Mouse monoclonal antibodies in biological research: strategies for high-throughput production

Mouse monoclonal antibodies have become key components in basic research as well as in the clinical laboratory. Being invaluable tools in many biological assays, they continue to be the primary choice in the research field, although the conventional technology used for hybridoma generation and screening is a still lengthy, time-consuming and low-throughput process. With the advent of genetic immunisation and the application of automation and microarray to the traditional biological assays, the monoclonal antibody field has been revolutionised. Here, we will briefly review the most relevant strategies which have made the manufacture of murine monoclonal antibodies a faster and high-throughput technology.     

Regulation of Anguillicola crassus (Nematoda) infrapopulations in their definitive host, the European eel, Anguilla anguilla

The parasitic nematode Anguillicola crassus was recently introduced into populations of the European eel, Anguilla anguilla. We investigated, under experimental conditions, the regulation of A. crassus infrapopulations. We tested the effects of (1) the resource-limited habitat of the parasite and (2) the coexistence of several developmental stages in its niche (the swim-bladder) on the composition of the infrapopulations. The results revealed that the respective effects of these factors differed substantially during the course of the infection. Third-stage larvae (L3s) establishment would not be constrained by the size of the swim-bladder. Their moult to fourth-stage larvae (L4s) would be accelerated as the number of L3s increased. The moulting time of L4s to adults would be reduced by males and would be constrained by the size of the swim-bladder. However, the moult of L4s to adults and their further development would be synchronized with those of the opposite sex. At the time of mating, the number of males and the body weight of adults would depend on the size of the swim-bladder. Soon after the laying of eggs, the developmental constraint on the late L3s would decrease. When adults die, constraints would cease and late larval stages would moult to become adults.     

Growth inhibition and differentiation induction in murine erythroleukemia cells by 4-hydroxynonenal

4-Hydroxynonenal (HNE) is one of the major end products of lipid peroxidation. Here we show that the exposure of murine erythroleukemia (MEL) cells to 1 μM HNE, for 10.5 h over 2 days, induces a differentiation comparable with that observed in cells exposed to DMSO for the whole experiment (7 days). The exposure of MEL cells for the same length of time demonstrates a higher degree of differentiation in HNE-treated than in DMSO-treated MEL cells. The protooncogene c-myc is down-modulated early, in HNE-induced MEL cells as well as in DMSO-treated cells. However, ornithine decarboxylase gene expression first increases and then decreases, during the lowering of the proliferation rate. These findings indicate that HNE, at a concentration physiologically found in many normal tissues and in the plasma, induces MEL cell differentiation by modulation of specific gene expression.     

Typing of Neisseria meningitidis isolates from patients with invasive disease by molecular analysis of porin genes

Polymerase Chain Reaction-Restriction Fragment Length Polymorphism assay (PCR-RFLP) for porA and porB genes of Neisseria meningitidis was applied to type 64 strains isolated from cases of invasive disease in Italy. The technique was also successfully used on non-viable strains and on cerebrospinal fluid samples from patients with meningococcal meningitis diagnosed by PCR. RFLP patterns were obtained for all strains, including those nontypeable or nonsubtypeable by the serological methods. The results confirmed the usefulness of the PCR-RFLP for porA and porB genes of Neisseria meningitidis in differentiating strains belonging to the same serological type and provided discriminative patterns in the increased proportion of non serotypeable/serosubtypeable strains circulating in Italy in recent years.     

Serial immunoprecipitation assays for interferon--(IFN)-beta antibodies in multiple sclerosis patients

We devised a sensitive, radioimmunoprecipitation assay (RIPA) for anti-interferon (IFN)-beta-binding antibody (BAB) detection. Our RIPA showed good agreement with a reference RIPA (mean difference, -3.2 +/- 10.6 AU), and detected BAB to both IFN-beta-1a and IFN-beta-1b. Neutralizing antibodies to IFN-b (NAB) were also determined with a standard method. BAB and NAB were measured in 393 serum samples from 77 multiple sclerosis (MS) patients treated with IFN-beta-1a or -1b, who were studied over two years, and subsequently classified as responders and non-responders. BAB were found at higher concentrations, and more frequently detected, in IFN-beta-1b- than in IFN-beta-1a-treated patients, and, at highest titres, preferentially in patients who were positive for NAB. However, in our series of MS patients, both titres and frequency of detection of BAB or NAB did not differ between IFN-b responders and non-responders.     

Ethanol Inhibits the Binding of Substance P to Rat Brain Cortex NK1 Receptors

The binding of ¹²⁵I-labeled substance P (SP) to rat brain cortex membranes has been studied Under control conditions and in the presence of ethanol. The binding of SP at low concentrations (20–1000 pM) gave two components, one with a K _D value of 80 pM and another one with a K _D of 500 pM. The higher-affinity component is due to NK₁ receptors, as confirmed by the inhibition Of the SP binding by the rodent NK1 specific agonist [Sar₉ Met(O₂)₁₁]SP. Ethanol (1.7 mM) added to the binding assays inhibited by more than 50% the specific binding at a very low SP concentration (20 pM); however, it had no effect at SP concentrations ranging from 50 to 120 pM. This suggests a decrease by ethanol of the affinity of SP to the NK₁ receptors involved in this binding component. The ethanol effect disappeared at [EtOH] 0.17 mM.     

Genetic mapping and QTL analysis of horticultural traits in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) using recombinant inbred lines

A set of 171 recombinant inbred lines (RIL) were developed from a narrow cross in cucumber (Cucumis sativus L.; 2n = 2x = 14) using the determinate (de), gynoecious (F), standard-sized leaf line G421 and the indeterminate, monoecious, little-leaf (ll) line H-19. A 131-point genetic map was constructed using these RILs and 216 F₂ individuals to include 14 SSRs, 24 SCARs, 27 AFLPs, 62 RAPDs, 1 SNP, and three economically important morphological [F (gynoecy), de (determinate habit), ll (little leaf)] markers. Seven linkage groups spanned 706 cM with a mean marker interval of 5.6 cM. The location of F and de was defined by genetic linkage and quantitative trait locus (QTL) analysis to be associated with SSR loci CSWCT28 and CSWCTT14 at 5.0 cM and 0.8 cM, respectively. RIL-based QTL analysis of the number of lateral branches in three environments revealed four location-independent factors that cumulatively explained 42% of the observed phenotypic variation. QTLs conditioning lateral branching (mlb1.1), fruit length/diameter ratio (ldr1.2) and sex expression (sex1.2) were associated with de. Sex expression was influenced by three genomic regions corresponding to F and de both on linkage Group 1, and a third locus (sex6.1) on linkage Group 6. QTLs conditioning the number of fruit per plant (fpl1.2), the number of lateral branches (mlb1.4) and fruit length/diameter ratio (ldr1.3) were associated with ll. The potential value of these marker-trait associations (i.e., yield components) for plant improvement is portended by the relatively high LOD scores (2.6 to 13.0) and associated R² values (1.5% to 32.4%) that are affiliated with comparatively few genetic factors (perhaps 3 to 10).Communicated by H.C. BeckerMention of trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable