Bovine Serum Albumin - an overview (2023)

General information

Bovine serum albumin is often used as protein supplement in cell culture media, and residual bovine serum albumin in some formulations has occasionally caused adverse effects. Symptoms are usually mild, such as itching and urticaria. An anaphylactic reaction has been described in a patient undergoing bone marrow transplantation after the bone marrow cells had been kept in bovine serum albumin. As a component of sperm processing media, bovine serum albumin has caused adverse effects after intrauterine insemination [1]. Bovine serum albumin has also been reported to cause membranous nephropathy in early childhood [2]. The risk of transmission of nvCJD by recombinant proteins is unknown, as cell cultures used for manufacturing them often contain bovine serum albumin [3].

General information

Bovine serum albumin is often used as protein supplement in cell culture media, and residual bovine serum albumin in some formulations has occasionally caused adverse effects. Symptoms are usually mild, such as itching and urticaria. An anaphylactic reaction has been described in a patient undergoing bone marrow transplantation after the bone marrow cells had been kept in bovine serum albumin. As a component of sperm processing media, bovine serum albumin has caused adverse effects after intrauterine insemination [1]. Bovine serum albumin has also been reported to cause membranous nephropathy in early childhood [2]. The risk of transmission of nvCJD by recombinant proteins is unknown, as cell cultures used for manufacturing them often contain bovine serum albumin [3].

Assessment of Cardiac Structure and Function

The primary goal of the echocardiographic examination remains assessment of cardiac structure and function. Each chamber, valve, and great vessel can be assessed qualitatively and quantitatively to define any alterations in size, geometry, and patency. Measurements of cardiac structures are typically made in various locations throughout the heart, and linear, area, or volumetric measures can be obtained. These methods are often complementary to one another. For example, although volumetric measurements of the left ventricle (see later) are generally considered best suited to characterize LV size, many laboratories continue to record linear cavity measurements, because there is extensive literature correlating these measures with outcomes in numerous disease states. Moreover, linear measures may be subject to less variability than area- or volume-based measures and therefore may be more reliable for assessing changes over time.

Tables 14.1 to 14.3 show established normal values on echocardiography. For LV linear dimensions and volume,Table 14.1 gives the normal ranges for the general population, but ideally one should take into account not only sex, but also body surface area (BSA) and age² (eFigs. 14.3and14.4). Current American Society of Echocardiography (ASE) consensus statements also provide partition values—that is, mild, moderate, and severely abnormal ranges—for LV size, mass, and ejection fraction and left atrial (LA) volume, but caution that the ranges were arrived at by experience-based consensus only, and that degree of abnormality does not necessarily connotate a direct correlation with outcomes or prognosis (Table 14.2). Normal values for LV parameters obtained with 3D echocardiography also exist and appear accurate and reproducible when image quality is good. In general, LV volumes calculated by 3D imaging are smaller than data generated from CMR data, but correlations with trends in sex and body surface area hold true.²

Albumin-based Stationary Phases

BSA and HSA are closely related proteins and, consequently, the chromatographic properties of the chiral stationary phases based on these proteins are similar. Sometimes the elution order is reversed between chiral stationary phases based on these proteins; on the HSA-based phases (S)-warfarin elutes before (R)-warfarin, whereas on the BSA-based phases the opposite elution order is observed.

Fluid Resuscitation

Fluid resuscitation should begin soon after the injury has occurred, in the ED before transferring to a burn center. For most children, theParkland formula is an appropriate starting guideline for fluid resuscitation (4 mL lactated Ringer solution/kg/% BSA burned). Half the fluid is given over the 1st 8 hr, calculated from the time of onset of injury; the remaining fluid is given at an even rate over the next 16 hr. The rate of infusion is adjusted according to the patient's response to therapy. Pulse and blood pressure should return to normal, and an adequate urine output (>1 mL/kg/hr in children; 0.5-1.0 mL/kg/hr in adolescents) should be accomplished by varying the IV infusion rate. Vital signs, acid-base balance, and mental status reflect the adequacy of resuscitation. Because of interstitial edema and sequestration of fluid in muscle cells, patients may gain up to 20% over baseline (preburn) body weight. Patients with burns of 30% BSA require a large venous access (central venous line) to deliver the fluid required over the critical 1st 24 hr. Patients with burns of >60% BSA may require a multilumen central venous catheter; these patients are best cared for in a specialized burn unit. In addition to fluid resuscitation, children should receive standard maintenance fluids (seeChapter 69).

During the 2nd 24 hr after the burn, patients begin to reabsorb edema fluid and to experience diuresis. Half the 1st day's fluid requirement is infused as lactated Ringer solution in 5% dextrose. Children <5 yr old may require the addition of 5% dextrose in the 1st 24 hr of resuscitation. Controversy surrounds whethercolloid should be provided in the early period of burn resuscitation. One preference is to use colloid replacement concurrently if the burn is >85% of total BSA. Colloid is usually instituted 8-24 hr after the burn injury. In children <12 mo old, sodium tolerance is limited; the volume and sodium concentration of the resuscitation solution should be decreased if the urinary sodium level is rising. The adequacy of resuscitation should be constantly assessed by means of vital signs as well as urine output, blood gas, hematocrit, and serum protein measurements. Some patients require arterial and central venous lines, particularly those undergoing multiple excision and grafting procedures, as needed, for monitoring and replacement purposes. Central venous pressure monitoring may be indicated to assess circulation in patients with hemodynamic or cardiopulmonary instability. Femoral vein cannulation is a safe access for fluid resuscitation, especially in infants and children. Burn patients who require frequent blood gas monitoring benefit from radial or femoral arterial catheterization.

Oral supplementation may start as early as 48 hr after the burn. Milk formula, artificial feedings, homogenized milk, or soy-based products can be given by bolus or constant infusion through a nasogastric or small bowel feeding tube. As oral fluids are tolerated, IV fluids are decreased proportionately in an effort to keep the total fluid intake constant, particularly if pulmonary dysfunction is present.

8.9.3.1.1 Bovine serum albumin

BSA-immobilized agarose was used for the enantioseparation of tryptophan: d- and l-tryptophan were clearly resolved using the CSP, and the d-form was eluted first, consistent with the previous binding studies in solution.¹ Later, silica gels and polymers were used for immobilization of BSA as the base materials.^24,25 Silica gels were mainly used, whereas the polymers used were hydroxyethylmethacrylate and poly(styrene-divinylbenzene) perfusion beads.^24,25 CSPs based on BSA were used for the separations of a variety of acidic and neutral enantiomers such as N-derivatized amino acids, aromatic amino acids, uncharged solutes, sulfoxides, and sulfoximine derivatives.^24,25

It was considered that BSA consisted of 582 amino acid residues. Recently, Hirayama et al.³² reported that Tyr156 was lacking in the previous sequence and that BSA consisted of 583 amino acid sequences. BSA-fragments were isolated and immobilized onto silica gels.²⁴ First, it was reported that CSPs based on a BSA-fragment(s) had less capacity and enantioselectivity than the intact BSA-based CSPs, being less stable. However, according to other reports, BSA fragment-based CSPs yielded higher enantioselectivity for lorazepam and benzoin because of the more bound amounts, and lower enantioselectivity for other compounds tested, compared with the intact BSA-based CSPs. These differences might arise from both the purification method and the immobilization procedures. The lower enantioselectivity might be due to changes in the globular structure of BSA-fragments or changes in the local environment around binding sites.

4.2 Preparation of 7% BSA/5mM cold palmitate mixture

Tip: If performing this assay for the first time, it may be useful to vary the amount of cold palmitate per reaction to find the ideal concentration of total palmitate per reaction for your sample, tissue of choice, and experimental conditions. To do this, use the 7% BSA control solution to adjust the concentration of cold palmitate. Typical concentrations of cold palmitate for tissue homogenates and cells are 0.1–0.5mM and 0.3–1.0mM, respectively.

3.23.5.3.2 Acrosome reaction induced by neoglycoproteins containing Man and GlcNAc

Several BSA-based ‘neoglycoproteins’ were shown to stimulate the acrosome reaction. The binding of Man,Flu-BSA results in a time-dependent receptor aggregation and the induction of acrosome exocytosis in capacitated sperm populations from fertile donors.²⁰⁰ N-acetyl-α-glucosamine (α-GlcNAc) and α-mannose (αMan-BSA) may interact with the putative receptor for one protein of zona pellucida: ZP3 in human spermatozoa.^{195,202–205} This binding results in the exocytosis of the sperm acrosome (AR).²⁰⁶ The GlcNAc-BSA-induced acrosome reaction is inhibited by a small ligand: N-acetylglucosamine (GlcNAc) and by a purified soluble hydrolase, β-N-acetylglucosaminidase. The induction of the AR with Man-BSA was inhibited by mannose, while soluble α-mannosidase was only partially effective. These data suggest that binding sites for GlcNAc and Man seem to be involved in the induction of the AR in human sperm. The induction of AR in human spermatozoa by GlcNAc-BSA could be used to predict their fertilizing ability in vitro.²⁰⁵

The induction of the acrosome reaction by GlcNAc-BSA and Man-BSA has been shown to involve voltage-dependent Ca²⁺ channels and a Gi-like guanine regulatory protein.^203,207 The GlcNAc-BSA- or Man-BSA-induced AR was completely inhibited by preincubation of spermatozoa with calcium antagonists, indicating a link between the binding of sugar residues of the neoglycoproteins and channel activation. The pretreatment of spermatozoa with Pertussis toxin (PTx) inhibits GlcNAc-BSA- or Man-BSA-induced AR, whereas cholera toxin has no effect. Therefore, the transduction mechanism for GlcNAc-BSA- and Man-BSA-induced AR involves G-proteins of the inhibitory type (GI).²⁰⁴

The effect of ‘peritoneal fluid’ on the human sperm acrosome reaction (AR) was tested.²⁰⁸ When the AR was induced by GlcNAc-BSA, pre-incubation with peritoneal fluid reduced (60%) the percentage of AR, while peritoneal fluid from either the endometriosis group or infertile patients without endometriosis caused no significant differences. These data indicated that peritoneal fluid possesses a protective factor which prevents premature AR.

BSA and cBSA

BSA (MW 67,000) and cationized BSA (cBSA) are highly soluble proteins containing numerous functional groups suitable for conjugation. Even after extensive modification with hapten molecules these carriers usually retain their solubility. The exception to this statement is when hydrophobic peptides or other sparingly soluble molecules are conjugated to the proteins. Modification of any carrier with numerous hydrophobic haptens may cause enough masking of the hydrophilic surface to result in precipitation. Depending on the degree of precipitation, such conjugates are often still useful in generating an immune response. To limit the production of insoluble complexes, however, the conjugation reaction can be scaled back to reduce the level of carrier modification and thus reduce or eliminate precipitation.

BSA possesses a total of 59 lysine ε-amine groups (with only 30–35 of these typically available for derivatization), one free cysteine sulfhydryl (with an additional 17 disulfides buried within its three-dimensional structure), 19 tyrosine phenolate residues, and 17 histidine imidazole groups. In addition, the presence of numerous carboxylate groups gives BSA its net negative charge (pI 5.1).

The highly positive charge of cBSA dramatically increases its immunogenicity. The positive character of the molecule aids in its binding to antigen-presenting cells (APCs) in vivo, the first step in antibody production. The protein thus gets incorporated into the APCs faster than molecules having lower pI values. It also gets processed at an accelerated rate, producing a quicker immune response, and one that occurs with greater concentrations of specific antibody (Domen et al., 1987; Muckerheide et al., 1987b; Apple et al., 1988; Domen and Hermanson, 1992; Chen et al., 2002).

Cationized BSA used as a carrier protein also induces a similar increase in the production of antibodies against any attached hapten molecules. Even when haptens are coupled through cBSA’s amine residues, the overall charge of the molecule remains basic enough to augment the immune response beyond that usually obtained using other carriers. This augmentation occurs even when the attached molecule is not merely a hapten, but a larger antigen macromolecule. Conjugation of a complete antigen (a molecule able to generate an immune response on its own) to cBSA causes an increased immune response against the antigen beyond that normally obtainable for the native antigen administered in unconjugated form (Domen and Hermanson, 1992). Bioconjugates of cBSA have been developed for polysaccharide-based glycoconjugate vaccines (Burtnick et al., 2012), for vaccines strategies in overcoming atherosclerosis (de Jager and Kuiper, 2011), and to make cationic cBSA-nanoparticles for treatment of Parkinson’s disease (Rodríguez et al., 2011).

After the boost, the group of mice receiving the AV–cBSA conjugate generated over twice the antibody response as the group receiving the peptide conjugated to native BSA.

A corollary to the use of cBSA as a carrier protein is that its increased immune response often abrogates the use of complete Freund’s adjuvant, which is a source of concern because of its potential side-effects in animals. A relatively innocuous mixture with alum is usually all that is required as adjuvant to result in good antibody production.

As mentioned previously for KLH, DMSO may be used to solubilize hapten molecules that are rather insoluble in aqueous environments. Conjugation reactions may be performed in solvent/aqueous phase mixtures to maintain some solubility of the hapten once it is added to a buffered solution. BSA remains soluble in the presence of up to 35% DMSO, becomes slightly cloudy at 40%, and precipitates at 45% (v/v).

4.1 Ligand-based screening