Development of monoclonal antibodies against creatine kinase MB2.

An increase of creatine kinase MB (CKMB) in serum has long been used as a marker for acute myocardial infarction (AMI). It is important with an early diagnosis and since the amount of the CKMB2 isoform rises above reference levels much earlier than the total creatine kinase amount, quantification of CKMB isoforms could be a feasible alternative for early analysis. The two CKMB isoforms differ by only one C-terminal lysine residue, which makes it difficult to separate one from the other. To overcome this problem, monoclonal antibodies were produced using unique peptides as antigen in hybridoma technology. Two peptides with 16 and 15 amino acids corresponding to the C-terminal end of the M-subunits of CKMB2 and CKMB1, respectively, were conjugated to keyhole limpet hemocyanin and used as antigens. Sixteen different monoclonal antibodies to these peptides were obtained and characterized. Their specificity was analyzed by immunoassay and 10 of the antibodies showed cross-reactive binding to creatine kinase. Surface plasmon resonance based biosensor analysis was used to determine affinity and kinetics towards the peptides and the epitopes of four of the antibodies were studied by means of phage display. Some of these antibodies have binding properties that might qualify them for use in the establishment of procedures allowing early diagnosis of AMI.

Prediction of affinity and kinetics in biomolecular interactions by affinity chromatography.

Computer simulation of affinity chromatography is a valuable tool for accurate prediction of column performance. In our study affinity pairs based on lectin and antibody interactions with carbohydrates have been used as model systems. In this well-characterized system we have demonstrated the usefulness of the simulation approach for determination of affinity and kinetics. These properties are typically difficult to obtain for many weakly interacting molecular species (i.e., when dissociation constants (K(D)) are greater than 10(-5) M). The influence of affinity and kinetics on peak broadening in affinity chromatography has also been investigated.

A lectin immunosensor technique for determination of alpha(1)-acid glycoprotein fucosylation.

The fucosylation of alpha(1)-acid glycoprotein (AGP), an acute-phase protein, is known to change in association with inflammatory diseases. Thus, fucosylation of AGP could be a potential diagnostic or prognostic marker. The change in fucosylation has previously been investigated using crossed affinoimmunoelectrophoresis, high-pH anion-exchange chromatography, and lectin ELISA. This study describes a surface plasmon resonance-based affinity biosensor assay for quantification of the fucosylation of AGP. Diluted EDTA plasma or serum was injected directly in a BIACORE 2000 biosensor. AGP was captured on the sensor surface using immobilized antibodies and a fucose-binding lectin from Aleuria aurentia was then used for the detection of fucosylation. The feature of the biosensor makes it possible to determine both the amount of bound AGP and the amount of bound lectin. Using a calibration curve it was possible to obtain a fucosylation ratio that was independent of AGP concentration. The assay was validated against a lectin ELISA and used to follow inflammation in patients with severe burns.

Origin of corticospinal neurones evoking monosynaptic excitation in C3--C4 propriospinal neurones in the cat.

Intracellular recording was made from propriospinal neurones (PNs) in the C3-C4 spinal cord segments in the cat (alpha-chloralose anaesthesia). The effect of electrical stimulation of corticospinal neurones (CSNs) in the cortex was investigated. Short C3-C4 PNs were identified by antidromic activation of their axons in the ventral horn in C6/C7 and in the lateral reticular nucleus. Long PNs were antidromically identified from Th12-13. In short PNs, monosynaptic excitory postsynoptic potentials (EPSPs) were elicited from the rostral part of the lateral sigmoid gyrus, the lateral part of the anterior sigmoid gyrus in area 4 gamma and in the adjacent area 6. Two subtypes of short PNs were identified. PNs of type I received monosynaptic EPSPs from the rostral part of the lateral sigmoid gyrus, the lateral part of the anterior sigmoid gyrus in area 4 gamma, which is from the same region as disynaptic cortical EPSPs were evoked in forelimb motoneurones. PNs of type II received monosynaptic EPSPs from regions slightly more rostrally in the anterior sigmoid gyrus in area 4 gamma and in the adjacent area 6, which is outside the region from which disynaptic EPSPs could be evoked in forelimb motoneurones. Long PNs received monosynaptic EPSPs, like the short PNs, by stimulation in the rostral part of the lateral sigmoid gyrus, the lateral part of the anterior sigmoid gyrus in area 4 gamma and in the adjacent area 6. In contrast, the long PNs also received monosynaptic EPSPs from area 3b near the border of area 1. The present results show segregation of the cortical control to functionally different premotoneuronal systems and suggest that this control could in part be separated for subtypes of short C3-C4 PNs.

Analysis of carbohydrates using liquid chromatography--surface plasmon resonance immunosensing systems.

An immunosensing system based on surface plasmon resonance (SPR) was used for on-line detection and characterization of carbohydrate molecules separated by high-performance liquid chromatography. These analytes, with or without serum, were continuously separated and analyzed in the combined liquid chromatography-surface plasmon resonance (LC-SPR) system. By using weak and readily reversible monoclonal antibodies, the SPR system allowed specific on-line monitoring of the substances. To increase the specificity of the immunosensor, nonrelevant antibodies were used as reference in a serial flow cell. The sensitivity of the LC-SPR system was dependent on molecular weight of the carbohydrate, affinity of binding, and design of the sensor.

Origin of corticospinal neurones evoking disynaptic excitation in forelimb motoneurones mediated via C3-C4 propriospinal neurones in the cat.

Intracellular recording was made from forelimb motoneurones in the cat (alpha-chloralose anaesthesia) during electrical stimulation of corticospinal neurones (CSNs) and their afferents in the contralateral cortex. Axons of the CSNs were stimulated in the contralateral pyramid. The corticospinal tract was transected at the C5/C6 segmental border in order to restrict transmission through the C3-C4 propriospinal neurones (C3-C4 PNs). Di- and trisynaptic cortical EPSPs could be evoked after transection of the corticospinal fibres in C5/C6 but not after a corresponding transection in C2/C3. Pyramidal stimulation elicited disynaptic EPSPs that were abolished after a C2/C3 transection. Disynaptic pyramidal EPSPs, mediated via C3-C4 propriospinal neurones could be facilitated by a single cortical stimulation. It is concluded that di- and trisynaptic cortical EPSPs and disynaptic pyramidal EPSPs are mediated via the same C3-C4 PNs. Cortical surface stimulation showed that di- and trisynaptic cortical EPSPs could be evoked from distinct spots in the lateral part of the anterior sigmoid gyrus (Sig. a) and/or in the rostral part of the lateral sigmoid gyrus (Sig. l). No cortical EPSPs or facilitation of pyramidal disynaptic EPSPs was evoked from the posterior part of the Sig. l, posterior sigmoid gyrus, coronal gyrus, lateral gyrus, suprasylvian gyrus and ectosylvian gyrus. It is concluded that the CSNs, which issue the command for visually guided target reaching with the forelimb via the C3-C4 PNs, originate in the lateral part of the Sig. a and in the rostral part of the Sig. l. A dual representation of the forelimb in the primary motor cortex of the cat has previously been proposed. The present results show that with respect to one identified interneuronal system like the C3-C4 propriospinal system, the CSNs may have their origin restricted to one region of the primary motor cortex.

Continuous weak-affinity immunosensing.

A multitude of weak biological interactions, either working alone or in concert, occur frequently throughout biological systems. We have used this natural feature of readily reversible interactions as the basis for continuous immunosensing. In a model system, a set of weak monoclonal antibodies directed towards a carbohydrate epitope was studied with the aid of surface plasmon resonance. Because the system requires no regeneration, it can be used as a truly on-line immunosensing device. This principle should have wide application in all areas where there is a need for the continuous evaluation of a molecule.

Interactions between neutrophil gelatinase-associated lipocalin and natural lipophilic ligands.

Neutrophils are activated by both paracrine molecules, e.g. platelet activating factor (PAF) and leukotriene B4 (LTB4), and the bacterial hydrophobic peptide N-formyl-Met-Leu-Phe (fMLP). Several mechanisms are involved in regulation of the activation, including receptor endocytosis and ligand breakdown. The interactions between the specific granule protein neutrophil gelatinase-associated lipocalin (NGAL), expressed in human neutrophils, and fMLP, PAF and LTB4, were investigated by weak affinity chromatography. NGAL was immobilised to a silica matrix and packed in a micro-column and the retention times of retarded ligands were measured and used to calculate the strength of the interactions. The association constants for fMLP were K(ass) = 0.85 x 10(3) M(-1) at 20 degrees C and 0.77 x 10(3) M(-1) at 37 degrees C, for LTB4 were K(ass) = 4.37 x 10(3) M(-1) at 20 degrees C and 3.27 x 10(3) M(-1) at 37 degrees C and for PAF were K(ass) = 25.4 x 10(3) M(-1) at 20 degrees C and 10.5 x 10(3) M(-1) at 37 degrees C. Other methods of detecting the interactions such as gel filtration, immunoprecipitation, photoactivated ligands and fluorescence quenching proved to be insufficient. The results demonstrate the superiority of weak affinity chromatography as a method of studying the interactions of the specific granule protein NGAL.

Affinity screening for weak monoclonal antibodies.

When selecting for monoclonal antibodies of a desired affinity, affinity chromatography can be a feasible alternative. This is of particular interest when low affinity monoclonal antibodies (dissociation constant (Kd) > 10(-4) M) are screened, as they are not easily recognised by traditional immunoassay procedures. In this study we have evaluated this approach by monitoring low affinity monoclonal antibodies on high performance liquid affinity chromatography columns with oligosaccharides, dinitrophenol and digoxin as immobilised antigen. Crude monoclonal antibodies in ascites or cell culture supematants, directed against these antigens, were retarded or adsorbed according to affinity or avidity on the antigen columns. Based on antibody retention, we were able to select hybridomas with the desired low affinity characteristics.

Weak affinity chromatography of small saccharides with immobilised wheat germ agglutinin and its application to monitoring of carbohydrate transferase activity.

In this work we have evaluated the potential to use wheat germ agglutinin (WGA) for weak affinity chromatography (WAC) of N-acetyl derivatives of mono-, di-, tri- and tetrasaccharides. WGA was used as a ligand in a high performance liquid affinity chromatography (HPLAC) system. Isocratic affinity chromatography was conducted where similar N-acetyl saccharides were separated according to their binding strength to WGA. Affinities are weak and lie typically in the mM range. For example, for 3'sialyllactose, the dissociation constant (Kd) was found to be 2.4 mM at 8 degrees C. It was interesting to note that the WGA-HPLC column can distinguish between the anomeric forms of N-acetylglucosamine. Weak affinity chromatography with immobilised WGA was used in an enzyme assay to detect the activity of GlcNAc-transferases.

New approach to steroid separation based on a low affinity IgM antibody.

IgM antibodies are often of low affinity (dissociation constant (Kd) > 10(-5) M) and therefore they are usually neglected as tools in, e.g., immunoassays. Previous studies have shown that low affinity biological interactions can be studied and exploited in affinity chromatography, biosensor technology and capillary electrophoresis. In this study we have demonstrated that IgM can be a useful ligand for analytical separation of antigens in weak affinity chromatography (WAC). A low affinity human monoclonal IgM antibody, directed at digoxin, was produced in a hybridoma cell culture, purified to homogeneity and immobilized onto an HPLC support. The IgM HPLC column displayed specific weak affinity retention in the 0.01-0.1 mM range as evaluated with digoxin and ouabain. The specificity was not affected when samples of ouabain in a crude environment of diluted serum were separated on the IgM column. These findings suggest an approach in immunoadsorbent technology where biomolecules can be analyzed and separated with weak affinity chromatography using IgM as a general affinity ligand.

Exploitation of a monoclonal antibody for weak affinity-based separation in capillary gel electrophoresis.

Weak biospecific recognition has been established for affinity separation in high performance liquid chromatography (HPLC). The use of weak affinity chromatography (WAC) has been limited previously by the insufficient separation efficiency achieved, allowing only some 1000 plates/m to be obtained. However, it has been shown that chiral drug separation can be performed with capillary affinity gel electrophoresis (CAGE) at considerably improved efficiency as compared with traditional chromatographic procedures. Our present study demonstrates the potential of weak affinity monoclonal antibodies as a generic method for immunologically based separations in capillary electrophoresis. Monoclonal antibodies were polymerized within a silica capillary and were used for the separation of structurally similar carbohydrate antigens. The results indicate that weak biospecific interactions can be utilized in a CAGE format to produce highly selective separation of the alpha- and beta-forms of p-nitrophenyl-labeled maltose. It remains to be seen, however, how efficient weak affinity separation in CAGE can be compared with affinity HPLC protocols. Details of typical separations and of the preparation of the antibody gel are presented.

Screening for weak monoclonal antibodies in hybridoma technology.

As the interest in weak-affinity antibodies has been widened by their introduction to various analytical techniques such as HPLC, capillary electrophoresis and biosensors, there has been a need for new screening/monitoring methods. In this study, weak-affinity chromatography was adopted to screen/monitor directly for monoclonal antibodies in ascites. Monoclonal antibodies against a carbohydrate antigen (maltohexaose) were used to evaluate this approach. In short, malthohexaose was immobilized on an HPLC support in such a configuration to allow, during HPLC, retardation of weak monoclonal antibodies. Based on the retention, the affinity or the avidity, as determined by the presence of multiple binding of the monoclonal antibody towards antigen, can be estimated. In this way it is possible to select clones of hybridomas that produce desired weak monoclonal antibodies. Adjustments in temperature (10-20 degrees C) were used to moderate the retention and hence affinity of the weak monoclonal antibodies during chromatography.

Use of weak monoclonal antibodies for affinity chromatography.

When using weakly interacting ligands in affinity chromatography, it is possible to take advantage of a true chromatographic process in the separation, as compared with traditional affinity chromatography which is rather an on/off process. In this work, weak monoclonal antibodies were immobilized on a silica and a perfusion-type support (POROS AL) and used for high-performance liquid affinity chromatography (HPLAC). Similar carbohydrate antigens were separated under isocratic conditions according to their weak interaction with the immobilized monoclonal antibody. The affinity of the antibodies was adjusted with temperature and pH to modify the separation. The productivity of the chromatographic system was increased with the immobilized perfusion support but at the expense of loss of plate numbers. This study clearly demonstrates the potential of weak affinity biological interactions as a basis for chromatographic separation.

Studies of interactions with weak affinities and low-molecular-weight compounds using surface plasmon resonance technology.

Interactions between the immobilized weak-affinity monoclonal IgG antibody 39.5, which is specific for the glucose-alpha 1,4-glucose motif, and various oligosaccharides were studied with surface plasmon resonance technology. The antibody was immobilized at high levels on the surface of the sensor chip and different concentrations of the analytes were injected at 25 and 40 degrees C. The 39.5 antibody exhibited specific binding to maltose, tetraglucose and maltotriose, with dissociation constants Kd in the range from 0.07 mM (25 degrees C) to 1.0 mM (40 degrees C). Association and dissociation rate constants (ka and kd) were rapid and baseline was obtained almost immediately after the end of each antigen injection. This excluded the need for a regeneration step but also made calculation of the kinetic values impossible. Owing to the weak affinity and the small size of the analytes (< 1000 Da), a careful design of control surfaces is demanded to exclude artefactual results.

Detection of low-molecular-weight heparin oligosaccharides (Fragmin) using surface plasmon resonance.

During the last decades there has been a growing realization of the central biological role that oligosaccharides and oligosaccharide-protein interactions play. One of the most striking examples is the use of heparin and low-molecular-weight heparin oligosaccharides (Fragmin) to modify blood coagulation. Several monoclonal antibodies directed against glycosaminoglycan structures have been produced. However, their clinical use is limited by the difficulty of detection systems for oligosaccharides. In the present study we used a monoclonal antibody directed against heparin oligosaccharides prepared by partial nitrous acid deamination of heparin. Using a biosensor (BIAcore), purified antibody was immobilized on sensor surfaces and binding of oligosaccharide was measured by surface plasmon resonance. Using this technique, it was possible to quantitate low-molecular-weight heparin oligosaccharides in nanomolar concentrations.