Immunoblotting and immunodetection.

This unit provides protocols for immunoblotting, which is used to identify specific protein sequences separated by electrophoresis and transferred to an appropriate membrane and recognized by a polyclonal or monoclonal antibody. After the proteins are separated on a gel, they are transferred to a membrane by electroblotting or a semidry transfer system. Proteins on the membrane can be visualized with the reversible stain Ponceau S to assess the completeness of transfer. Then the blot is analyzed with antibodies. The primary antibody is specific for the protein(s) of interest; the secondary antibody (an anti-Ig) is conjugated to horseradish peroxidase or alkaline phosphatase and detected colorimetrically or by chemiluminescence. The membrane can be stripped and reused for other probes.

Conjugation of enzymes to antibodies.

Conjugation of enzymes to antibodies involves the formation of a stable, covalent linkage between an enzyme [e.g., horseradish peroxidase (HRPO), urease, or alkaline phosphatase] and an antigen-specific monoclonal or polyclonal antibody in which neither the antigen-combining site of the antibody nor the active site of the enzyme is functionally altered. This unit describes procedures for cross-linking HRPO, urease or alkaline phosphatase to immunoaffinity-purified monoclonal or polyclonal antibodies (IgG).

Purification of monoclonal antibodies.

The uses of monoclonal antibodies as enzyme conjugates and as immunoaffinity reagents require their purification from the crude ascites fluid. This unit provides protocols for purification using protein A-Sepharose chromatography and affinity chromatography, which are superior to ammonium sulfate precipitation, gel filtration, or ion-exchange chromatography for the preparation of contaminant-free antibody.

Immunization of mice.

In the protocols in this unit, antigen is prepared for injection either by emulsifying an antigen solution with Freunds adjuvant or by homogenizing a polyacrylamide gel slice containing the protein antigen. Mice are immunized at 2- to 3-week intervals. Test bleeds are collected 7 days after each booster immunization to monitor serum antibody levels. Mice are chosen for hybridoma fusions when a sufficient antibody titer is reached.

Preparation of myeloma cells.

In this unit, myeloma cells are cultured to ensure their sensitivity to the HAT selection medium used after cell fusion. Cell culture conditions are adjusted such that the Sp2/0 cells are in the log phase of growth and exhibit high viability at the time of collection for fusion. A support protocol is provided to determine the number of viable cells present in the cell culture.

Preparation of mouse feeder cells for fusion and cloning.

To maximize the yield of hybrids from fusion and cloning procedures, feeder cells are required to be cocultured with the hybrids, while hybrid cell density is low. Mouse peritoneal cells, most of which are macrophages, have been found to be convenient and effective feeder cells which are a source of soluble growth factors for hybridoma cells.

Fusion of myeloma cells with immune spleen cells.

Liquid nitrogen storage is the method of choice for long-term safekeeping of hybridoma cell lines. Frozen aliquots of originally isolated hybridomas provide insurance against loss of antibody production and vigor during culture. There are many variations of cell freezing methods in use. However, for freezing and recovering hybridomas and lymphoid cells in general, the protocol described in this unit is simple and has been successful.

Cloning of hybridoma cell lines by limiting dilution.

Cloning by limiting dilution is a method based on the Poisson distribution. Dilution of cells to an appropriate number per well can maximize the proportion of wells that contain one single clone. Two or more cloning procedures are carried out until >90% of the wells containing single clones are positive for antibody production.

Freezing and recovery of hybridoma cell lines.

Liquid nitrogen storage is the method of choice for long-term safekeeping of hybridoma cell lines. Frozen aliquots of originally isolated hybridomas provide insurance against loss of antibody production and vigor during culture. There are many variations of cell freezing methods in use. However, for freezing and recovering hybridomas and lymphoid cells in general, the protocol described in this unit is simple and has been successful.

An accelerated enzyme immunoassay for human choriogonadotropin in urine, involving reflow of specimen through capillary tubes.

A very rapid and sensitive assay for human choriogonadotropin (hCG) has been developed involving two beta-subunit-specific monoclonal antibodies. In the assay the test specimen is passed backward and forward (reflow) through a monoclonal-antibody-coated capillary tube for 1 min, then incubated for 1 min with a second monoclonal antibody conjugated to urease (EC 3.5.1.5). After addition of a urease substrate solution, 10 int. units of hCG per liter can be detected visually within 5 min, which compares very favorably with other currently available hCG assay procedures. Advantages of the reflow/capillary tube assay system and optimization of the test procedure are discussed.

Production of IgG-producing hybridomas by in vitro stimulation of murine spleen cells.

An in vitro stimulation protocol has been established which allows production of IgG-secreting murine hybridomas. This procedure has been examined using jack bean urease and human luteinizing hormone as antigens. Parameters which have been optimized include selection of media and serum supplements, thymocyte-conditioned media, antigen dosage, length of stimulation and the effect of medium changes during stimulation and additions of polyclonal mitogen. Murine spleen cells (1 X 10(8) in 10 ml) were incubated with varying doses of jack bean urease and human luteinizing hormone in a six-well plate in supplemented DMEM with 5% normal rabbit serum and 10% thymocyte-conditioned media. Following 5 and/or 8 days stimulation, the spleen cells were fused with SP2/0 cells and plated in 96-well plates. Stable hybridomas were obtained for both antigens from over 25% of the wells identified in initial screening for specific antibody production. All monoclonal antibodies obtained in the LH stimulation experiments, with one exception, were of the IgM isotype. A large number of IgG-producing hybridomas were isolated following prolonged (8 day) stimulation with high concentrations of urease, during which time the medium remained unchanged. Addition of polyclonal mitogen (E. coli lipopolysaccharide) at 10 micrograms/ml markedly increased the production of hybridomas secreting anti-urease, but most were of IgM class.

A rapid quantitative capillary tube enzyme immunoassay for human chorionic gonadotropin in urine.

A quantitative capillary tube enzyme immunoassay (CTEIA) method for the determination of human urinary chorionic gonadotropin (hCG) has been developed. The method utilizes an antibody-coated capillary tube through which the test fluid is passed and a urease-labelled second antibody in an immunometric format. Any hCG in the test solution is 'captured' by the immobilized antibody which is hybridoma derived and specific for the beta-subunit of hCG. The second hCG-specific antibody, conjugated to the enzyme urease, is used to detect the captured hCG on the internal surface of the capillary tube. The amount of urease bound to the surface is determined by the introduction of a substrate solution containing urea and the pH indicator bromothymol blue. The rate of colour change, from yellow to blue, caused by the release of ammonia from urea by urease, is determined in a spectrophotometer using a cell holder adapted to accommodate capillary tubes. The initial rate of absorbance change is directly proportional to the concentration of hCG in the sample in the range 0-100 mIU/ml. The test can detect concentrations of hCG as low as 10 mIU/ml in a total elapsed time of 5 min.

Immune response and reactions to various dose regimens for raising hyperimmune antisera in sheep.

To determine the optimum procedure for raising hyperimmune sera to tetanus toxin, three adjuvants, four antigen preparations and two routes of administration in various combinations were investigated in sheep. Oil-in-water adjuvants alone or in combination with aluminum gels were superior to aluminium gels on their own. This disadvantage of aluminium gels was partially but not completely abrogated when the frequency of doses was increased to three per week. Intensity of local reaction was strongly correlated with immune response; the more immunogenic a dose, the more reactive. Reactivity of oily adjuvants could be lessened by use of a more suitable route of administration, thus oily adjuvants appeared suitable for use when administered by the intraperitoneal route even though moderate to severe reactions resulted from subcutaneous injections. Of other variables investigated, toxin did not confer any advantage over toxoid as an immunogen, purified toxoid was a significantly better immunogen than unpurified toxoid and two large bleeds (30% of total blood volume each) every six weeks rather than 20 ml test bleeds did not affect the titre of the hyperimmune serum produced.

A new rapid semi-quantitative enzyme immunoassay suitable for determining immunity to tetanus.

A sensitive enzyme immunoassay for rapidly assessing a patient's state of immunity to tetanus is described. The test, which uses 50 microliter sample of blood, plasma or serum, is done in a capillary tube and, by comparison with two adjacent reference tubes containing standardised sera, places immunity to tetanus in one of three categories--low-negative (less than 0.01 IU/ml), intermediate (0.01-1.28 IU/ml) or high (greater than 1.28 IU/ml). In a study of 90 clinical specimens assayed both by toxin neutralisation bioassay and capillary enzyme immunoassay the enzyme immunoassay accurately assessed the state of immunity to tetanus of the patients concerned.

Enzyme immunoassay for antibodies to membrane associated antigen of varicella zoster virus.

An in situ enzyme immunoassay to viral membrane antigen was developed to enable the specific estimation of antibodies to varicella zoster (VZ) virus. The technique was compared with a modified fluorescent antibody to membrane antigen (FAMA) procedure and with the complement fixation (CF) test by parallel assay of 352 plasma samples. The enzyme immunoassay (EIA) procedure showed very good correlation with the modified FAMA procedure, and both were far more specific than the CF test. This specificity was achieved by the use, in the EIA, of VZ virus-infected cells grown and fixed in situ with glutaraldehyde. Thus the only virus antigens accessible to antibody were the VZ-specific antigens expressed at the cell membrane, cross-reactions with herpes simplex virus antibodies thereby being avoided.

Monoclonal antibodies against hepatitis A virus.

Three monoclonal antibodies (K2-4F2, K3-2F2, and K3-4C8) of the immunoglobulin G2a class were raised against hepatitis A virus. The specificity of these antibodies was confirmed by immune electron microscopy, solid-phase radioimmunoassay, and in vitro neutralization in cell culture. Binding studies suggested that they all recognize closely related antigenic determinants. These monoclonal antibodies should prove to be of great value as diagnostic and research reagents.

A rapid semi quantitative capillary enzyme immunoassay for digoxin.

A rapid and sensitive enzyme immunoassay (EIA) which does not require highly trained personnel or specialised instrumentation is described for the estimation of digoxin in serum, plasma or whole blood samples. The method is based on the ability of digoxin in a clinical sample to inhibit the binding of urease-conjugated sheep-antidigoxin immunoglobulin to a glass capillary tube coated internally with a human serum albumin-digoxin conjugate. The bound enzyme activity can then be measured using a substrate solution containing urea and a pH indicator, most suitably bromocresol purple. The enzymic hydrolysis of urea produces ammonia which causes a vivid yellow to purple colour change in the pH indicator. Plasma samples from 92 patients receiving digoxin were screened in parallel with reference plasma containing 1.3 or 3.8 nmol/l digoxin. The results were available within a total test time of 30 min, and showed excellent correlation with those obtained by radioimmunoassay.