similarly, SAA isotype-specific monoclonals could have been selected by a similar approach.

The Invariant Peptide Clusters of Serum Amyloid A Are Humoral Checkpoints for Vital Innate Functions… http://dx.doi.org/10.5772/intechopen.91983 79

**Figure 5.** Structure of SAA and epitope-mapped AA monoclonal antibodies. The structure of SAA 1–104 and its fragments AA 1-76 and SL-77-104 with invariant (in red) and variant (white in between the red) peptides and epitope mapped of five monoclonal AA/SAA antibodies. There are three species-independent (mc4, mc21 and mc29), and two variant as well as species-specific (mc1 and mc20) monoclonal antibodies against AA and SAA. The positions of epitopes on the α-helical coils 1–4 and the contribution of the invariable (red) and variable peptides are also visible.

### **4.2. Identification of functional SAA epitopes by the monoclonals: finding their use and detecting their function**

### *4.2.1. Diagnostic application*

Monoclonals (see **Table 1**) are being applied for classification of AA amyloid in tissue sections when a recent amyloidosis was diagnosed in a patient or in an animal. Every amyloid has to be classified for therapeutic and prognostic purposes. This is exemplified in exploiting the generic monoclonals mc4 and mc29 (in Refs. [23, 24]) by either the immunohistochemical classification (IHC) on paraffin section of animal AA amyloidosis [23, 24] or by the immunoelectron microscopic classification (EM) of human AA amyloid on ultrathin sections [25].

### *4.2.2. The structure of SAA*

Therefore, another strategy for the epitope mapping has been worked out that is the cooperative precipitation with either the antigens AA or SAA in 1.5% agarose gel. Applied were various combinations of two different monoclonals on one antigen, respectively. A precipitation showed that the two given monoclonals react with two epitopes. This approach resulted in precipitations and the epitope could be estimated roughly in some of the antibodies (unpublished). This has been expected since all the AA/SAA antibodies were selected by reactivity with amyloid in tissues. Finally, precipitation with SAA but not with AA pointed to a monoclonal against the SL peptide (see **Figure 5**, aa 77–104). Similarly, SAA isotype-specific monoclonals could have been

**Isotype Quality References (selected)**

**Usage EM**

+++ +++ [17, 20–28]

++ +++ [17, 20–22, 25, 31]

+++ +++ [17, 21, 24–31, 33–36]

Available from Dako, Denmark.

**Usage IHC**

**Epitope and SAA peptides**

mc 4 34 IgG 1 κ 19–31 +++ +++ [17, 20–25, 29, 30] mc 8 38, 57 IgG 3 κ 25–76 ++ 0 [20, 22, 25] mc 9 39, 41, 53 IgG 1 κ +++ 0 [20, 22, 25, 31] mc 12 40, 42 IgG 2b κ 25–76 ++ 0 [20, 22, 25, 31] mc 13 43, 54 IgG 1 κ +++ ++ [20, 22, 24, 25]

7–15

mc 2 ++ [22]

selected by a similar approach.

**For notes** **Clone no Internal lab K-Nr.**

78 Infectious Process and Sepsis

mc 20 50, 28 60

mc 1\* 17 IgG 2a κ 5–16

mc 3 33 +

mc 15 45 + mc 17 47 +

mc 23 63 IgG 1 κ + mc 25 55, 124, 125, 126 ++ mc 27 77, 127 IgM κ ++

mc 30 130 IgG 1 κ +++

Explanations: IHC, immunohistochemistry; EM, immunoelectron histochemistry.\*

mc 29 129 IgG 1 κ 28–40

**Table 1.** Monoclonal AA and SAA antibodies [20].

IgG 2a κ 60–75

mc 28 58, 128 +++ +

25–76

mc 21 65 IgG 1 κ 33-42 +++ [32] mc 22 70 IgG 2b κ + 0 [25]

25–76

mc 31 131 IgG 1 κ + 0 [25]

The SAA1 and SAA2 proteins are presented in **Figure 5** as a continuous string with 1–104 amino acids. SAA consists of two parts, the N-terminal AA 1–76 polypeptide, which causes AA amyloidosis in humans [11] and animals under unfavorable inflammatory conditions [23, 24], and the C-terminal SL 77–104 polypeptide, whose function is stabilizing the two double coils [39, 40]. **Figure 5** was constructed using data from the USCS Genome Browser (GRCH38hg38) Assembly, as reported by 2.

The structure of SAA consists of four α-helical coils, 1–4, with 1–27, 32–47, 50–69 and 73–88 aa in length, respectively, followed by a tail after the 4th coil. These four coils are arranged in two antiparallel double coils, whereby 1 joins 3 and 2 joins 4 [39]. Each α-helix and its tail contain alternating blocks of twenty invariable peptides (**Figure 5**, in red, numbered 1–20). The variable peptides that can be species specific are the white, unstained sites in between the invariable peptides. Variables are also the peptides joining the coils, which represent the turns. In addition, the tail is winding around these double coils for stabilization [39, 40]. This conformation of SAA with the short-distance, alternating peptides in red and white would need more exact three-dimensional analyses for identifying the proposed discontinuities of peptides based on the partial un-reactivity of the monoclonals with linear peptides (see above).

Recombinant SAA shows the same binding to HDL and the same temperature release of SAA from HDL in vitro [21]. When separated from HDL under febrile temperatures, it can rebind again to HDL at body (or lower) temperatures. The binding is therefore reversible except when the temperature is above 41°C for some time, when it probably aggregates irreversibly [34]. The temperature-dependent mechanism has been proposed for activating the SAAs (see **Figures 2**–**4**). When fever is systemic, the free SAA load is part of the systemic APR. When local febrile temperatures are induced by local injuries or infection, a local APR is induced with local SAA. This can also apply for local tumors (see below). How this acts is not fully known. Where is the mc1 epitope located, considering that three invariant peptides are located within the 5–16 peptide stretch? Since the specificity is human specific, these peptides should be among the 2–3 variant areas; those are probably the white areas of the 5–16 peptide stretch; see **Figure 5**. In addition, this mc1 epitope (aa 5–16) has an overlap of 7 [5–11] of the 11 aa residues with the presumptive lipid-binding site (aa 1–11) (established by Turnell et al. [41]). Finally, mc1 binds very reliably to human (and some primate) AA in fixed paraffin sections and in ultrathin sections for EM [25], and not to SAA-HDL in serum at lower body temperatures (see above). Therefore, this murine monoclonal anti-AA mc1 has become a standard for examining human AA and SAA (available from Dako).

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In tissue sections, this monoclonal (see **Table 1** and **Figure 5**) reliably binds to human AA amyloid and is being used for diagnostic purposes. It binds to the synthetic peptide aa 60–75 of SAA, which is located at the longest variable peptide stretch of SAA and located at the C-terminal half of the third α-helical coil, and, to a minor extent, at the small N-terminal part of the fourth coil, which contains the first invariable peptide no. 12. We do not know whether

These antibodies demonstrated immunohistochemical, species-independent binding to most AA-type amyloids of the vertebrates (see above). They were therefore directed against the invariant peptides of SAA, which are located on the first and second α-helical coils. Their extent and their differences are depicted in **Figure 5**. The clone mc4 reacts largely with the invariant peptide no. 4–6 on coil 1. This clone binds differently as compared to mc21 and mc29, both of which bind to the 7th peptide of coil 2, the largest invariant peptide of SAA. While mc21 seems to be only reacting with peptide no. 7, the monoclonal mc29 extends to the variant joining peptide area (aa 28–32) that is between coil 1 and coil 2. This may explain the additional, partial binding of mc29 to the variable peptides. In addition, it binds to most of the

This series of monoclonal antibodies has been probed and exerted to establish a monoclonal

*4.2.4.2. mc20*

this mini peptide is part of the mc20 paratope.

animal AA amyloids tested (see below).

*4.2.4.4. mc1, mc4, mc13, mc29 and mc31*

micro-ELISA for quantification of SAA [42].

*4.2.4.3. mc4, mc21 and mc29*

### *4.2.3. The invariant peptide clusters of SAA and the binding sites of the SAA monoclonal antibodies*

The monoclonal antibodies can be divided into three categories by the kind of epitope onto which they bind. In (a), they bind to species-specific epitopes and could perhaps also be detected with polyclonal antibodies [37]; in (b), they bind to AA amyloid in tissue section, but not to synthetic linear peptides, and are probably reacting with discontinuous epitopes of the SAA, and in (c), they bind to species independent, i.e., the invariant peptides and epitopes of AA and SAA, which are almost identical throughout the vertebrates (reviewed in [1, 2]). These special antibodies can also be called "generic" AA/SAA antibodies. Generic SAA antibodies are mc4, mc21 and mc29 (the latter contains also an additional specificity; see later). The two monoclonals that functionally bind to variable epitopes are mc1 and mc20 and belong to a category in (a). All monoclonals and their known binding synthetic peptides are listed in **Table 1**, together with their binding to patients' and animals' AA amyloid in formalin-fixed paraffin tissue sections [20, 23, 24] and ultrathin sections for EM [25].

The binding sites of the monoclonals to SAA are shown in **Figure 5**. The invariant parts of SAA contain ancient peptide clusters preserved during their evolution from the lampreys (over 500 Mio years without hardly any changes, Wikipedia) to the mammals, including humans. Again, we as humans have the invariable peptides of SAA in common with all vertebrates and the lamprey. Therefore, these special peptides have to be of utmost importance for mechanisms related to the proteostasis of many systems. They become extremely activated when in imbalance, exemplified by injuries and inflammation or bacterial infection, and in the event that their activation cannot be resolved. This can result in a sepsis. Thus, one can assume with some likelihood that a single amino acid exchange in these 19 invariant areas must not have been accepted throughout evolution. Indications are in the literature that natural SAA behaves differently as compared to recombinant SAA or SAA with a single amino acid change or exchange [1, 2]. The importance of the proteins of the SAA family for survival can therefore hardly be overestimated and the phrase that SAA is "the hub in the interaction network" [40] can express this eminent role of the SAA.

### *4.2.4. Properties of the individual monoclonals against AA presented here*

### *4.2.4.1. mc1*

This monoclonal antibody mc1 (see **Table 1** and **Figure 5**) is of interest since it binds to the most N-terminally positioned human-specific epitope on SAA (aa 5–16), but only when it is devoid of HDL. Thus, SAA can be distinguished from SAA-HDL through the failure of mc1 to bind to the complex of SAA-HDL, since HLD conceals the mc1 epitope of SAA [15]. Recombinant SAA shows the same binding to HDL and the same temperature release of SAA from HDL in vitro [21]. When separated from HDL under febrile temperatures, it can rebind again to HDL at body (or lower) temperatures. The binding is therefore reversible except when the temperature is above 41°C for some time, when it probably aggregates irreversibly [34]. The temperature-dependent mechanism has been proposed for activating the SAAs (see **Figures 2**–**4**). When fever is systemic, the free SAA load is part of the systemic APR. When local febrile temperatures are induced by local injuries or infection, a local APR is induced with local SAA. This can also apply for local tumors (see below). How this acts is not fully known. Where is the mc1 epitope located, considering that three invariant peptides are located within the 5–16 peptide stretch? Since the specificity is human specific, these peptides should be among the 2–3 variant areas; those are probably the white areas of the 5–16 peptide stretch; see **Figure 5**. In addition, this mc1 epitope (aa 5–16) has an overlap of 7 [5–11] of the 11 aa residues with the presumptive lipid-binding site (aa 1–11) (established by Turnell et al. [41]). Finally, mc1 binds very reliably to human (and some primate) AA in fixed paraffin sections and in ultrathin sections for EM [25], and not to SAA-HDL in serum at lower body temperatures (see above). Therefore, this murine monoclonal anti-AA mc1 has become a standard for examining human AA and SAA (available from Dako).

### *4.2.4.2. mc20*

represent the turns. In addition, the tail is winding around these double coils for stabilization [39, 40]. This conformation of SAA with the short-distance, alternating peptides in red and white would need more exact three-dimensional analyses for identifying the proposed discontinuities of peptides based on the partial un-reactivity of the monoclonals with linear

The monoclonal antibodies can be divided into three categories by the kind of epitope onto which they bind. In (a), they bind to species-specific epitopes and could perhaps also be detected with polyclonal antibodies [37]; in (b), they bind to AA amyloid in tissue section, but not to synthetic linear peptides, and are probably reacting with discontinuous epitopes of the SAA, and in (c), they bind to species independent, i.e., the invariant peptides and epitopes of AA and SAA, which are almost identical throughout the vertebrates (reviewed in [1, 2]). These special antibodies can also be called "generic" AA/SAA antibodies. Generic SAA antibodies are mc4, mc21 and mc29 (the latter contains also an additional specificity; see later). The two monoclonals that functionally bind to variable epitopes are mc1 and mc20 and belong to a category in (a). All monoclonals and their known binding synthetic peptides are listed in **Table 1**, together with their binding to patients' and animals' AA amyloid in formalin-fixed

The binding sites of the monoclonals to SAA are shown in **Figure 5**. The invariant parts of SAA contain ancient peptide clusters preserved during their evolution from the lampreys (over 500 Mio years without hardly any changes, Wikipedia) to the mammals, including humans. Again, we as humans have the invariable peptides of SAA in common with all vertebrates and the lamprey. Therefore, these special peptides have to be of utmost importance for mechanisms related to the proteostasis of many systems. They become extremely activated when in imbalance, exemplified by injuries and inflammation or bacterial infection, and in the event that their activation cannot be resolved. This can result in a sepsis. Thus, one can assume with some likelihood that a single amino acid exchange in these 19 invariant areas must not have been accepted throughout evolution. Indications are in the literature that natural SAA behaves differently as compared to recombinant SAA or SAA with a single amino acid change or exchange [1, 2]. The importance of the proteins of the SAA family for survival can therefore hardly be overestimated and the phrase that SAA is "the hub in the interaction network" [40]

This monoclonal antibody mc1 (see **Table 1** and **Figure 5**) is of interest since it binds to the most N-terminally positioned human-specific epitope on SAA (aa 5–16), but only when it is devoid of HDL. Thus, SAA can be distinguished from SAA-HDL through the failure of mc1 to bind to the complex of SAA-HDL, since HLD conceals the mc1 epitope of SAA [15].

*4.2.3. The invariant peptide clusters of SAA and the binding sites of the SAA monoclonal* 

paraffin tissue sections [20, 23, 24] and ultrathin sections for EM [25].

*4.2.4. Properties of the individual monoclonals against AA presented here*

can express this eminent role of the SAA.

*4.2.4.1. mc1*

peptides (see above).

80 Infectious Process and Sepsis

*antibodies*

In tissue sections, this monoclonal (see **Table 1** and **Figure 5**) reliably binds to human AA amyloid and is being used for diagnostic purposes. It binds to the synthetic peptide aa 60–75 of SAA, which is located at the longest variable peptide stretch of SAA and located at the C-terminal half of the third α-helical coil, and, to a minor extent, at the small N-terminal part of the fourth coil, which contains the first invariable peptide no. 12. We do not know whether this mini peptide is part of the mc20 paratope.

### *4.2.4.3. mc4, mc21 and mc29*

These antibodies demonstrated immunohistochemical, species-independent binding to most AA-type amyloids of the vertebrates (see above). They were therefore directed against the invariant peptides of SAA, which are located on the first and second α-helical coils. Their extent and their differences are depicted in **Figure 5**. The clone mc4 reacts largely with the invariant peptide no. 4–6 on coil 1. This clone binds differently as compared to mc21 and mc29, both of which bind to the 7th peptide of coil 2, the largest invariant peptide of SAA. While mc21 seems to be only reacting with peptide no. 7, the monoclonal mc29 extends to the variant joining peptide area (aa 28–32) that is between coil 1 and coil 2. This may explain the additional, partial binding of mc29 to the variable peptides. In addition, it binds to most of the animal AA amyloids tested (see below).

### *4.2.4.4. mc1, mc4, mc13, mc29 and mc31*

This series of monoclonal antibodies has been probed and exerted to establish a monoclonal micro-ELISA for quantification of SAA [42].
