Avidin-HRP conjugates in biotin-avidin immunoenzyme cytochemistry

Summary Avidin-HRP conjugates were prepared, analysed and tested for avidin-biotin immunocytochemistry. Suitable biotinylation of enzymes, antigens and antibody was obtained by reacting biotin at equimolar ratio to epsilon aminogroups in proteins. The avidin-biotin interaction was used for immunocytochemical detection of phenomena in the field of immunology, i.e. immune complex trapping, specific antibody forming cells and in serology for the cytochemical detection of human auto-antibodies to basement membrane components. Avidin-HRP conjugation using the two step glutaraldehyde method gave a very small amount of monomeric, low molecular weight conjugate with excellent performance. Avidin-HRP conjugation using the periodate method was modified at two points. The first modification concerns the molar ratio of avidin to HRP in the reaction mixture which was brought to about equimolarity. The second modification concerns the periodate concentration which was decreased five fold, ten fold and twenty fold. Decreasing the periodate concentration decreased the amount of polymeric conjugate. Optimal amounts of monomeric, low molecular weight conjugate were obtained with a ten fold decrease of the periodate concentration. Comparable cytochemical results were obtained with monomeric conjugates obtained using both preparation methods.In honour of Professor P. van Duijn     

Peroxidase labelled monoclonal antibody against light chains of human cardiac myosin.

Monoclonal antibody against light chains of human cardiac myosin (MLC) was labelled with horseradish peroxidase. The conjugation was performed by two different methods with glutaraldehyde and periodate respectively. The binding activities of the conjugates were tested by enzyme linked immunosorbent assay (ELISA) on the microtitration plates with immobilized MLC (1-1000 ng per well). A comparison of both methods revealed their universal suitability for the preparation of conjugates as well as their applicability. The use of conjugates shortens the time needed and improves the ELISA method for MLC estimation. Specific advantages of the glutaraldehyde and the periodate method concern diverse details.     

Avidin-HRP conjugates in biotin-avidin immunoenzyme cytochemistry

Avidin-HRP conjugates were prepared, analysed and tested for avidin-biotin immunocytochemistry. Suitable biotinylation of enzymes, antigens and antibody was obtained by reacting biotin at equimolar ratio to epsilon aminogroups in proteins. The avidin-biotin interaction was used for immunocytochemical detection of phenomena in the field of immunology, i.e. immune complex trapping, specific antibody forming cells and in serology for the cytochemical detection of human auto-antibodies to basement membrane components. Avidin-HRP conjugation using the two step glutaraldehyde method gave a very small amount of monomeric, low molecular weight conjugate with excellent performance. Avidin-HRP conjugation using the periodate method was modified at two points. The first modification concerns the molar ratio of avidin to HRP in the reaction mixture which was brought to about equimolarity. The second modification concerns the periodate concentration which was decreased five fold, ten fold and twenty fold. Decreasing the periodate concentration decreased the amount of polymeric conjugate. Optimal amounts of monomeric, low molecular weight conjugate were obtained with a ten fold decrease of the periodate concentration. Comparable cytochemical results were obtained with monomeric conjugates obtained using both preparation methods.     

Comparative study of conjugation between horseradish peroxidase and D-cytochrome b5. Incorporation into subcellular membranes

Horseradish peroxidase was conjugated to D-cytochrome b5 by three different two-step methods. The yield of conjugates based on the peroxidase enzymatic activity recovered after gel filtration was very low in the glutaraldehyde method, but higher in the N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and periodate methods. The molecular size of the conjugates was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Monomeric conjugates were mostly formed via the glutaraldehyde and SPDP methods in the presence of appropriate molar ratios of proteins. Most of the conjugates formed via the periodate method were polymers. The conjugate preparations of the three methods could be incorporated into microsomal membranes. Conjugate polymers, however, appeared less able to be incorporated then monomers. There was a nonpreferential incorporation of free or conjugated D-cytochrome b5 contained in the conjugate preparation of the glutaraldehyde method. In conclusion, this study gives preference to the glutaraldehyde method for the preparation of conjugates that will subsequently be used as an in vivo marker of the D-cytochrome b5 incorporation into membranes.     

Peroxidase-conjugate chromatography isolation of conjugates prepared with glutaraldehyde or periodate using polyacrylamide-agarose gel.

Fractionation abilities of polyacrylamide-agarose gel (Ultrogel) and dextran gel (Sephadex) column chromatography were compared in isolating horseradish peroxidase conjugates, prepared using two different methods. Utrogel AcA-44 provides an efficient separation of monomer conjugated and nonconjugated immunoglobulins resulting from the two-step glutaraldehyde procedure, Sephadex G-200 does not. Both types of columns eluted the polymer conjugates resulting from the periodate procedure in the void volume; these were hardly isolated from the small amount of monomer conjugate. Unreacted horseradish peroxidase, present in very low quantities after the efficient periodate method and in large amounts after the glutaraldehyde procedure, was separated by both gel types.     

The effects of glutaraldehyde treatment and horesradish peroxidase conjugation on the immunoreactivity of bovine myelin encephalitogenic protein.

The effects of the immunoreactivity of bovine myelin encephalitogenic protein (EP) of treatment with glutaraldehyde and conjugation with horeradish peroxidase (HRP) were investigated by complement fixation and immunohistochemistry. Both glutaraldehyde treatment and HRP conjugation of EP decreased but did not abolish the reactivity of EP with rabbit anti-EP. Conjugation of EP to HRP by the two-step method of Avrameas had less detrimental effects on the immunochemical reactivity of EP than did the one-step procecure. The use of EP-HRP conjugates to probe for antibodies to EP is an immunochemically sound system with the major limitation that the number of antibody-producing cells is likely to be underestimated due to the failure to detect cells producing low levels of antibody or antibodies directed toward determinants altered by the modification procedures.     

Immunological detection of adulteration of ground meats by meats of other origins

Specific and sensitive assays for immunoglobulin G in pork, chicken, and beef were developed using polyester cloths coated with respective anti-immunoglobulin G antibodies. The captured immunoglobulin G proteins were detected by their reactions with the respective IgG antibody horseradish peroxidase conjugates and a chromogenic peroxidase substrate. This sensitive assay takes only 30 minutes and can detect the adulteration of ground meats.     

Preparation of immunoenzyme conjugates of beta-lactamase from Bacillus licheniformis 749/c and horseradish peroxidase with human antibodies to HIV-1]

By using three different linkage methods with carbodiimide, glutaraldehyde and periodite, immunoenzyme conjugates of beta-lactamase from Bacillus licheniformis 749/c and horse radish peroxidase with human antibodies to HIV-1 were prepared. The human antibodies were purified by the affinity procedure on Protein-A-Sepharose 6B. The conjugates were tested in a solid phase immunoenzymatic system for the HIV-1 antigen. It was shown that the conjugates prepared by the carbodiimide linkage method had the highest titer, the beta-lactamase conjugate being superior by its titer to the respective peroxidase conjugate. In the lyophilized state the conjugates prepared with the carbodiimide linkage method were stable.     

Conjugation of horseradish peroxidase to staphylococcal protein A with benzoquinone, glutaraldehyde, or periodate as cross-linking reagents

Horseradish peroxidase was conjugated to Staphylococcal protein A by three different two-step procedures using an increasing excess of peroxidase in the second step reaction. The yield of conjugated protein A was analyzed by SDS-polyacrylamide gel electrophoresis. Conjugation of peroxidase to protein A with benzoquinone or glutaraldehyde as cross-linking reagents at a 3- to 4-fold molar excess of peroxidase resulted in a high yield of coupled protein A with conjugates of low molecular size. Conjugation of peroxidase to protein A by the periodate method resulted in a high yield of coupled protein A with polymeric conjugates of large molecular size. Based on these results, conjugates produced with glutaraldehyde as cross-linking reagents were further analyzed. The capacity of the conjugates to precipitate human immunoglobulin evaluated by radial immunodiffusion was found to be reduced to about 50% of that of native protein A. Conjugates produced with glutaraldehyde as cross-linking reagent retained 70% of the enzyme activity of native peroxidase.     

Isolation and use in virological research of a peroxidase conjugate with protein A

On the basis of proteins A produced at the Mechnikov Central Research Institute for Vaccines and Sera (Moscow) and obtained from Pharmacia AB (Sweden), several batches of peroxidase conjugate have been prepared by a modified method of periodate oxidation. The preparations thus obtained have been evaluated by means of the corresponding enzyme immunoassay systems for the detection of antibodies to herpes simplex and measles viruses. The results of this investigation indicate that serum antibody titers, determined in the assays with antispecific conjugates and with protein A-based based conjugates, coincide. The comparative study of conjugates prepared on the basis of Soviet and imported proteins A has demonstrated their similar activities and specificities.     

Improved procedure for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide.

The procedures for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide were improved in several respects as compared with the previous methods (Eur. J. Biochem. 62, 285--292, 1976; J. Immunol. 116, 1554--1560, 1976). Maleimide residues were efficiently introduced into antibodies under an atmosphere of nitrogen; the average number of maleimide residues introduced into IgG and Fab' antibodies were 0.78 (0.65--0.86) and 0.86 (0.80--0.95) per molecule, respectively. The conjugation with the enzyme was performed at 4 degrees C at pH 6.5 for 15 or more hours. The conjugates were almost completely separated from unreacted IgG and Fab' by gel filtration. When the recoveries of IgG, Fab', and beta-D-galactosidase in the conjugates were 23-29, 35-44, and 99%, respectively, the average numbers of IgG and Fab' molecules conjugated with the enzyme were 1.5-1.7 and 2.1-2.8 per molecule, respectively. There was no significant impairment of beta-D-galactosidase activity or the activity of anti-human IgG antibody to bind to human IgG upon conjugation. However, the conjugate preparation was heterogeneous, and one-third of each preparation consisted of aggregated conjugates less useful in sandwich enzymoimmunoassay than the remaining material. The conjugate with Fab' antibody gave lower control values in sandwich enzymoimmunoassay with silicone rubber as a solid phase than that with IgG antibody.     

Purification of horseradish peroxidase conjugated IGG by affinity chromatography. Comparison of glutaraldehyde and periodate conjugation]

By affinity chromatography of the coupling mixture on Concanavalin-A-Sepharose unlabeled IgG is completely removed. HRP conjugated IgG is then separated from free HRP by gelfiltration. Glutaraldehyde conjugation yielded 20 to 68% unlabeled IgG, depending on the duration of glutaraldehyde addition, and periodate conjugation yielded 10 to 28% unlabeled IgG. By this method fractions of conjugates with 3fold spec. act. were obtained by the glutaraldehydemethod and with 1.6fold spec. act. by periodate method as compared with gelfiltration. The periodate method guarantees a 3fold higher yield of HRP-labeled IgG compares with glutaraldehyde method. Both methods produce conjugates of the same spec. act. although the denaturation of HRP is much greater at periodate conjugation. When applying of affinity chromatography and gelfiltration. Crude HRP with 7% spec. act. of purified HRP resulted in conjugates having a spec. act. of 85% of those with purified HRP.     

Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.     

Design and construction of novel molecular conjugates for signal amplification (I): conjugation of multiple horseradish peroxidase molecules to immunoglobulin via primary amines on lysine peptide chains

Immunoconjugates are widely used for indirect detection of analytes (such as antibodies or antigens) in a variety of immunoassays. However, the availability of functional groups such as primary amines or free sulfhydryls in an immunoglobulin molecule is the limiting factor for optimal conjugation and, therefore, determines the sensitivity of an assay. In the present study, an N-terminal bromoacetylated 20 amino acid peptide containing 20 lysine residues was conjugated to N-succinimidyl-S-acetylthioacetate (SATA)-modified IgG or free sulfhydryl groups on 2-mercaptoethylamine (2-MEA)-reduced IgG molecules via a thioether (S---CH₂CONH) linkage to introduce multiple reactive primary amines per IgG. These primary amines were then covalently coupled with maleimide-activated horseradish peroxidase (HRP). The poly-HRP–antibody conjugates thus generated demonstrated greater than 15-fold signal amplification upon reaction with orthophenyldiamine substrate. The poly-HRP–antibody conjugates efficiently detected human immunodeficiency virus (HIV)-1 antibodies in plasma specimens with significantly higher sensitivity than conventionally prepared HRP–antibody conjugates in an HIV-1 solid-phase enzyme immunoassay and Western blot analysis. The signal amplification techniques reported here could have the potential for development of highly sensitive immunodiagnostic assay systems.     

Commercial horseradish peroxidase degrades myelin encephalitogenic protein during coupling for immunohistochemical studies.

Conjugates of myelin encephalitogenic basic protein (EP) and commercial horseradish peroxidase (HRP) have been used for immunohistochemical demonstrations of anti-EP antibody in animals with experimental allergic encephalomyelitis. We performed gel electrophoresis studies on EP-HRP conjugates prepared with glutaraldehyde and on mixtures of EP and HRP incubated without glutaraldehyde. The results show that under conditions of one-and two-step coupling HRP causes rapid loss of the native EP band, apparently due to EP degradation. The EP-HRP mixtures are not encephalitogenic in rabbits, or encephalitogenic activity is lost during processing. The immunohistochemical reactivity of conjugates, however, signals some preservation of antibody-combining sites. The mechanism of the HRP effect on EP is unknown. The possibilities of a contaminating proteinase or direct peroxidatic attack are suggested. Until this action of HRP can be overcome, the effect of coupling procedures on the biological activities of EP will be difficult to assess, and EP-HRP conjugates cannot be expected to reveal sites that may bind encephalitogenic portions of the EP molecule.     

Effect of periodate oxidation on properties of antibodies

The subject of the present research is the investigation of the influence of sodium periodate on the properties of immunoglobulin G molecules. It is shown that 100 and 300 M of periodate cause a slight enhancement of the sedimentation coefficient which is a result of the higher protein density. However high concentrations (2000 M) of periodate decrease sedimentation coefficient considerably and disturb the protein structure homogeneity. Studies of the immunologic activity in the periodate-treated antibodies by the reaction of passive hemagglutination showed that in low concentrations it did not decrease significantly the activity but with an increase in the concentration up to 2000 M the activity lowered. The conjugation of antibodies with the enzyme markers was fulfilled due to periodate oxidation. The conjugates obtained were successfully used for improving sensitivity of the precipitation line in immunologic tests.     

Efficient strategies for the conjugation of oligonucleotides to antibodies enabling highly sensitive protein detection

Three methods for the conjugation of oligonucleotides to antibodies and the subsequent application of these conjugates to protein detection at attomole levels in immunoassays are described. The methods are based on chemical modification of both antibody and oligonucleotide. Aldehydes were introduced onto antibodies by modification of primary amines or oxidation of carbohydrate residues. Aldehyde- or hydrazine-modified oligonucleotides were prepared either during phosphoramidite synthesis or by post-synthesis derivatization. Conjugation between the modified oligonucleotide and antibody resulted in the formation of a hydrazone bond that proved to be stable over long periods of time under physiological conditions. The binding activity of each antibody-oligonucleotide conjugate was determined to be comparable to the corresponding unmodified antibody using a standard sandwich ELISA. Each oligonucleotide contained a unique DNA sequence flanked by universal primers at both ends and was assigned to a specific antibody. Highly sensitive immunoassays were performed by immobilizing analyte for each conjugate onto a solid support with cognate capture antibodies. Binding of the antibody-oligonucleotide conjugate to the immobilized analyte allowed for amplification of the attached DNA. Products of amplification were visualized using gel electrophoresis, thus denoting the presence of bound analyte. The preferred conjugation method was used to generate a set of antibody-oligonucleotide conjugates suitable for high-sensitivity protein detection.     

高碘酸钠和戊二醛交联法构建的R-藻红蛋白-C-藻蓝蛋白交联物能量传递效率的比较研究

从单细胞蓝藻钝顶螺旋藻中纯化C-藻蓝蛋白,从海洋红藻多管藻纯化R-藻红蛋白.分别用高碘酸钠氧化法和戊二醛法将二者共价连接为R-藻红蛋白-C-藻蓝蛋白交联物,再用Sephadex G-200柱层析纯化.光谱分析表明,用两种方法构建的共价交联物都可以将激发能从R-藻红蛋白传递到C-藻蓝蛋白.二者相比,高碘酸钠氧化法构建的共价交联物的能量传递效率更高.     

Indirect immunofluorescence colony staining method for detecting bacterial pathogens of tomato.

An indirect immunofluorescence colony staining method was developed for the detection of important seed-borne bacterial pathogens of tomato. The method involves the use of specific antiserum for initial binding of target bacteria and visualization of positive colonies with a commercially available secondary antiserum conjugated with FITC and observed under a fluorescence microscope. The indirect method is especially suitable for laboratories, seed companies, and quarantine stations which have no facilities for conjugation of primary antiserum. It is more economical and overcomes the problems generally encountered with variable conjugate quality in new batches of conjugates prepared from the same stock of primary antiserum. The assay is easy to perform and results can be easily assessed by visual scoring or image analyser. Results are available in 4-5 days as compared to 30-45 days in traditional methods. The resulting bacterial culture can be tested by PCR or host infectivity and a culture can be stored for future reference. Used in combination with highly specific antibodies (commercially available monoclonal and recombinant antibodies) it can be used as a very sensitive detection tool and has application potential in localization studies as well. Choosing the right secondary conjugate is however necessary to get best results in the assay.     

Production of monomeric antigen-enzyme conjugate to study requirements for follicular immune complex trapping

Summary Studies concerning the localization of immune complexes in lymphoid follicles and the involvement of these trapped immune complexes in the regulation of the immune response have thus far been performed with poorly defined complexes in terms of size and composition. For that reason, the minimum requirements for trapping in terms of number of antigen- and antibody molecules present in immune complexes could not be determined. We here describe the production and in vivo use of a monomeric HSA-HRP antigen-enzyme conjugate, readily demonstrable in cryostat sections and ELISA. This conjugate was obtained by combining the glutaraldehyde coupling-method with chromatography to fractionate monomeric and multimeric constituents.SDS-PAGE analysis showed that the conjugate consisted of a single molecular species of 109 kDa, whereas the often used periodate oxidation coupling method yielded a heterogeneous population of multimeric, oligomeric and monomeric molecules.We investigated the minimal size requirements for the composition of immune complexes to be trapped in murine spleen follicles using three different conjugates (monomeric HSA-HRP, multimeric HSA-HRP and multimeric HSA-HRP-Penicillin) and a panel of anti-HSA and anti-Penicillin monoclonal antibodies. We demonstrate that the smallest immune complexes, consisting of one antibody and two conjugate molecules, do not localize in splenic follicles. Immune complexes prepared with a single monoclonal antibody localize in follicles only if the epitope recognized occurs repeatedly on the antigen.The relevance of these results for physiological follicular trapping of protein antigens is discussed. The described method for the production of monomeric enzyme-labelled protein applicable in histochemistry and ELISA should prove useful to prepare other conjugates of defined size for studies of trapping and other applications.Abbreviations FDC follicular dendritic cell - HRP horseradish peroxidase - HSA human serum albumin - HY anti-HSA hyperimmune serum - MAb monoclonal antibody - Pen penicillin