Monoclonal and engineered antibodies for human parenteral clinical use: regulatory considerations.
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Antibodies, Monoclonal↗
Biomedical subjects
T Hoffman
Publications and source records attributed to T Hoffman.
Transduction of a signal for arachidonic acid metabolism by untriggered CSF-1 receptor induces an opposite effect to that induced by CSF-1 receptor and its ligand: separate regulation of phospholipase A2 and cyclooxygenase by CSF-1 receptor/CSF-1.
The mouse hematopoietic cell line, 32D, was transfected with c-fms, which encodes for the CSF-1 receptor, a tyrosine kinase (TK). In the absence of CSF-1, transfected cells show moderate levels of arachidonic acid (AA) release and produce a substantial amount of prostaglandin E2 (PGE2) in comparison with the original cell line. Exposure of transfected cells to CSF-1, while inducing a substantial increase in arachidonate release, nevertheless resulted in inhibition of PGE2 production. Addition of ST638, a tyrosine kinase inhibitor, to cells transfected with c-fms in the absence of CSF-1 inhibited PGE2 production within 10-60 min. Its addition to the same cells in the presence of CSF-1 induced an opposite effect, but required longer treatment (24 h). In either cell type, AA release was not affected by this agent. These data indicate that CSF-1 may regulate cyclooxygenase activity. The different effect of CSF-1 receptor on PGE2 production in the presence or absence of CSF-1 and the opposite effect of a tyrosine kinase inhibitor on PGE2 suggest that both the receptor alone or the receptor-ligand complex may transduce an active, but different, signal through tyrosine phosphorylation. CSF-1 receptor and CSF-1 may exert separate, but related, effects on phospholipase A2 and cyclooxygenase activity which, in concert, or along with other tyrosine kinases, regulate prostaglandin production.
Animals↗
Brucella abortus stimulates human T cells from uninfected and HIV-infected individuals to secrete IFN gamma: implications for use of Brucella abortus as a carrier in development of human vaccines.
Brucella abortus has been characterized as a T-independent type 1 antigen/carrier in human and murine antibody responses. In this report it is shown that BA can activate human CD3+ T cells to secrete interferon-gamma (IFN gamma). Unlike mitogens, such as phytohemagglutinin, this stimulation was associated with minimal T-cell proliferation or upregulation of interleukin-2 (IL-2) receptor. Monocytes inhibited BA-mediated IFN gamma secretion since their removal resulted in increased responses, whereas adding monocytes back to cultures caused inhibition. BA elicited IFN gamma from CD4+ and CD8+ T cells, although CD4+ T cells secrete significantly more (p less than 0.05) IFN gamma than CD8+ T cells. The ability of BA to elicit IFN gamma from human T cells was inhibited in the presence of anti-Tac, suggesting that BA also induces IL-2 secretion and that IL-2 is involved in BA-mediated IFN gamma secretion. Detectable IL-2 secretion was induced by BA in the presence of anti-Tac. Exogenous IL-2 acted synergistically with BA to enhance IFN gamma secretion, suggesting that the amount of IL-2 released by BA alone was insufficient for optimal IFN gamma release. Furthermore, addition of IL-2 to T cells from individuals with poor or absent responses to BA, including individuals infected with HIV-1, restored their ability to secrete IFN gamma in response to BA. These data indicate that BA is capable not only of activating human B cells but can also induce T cells, probably of the TH1 phenotype, to secrete IFN gamma.(ABSTRACT TRUNCATED AT 250 WORDS)
Brucella abortus↗
Intracranial abnormalities requiring emergency treatment: identification by a single midline tomographic slice versus complete CT of the head.
We retrospectively reviewed 89 noncontrast computerized tomographic scans of the head in trauma patients to determine whether a single midline image could reliably identify intracranial lesions significant enough to warrant emergency surgery. We found that a midline cut was able to detect 92% of all lesions for which emergency neurosurgical evacuation was considered necessary, though it detected only 71% of the more subtle abnormalities.
Brain↗
Inflammatory mediator release from human monocytes via immobilized Fc receptors. Its potential role in adverse reactions to systemic monoclonal antibody therapy.
Human monocytes released superoxide anion, IL-1, and TNF subsequent to binding of their Fc receptor I to murine IgG2a or rabbit IgG. Fc receptor II binding to murine IgG2b or IgG1 had similar consequences. Immobilized murine monoclonal antibodies, IgG2a anti-CD3 (OKT3) or IgG1 anti-CD44 also induced superoxide anion and monokine production. Monocytes bound OKT3 via FcRI and responded to immobilized OKT3 by inflammatory mediator release in the absence of T cells. These results suggest that direct interaction of immunoglobulins with monocytes via FcR may represent an important phase of the pathophysiology of adverse reactions to systemic monoclonal antibodies.
Humans↗
Lipopolysaccharide (LPS) from Brucella abortus is less toxic than that from Escherichia coli, suggesting the possible use of B. abortus or LPS from B. abortus as a carrier in vaccines.
Brucella abortus may be useful as a component of vaccines. This is because it possesses several unique properties as a carrier that enable it to stimulate human B cells even in the relative absence of T cells. Human immunodeficiency virus type 1 proteins conjugated to B. abortus could induce neutralizing antibodies against human immunodeficiency virus type 1. Recently we showed that the characteristics of lipopolysaccharide (LPS) derived from B. abortus are similar to those of the whole bacterium in that the LPS acts as a T-independent type 1 carrier in mice. In this study we wanted to determine whether LPS derived from B. abortus is associated with the adverse effects seen with other bacterial endotoxins. LPS purified from B. abortus by butanol extraction was shown to have less than 2% (wt/wt) contamination by protein and less than 1% (wt/wt) contamination by nucleic acids and to contain 1% (wt/wt) ketodeoxyoctanic acid. Compared with LPS derived from Escherichia coli, B. abortus LPS was 10,000-fold less potent in eliciting fever in rabbits, 268-fold less potent in killing D-galactosamine-sensitized mice, and 1,400-fold and 400-fold less potent in inducing interleukin-1 beta and tumor necrosis factor alpha production, respectively. These results suggest that B. abortus LPS is much less likely than the LPS from E. coli to evoke endotoxic shock; therefore, it may be feasible to incorporate B. abortus as a component of vaccines.
Animals↗
A role for guanine-nucleotide-binding proteins in mediating T-cell-receptor coupling to inositol phospholipid hydrolysis in a murine T-helper (type II) lymphocyte clone.
Perturbation of the T-cell receptor (TCR) complex is followed by the rapid hydrolysis of inositol phospholipids (InsPL) by phospholipase C (PLC), producing diacylglycerol and inositol phosphates, which act as second messengers in signal transduction. The mechanism coupling the TCR to InsPL hydrolysis is not clearly defined, and no information is available on this mechanism in the CD4+ helper subset of T-lymphocytes (Th). We have tested the hypothesis that guanine-nucleotide-binding proteins (G-proteins) may couple the TCR to PLC in a murine Th type II (Th2) cell clone. Cell permeabilization with streptolysin O (SLO) or tetanolysin (TL) was used to allow membrane-impermeable nucleotides access to intracellular sites of action. Exposure of permeabilized Th2 cells to guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), a non-hydrolysable GTP analogue, resulted in a 2.1-2.5-fold increase in inositol phosphate generation. Similarly, perturbation of the TCR with the monoclonal antibody 145.2C11 (directed against the epsilon-chain of the CD3 component of the TCR) resulted in a 3.1-4.2-fold increase in InsPL hydrolysis by permeabilized cells. Both lysins were similarly effective in allowing GTP gamma S induction of InsPL hydrolysis, but TL-permeabilized cells responded better to TCR perturbation than SLO-treated cells. A role for G-proteins in TCR coupling to PLC was further supported by the inhibition of TCR-induced InsPL hydrolysis by guanosine 5'-[beta-thio]diphosphate (GDP beta S), a guanine nucleotide analogue that inhibits G-protein function. ATP was required for TCR-mediated InsPL hydrolysis, and potentiated GTP gamma S-induced hydrolysis. Other nucleotides (i.e. CTP, GDP, GTP, ITP) did not affect the response. These data indicate that G-proteins may contribute to the regulation of PLC activation in Th2 cells, coupling it to the TCR.
Adenosine Triphosphate↗
Down-regulation of surface FcRI and decrease in antibody-dependent cellular cytotoxicity of cultured monocytes. Reversal by monensin or cytochalasin-D.
To investigate the mechanisms involved in regulation of antibody-dependent cellular cytotoxicity (ADCC) mediated by human monocytes, 51Cr-labeled sheep red blood cells (RBC) were used as target cells in vitro. Monocytes incubated overnight at 37 degrees C before addition of SRBC and antibody exhibited a significant decrease in ADCC activity compared with freshly isolated cells. This pattern was observed with monocytes from all donors tested, regardless of the media used for culture. Supernatants from monocyte cultures did not inhibit the cytotoxic ability of fresh monocytes and cycloheximide, a protein synthesis inhibitor, could not reverse ADCC suppression in cultured monocytes, indicating that the alteration in ADCC is probably not due to inhibitory molecules secreted or synthesized during incubation. A correlation between the decrease in the number of surface FcRI and loss in ADCC ability of cultured monocytes was found. One mechanism for the reduced FcRI expression of 1-day-old monocytes may be rapid internalization that exceeds the rate of reexpression, because cytochalasin-D or monensin, each of which inhibits receptor internalization, maintained FcR expression as well as ADCC ability of cultured monocytes. These data illustrate mechanisms whereby alteration in the number of receptors may underlie loss of receptor-mediated functions, or be involved in augmentation of their biologic activity. The findings that important monocyte functions change under conditions of storage or culture have relevance to in vitro testing of various immune functions of monocytes performed clinically to monitor or guide therapy.
Animals↗
Calcium-dependent eicosanoid metabolism by concanavalin A-stimulated human monocytes in vitro. Synergism with phorbol ester indicates separate regulation of leukotriene B4 synthesis and release.
Human monocytes obtained by counter-current centrifugal elutriation released arachidonic acid when challenged in vitro with Con A, as well as with other soluble (PMA or ionomycin) or particulate stimuli (serum-treated zymosan). Cyclo-oxygenase metabolites were the principal eicosanoids detected in the supernatants of Con A-stimulated, [3H]arachidonate-labeled monocytes, 5-Lipoxygenase (5-LO) products, such as leukotriene B4 (LTB4), were conspicuously absent. Release of arachidonate and its metabolites in response to Con A was dependent on the presence of extracellular Ca2+, but not Mg2+. In contrast to serum-treated zymosan challenge, which resulted in increased inositol trisphosphate and LTB4 release, Con A-induced inositol phospholipid hydrolysis in monocytes was limited to phosphatidylinositol or phosphatidylinositol monophosphate. Despite an inability to augment LTB4 release, Con A or PMA induced a loss of 5-lipoxygenase from a cytosolic compartment that was similar to that achieved with a calcium ionophore (ionomycin), a potent stimulus for LTB4 generation. When cell-associated LTB4 was evaluated, evidence for increased LTB4 production was obtained in response to either stimulus (PMA greater than Con A). In combination, however, PMA and Con A treatment resulted in monocyte LTB4 release comparable with that observed with the calcium ionophore or STZ. LTB4 release in response to all stimuli tested was inhibited by MK-886, a drug that binds to 5-lipoxygenase-activating protein. These results indicate the following: 1) Phospholipase A2 activation and attendant arachidonic acid release induced by agents that increase intracellular Ca2+ and/or generate diacylglycerol results in increased synthesis and release of PG and increased synthesis of leukotrienes, but not necessarily leukotriene release. 2) 5-LO translocation, which may occur independently of increased intracellular Ca2+, may be necessary for LTB4 generation but is insufficient for its release. 3) 5-Lipoxygenase-activating protein activity is necessary for 5-LO activation and LTB4 release in response to all stimuli investigated here. 4) Phorbol ester, an activator of protein kinase C, may synergize with agents such as Con A (which by themselves induce a minimal intracellular Ca2+ rise), so as to result in the release of LTB4. Thus, Con A may represent a class of surface receptor-aggregating agents that initiates inflammatory changes or immunomodulation associated with liberation of PG and might predispose to release of other inflammatory mediators, such as leukotrienes, in the presence of additional signals including protein kinase activation.
Arachidonate 5-Lipoxygenase↗
Stimulatory effect of counterflow centrifugal elutriation in large-scale separation of peripheral blood monocytes can be reversed by storing the cells at 37 degrees C.
Transfusion of peripheral blood monocytes may be of benefit as adjuvant treatment of leukopenic patients with serious infections. To study the feasibility of this approach, a large-scale monocyte separation procedure employing leukapheresis, density gradient centrifugation, and counterflow centrifugal elutriation was established. By processing 5 to 6 liters of normal donor blood, it was possible to obtain a mean of 1.1 x 10(9) (range: 0.5-1.7 x 10(9) cells) of mononuclear cells, of which 89% (range: 82-94%) were monocytes by Wright's stain morphology. When the elutriation was performed in XVIVO-10, a commercially available, serum-free medium developed for adoptive immunotherapy, spontaneous secretion of superoxide by the monocytes was significantly higher than for monocytes elutriated in Hanks' balanced salt solution without calcium and magnesium or non-elutriated peripheral blood mononuclear cells. This stimulated state of the monocytes was observed both immediately after elutriation and after overnight storage at 4 degrees C, and it was not affected by the type of storage vessel used. Overnight storage of the monocytes at 37 degrees C resulted in a reversal of the stimulated state of the cells. Monocytes elutriated in XVIVO-10 and kept overnight at 4 degrees C released high amounts of arachidonic acid. A subsequent decrease in this release was seen after additional storage at 37 degrees C for 18 hours. These observations demonstrate that separation and storage variables have important effects on the state of stimulation of monocytes. Further investigations of such variables may suggest improved procedures for preparation and storage of these cells, as well as possible ways to stimulate monocytes prior to transfusion.
Arachidonic Acid↗
Immunogenicity of lipopolysaccharide derived from Brucella abortus: potential as a carrier in development of vaccines for AIDS.
In view of its unique ability to stimulate human B cells, we have considered using Brucella abortus (BA) as a carrier for human vaccines. Recently we showed that HIV-1 coupled to BA, but not unconjugated HIV-1, was able to stimulate murine responses even in the relative absence of CD4+ T cells. This result suggested that HIV-BA may be useful in boosting the immunity of individuals infected with HIV-1 and who have impaired CD4+ T cell function. In order to refine this carrier we purified lipopolysaccharide (LPS) from BA and examined its effects on immune responses. Similar to LPS from E. coli (LPS-EC), LPS-BA was capable of stimulating mouse B cells to proliferate. In addition, LPS-BA could activate mouse spleen cells to secrete antibodies in vitro. Isotype analysis revealed that IgM and all the IgG subclasses were elicited. When comparing these responses to those of LPS-EC, LPS-BA induced a greater percentage of IgG2a and LPS-EC evoked more IgG3. IgG2a is probably important in protection against murine viral infection. LPS-BA was haptenated with trinitrophenol TNP-LPS (BA) and tested for carrier effect. Similar to TNP-BA and TNP-LPS (EC), TNP-LPS (BA) triggered anti-TNP antibody of the IgM and all IgG subclasses. In contrast, TNP-ficoll induced mainly IgM and only small amounts of IgG3. These results suggest that LPS-BA, like intact BA, behaves as a T-independent type 1 carrier, and as such may be advantageous as a carrier for human vaccines development.
AIDS Vaccines↗
Selective inhibition of PGE2 production in cells transfected with c-fms encoded CSF-1 receptor genes by the tyrosine kinase inhibitor, ST638.
In this study, we investigated the effect of ST638, a novel tyrosine kinase inhibitor, on TPA and ionomycin-stimulated PGE2 production after transfection of c-fms encoded CSF-1 receptor (CSF-1R) DNA (with or without transforming activity) into the myeloid progenitor cell line, 32D. ST638 inhibited prostaglandin E2 (PGE2) production induced after transfection of normal c-fms into 32D cell line, but failed to inhibit PGE2 production induced in 32D cells transformed with c-fms containing a point mutation at tyrosine 969 in the intracellular domain (substitution with phenylalanine) and at leucine 301 in the extracellular domain (substitution with serine). The selective effect on PGE2 production in normal c-fms-bearing cells by ST638 may indicate the presence of different induction pathways, one sensitive to ST638 and the other not. Alternatively, the phosphorylation site at tyr969 in c-fms may be a site of ST638 action, and upon its removal from the transforming c-fms, ST638 loses its inhibitory effect.
Arachidonic Acid↗
Augmentation of monocyte-mediated antibody-dependent cellular cytotoxicity by protein synthesis inhibitors: evidence for an endogenous regulatory mechanism.
Protein synthesis inhibitors, cycloheximide and puromycin, were used in cytotoxic assays employing human peripheral blood monocytes as effectors and sheep erythrocytes as target cells. ADCC could be initiated and could also achieve its full lytic activity in the absence of new protein synthesis. Furthermore, an augmentation of ADCC was observed in the presence of protein synthesis inhibitors. This augmentation was due to an increase in the cytotoxic ability of effector cells rather than a change in the lytic susceptibility of the target. Enhanced cytotoxic potential could not be attributed to an increase in the expression of FcRI but could be due to increased availability of antibody for mediating ADCC as a result of reduced numbers of FcRII. Suppression of prostaglandin-E2 release by monocytes was noted in the presence of cycloheximide, possibly as a result of inhibition of synthesis of cyclooxygenase. However, prostaglandin-E2 and other arachidonic acid metabolites did not appear likely to play a role in negatively regulating human monocyte ADCC since neither cytotoxicity nor cycloheximide-induced augmentation was affected by the presence of exogenous prostaglandin-E2 or arachidonic acid. Cycloheximide was found to induce the secretion of superoxide anions by monocytes, but a role for reactive oxygen species in cycloheximide-induced augmentation of ADCC could not be established by experiments involving the use of catalase or superoxide dismutase. These results raise the possibility that a rapidly turning over protein which negatively regulates monocyte-mediated ADCC exists.
Antibody-Dependent Cell Cytotoxicity↗
Absence of modulation of monokine production via endogenous cyclooxygenase or 5-lipoxygenase metabolites: MK-886 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2- dimethylpropanoic acid), indomethacin, or arachidonate fail to alter immunoreactive interleukin-1 beta, or TNF-alpha production by human monocytes in vitro.
Human peripheral blood monocytes exposed to MK-886 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2- dimethylpropanoic acid) at doses which abolish formation of 5-lipoxygenase metabolites showed unaltered interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) levels in response to phorbol ester, concanavalin A, serum-treated zymosan, or lipopolysaccharide. Indomethacin (10 microM), alone or in combination with MK-886, also failed to modulate monokine production in response to any stimulus. Exogenous arachidonate (3-30 microM) which augmented the formation of PGE2 and LTB4 in the absence of stimulation, also had no effect on monokine production. LPS-induced IL-1 and TNF production occurred despite stimulation of PGE2 synthesis. The results make a role for endogenous prostaglandins and leukotrienes in the regulation of monocyte IL-1 beta and TNF-alpha production unlikely. These data also indicate that MK-886, a novel inhibitor of 5-lipoxygenase product formation, is a potentially useful leukotriene inhibitor which does not affect monokine production.
Analysis of Variance↗
Production of a novel antigen by conjugation of HIV-1 to Brucella abortus: studies of immunogenicity, isotype analysis, T-cell dependency, and syncytia inhibition.
In the present study inactivated human immunodeficiency virus type 1 (HIV-1) was conjugated to Brucella abortus and tested for immunogenicity in normal and anti-L3T4-treated BALB/c mice. HIV-BA was more immunogenic than uncoupled HIV in normal mice, since 6-fold less virus in HIV-BA preparations elicited higher titer responses than HIV-1 alone. Furthermore, the HIV-BA antibody response reached higher levels before the HIV-1 response. Immunoblot analysis showed that most of the HIV-1 antigens were recognized by antibodies induced by either HIV-1 or HIV-BA. Isotype analysis revealed that HIV-1 induced similar levels of IgG1 and IgG2a antibodies, whereas the IgG2a responses to HIV-BA were more pronounced than the IgG1 response. These different IgG subclass patterns suggest that conjugation of HIV-1 to BA changed the immunogenic nature of HIV-1. The requirement for helper T cells was examined by immunizing mice that were depleted of CD4+ T cells by in vivo anti-L3T4 treatment. Under these conditions the IgG responses to HIV-1 were completely eliminated. Although HIV-BA antibody responses were markedly reduced in anti-L3T4-treated mice, anti-HIV-1 antibodies, mainly of the IgG2a isotype, were produced. The antibodies generated by HIV-1 and HIV-BA immunization were also tested for their ability to inhibit syncytia formed by infecting CD4 + CEM cells with gp160 vaccinia. Sera from normal mice, immunized with either HIV-1 or HIV-BA were capable of inhibiting syncytia. In contrast, following anti-L3T4 treatment, only mice immunized with HIV-BA, but not HIV-1, produced antibodies capable of inhibiting syncytia.
Animals↗
The chemistry of acetaldehyde-protein adducts.
Acetaldehyde reacts with a variety of proteins to form both stable and unstable adducts. Unstable adducts are readily reversible and are largely represented by Schiff bases. Stable adducts are essentially irreversible products that are characterized by their resistance to various treatments. The structures of stable adducts have not been established, but it appears that the epsilon-amino group of certain lysines and the amino group of N-terminal amino acids participate in aldehyde binding. Stable adducts can form in the absence of a reducing agent, such as sodium cyanoborohydride, and are distinctly different than the ethylated amino groups formed under reductive conditions. Upon prolonged incubation, stable binding of acetaldehyde results in the formation of fluorescent products, and Schiff bases serve as intermediates of these advanced stable products. It is likely that several acetaldehyde molecules and perhaps at least two amino groups participate in the formation of these stable fluorescent products. Such a reaction mechanism could account for both inter-and intra-molecular cross-linking of proteins. Furthermore, it is likely that stable adducts are represented by multiple products whose structures may vary, depending upon the particular target protein. A reaction mechanism involving an aldol condensation and generation of a crotonaldehyde Schiff base is proposed as an essential step in stable adduct formation.
Acetaldehyde↗
Differential changes in lipid metabolism of myeloid and lymphoid cell lines induced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA).
Treatment of the myeloid cell lines, U-937 or HL-60, with 10 nM of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), for 24 h increased the rate of incorporation of [3H]glycerol into total chloroform extracts. A proportionally greater labeling of the non-polar lipid (NL) fraction compared to the polar, phospholipid (PL), fraction was observed. Chromatographic analysis showed a 6-fold increase in the labeling of triacylglycerols (TAG), a 2-fold increase in diacylglycerols, and no changes in monoacylglycerols. PL labeling showed a 3-fold increase in phosphatidylcholine (PC). The effect of TPA on TAG labeling was selectively observed in myeloid cell lines. No such a change was found in the lymphoid cell line. MOLT-3, which did respond to TPA with increased PC labeling. Incorporation of [3H]arachidonic acid (AA) into TAG by U-937 cells was selectively increased (2.5-fold) after treatment with TPA for 24 h. Treatment of U-937 cells with TPA in serum-free medium resulted in no increased labeling of TAG. These studies suggest that changes in TAG metabolism may be characteristic of myeloid differentiation and depend on the presence of serum factor(s).
Arachidonic Acid↗
High-performance liquid chromatographic separation of inositol phosphate isomers employing a reversed-phase column and a micellar mobile phase.
Surfactants have been employed in high-performance liquid chromatography (HPLC) for the separation of ionic and non-ionic compounds. We have developed a method employing a reversed-phase column and a mobile phase containing a surfactant, hexadecyltrimethylammonium hydroxide (HDTMA+OH-), for the separation of several inositol phosphate positional isomers. Various parameters were studied for their effect on the chromatographic capacity factor (k'). They included the concentration of HDTMA+OH-, the pH of the bulk micellar suspension and the addition of inorganic salts to the mobile phase. Resolution of the inositol monophosphates was controlled by a mixed mechanism, where the predominant elements were electrostatic forces and the formation of micelles. The elution of the inositol polyphosphate isomers was obtained by increasing the amount of a non-polar solvent, in agreement with an ion-pairing process. This method represents an alternative to ion-exchange HPLC. If offers a practical advantage when detection of radiolabeled samples by in-line radioactive flow detectors is required, because low-quenching solvents with good miscibility with scintillant fluids are employed. The analysis of various chromatographic conditions, the system reproducibility and its application to the analysis of biological samples are described.
Chromatography, High Pressure Liquid↗