Prostatic adenocarcinoma. Evaluation of immunoreactivity to monoclonal antibody B72.3

Monoclonal antibody B72.3 reacts with a tumor-associated glycoprotein designated TAG-72 that is expressed in many adenocarcinomas but not in normal tissues. The authors evaluated the immunoreactivity of B72.3 to benign, hyperplastic prostate, and to primary adenocarcinoma of the prostate to determine the frequency of TAG-72 production by benign and malignant prostatic epithelium. Focal cytoplasmic staining of gland cells was seen in 19 of 20 cases of glandular hyperplasia, and weak, homogeneous staining of secretions was seen in five cases. In contrast, 27 of the 35 (77%) adenocarcinomas studied showed at least focal intense staining of secretions, and 30 (86%) of the tumors showed some cytoplasmic immunostaining with B72.3. Positive staining occurred in all of the well-differentiated adenocarcinomas (100%) but was seen less often in moderately differentiated (82%) and poorly differentiated adenocarcinomas (58%). Because benign gland cells may express the TAG-72 antigen, immunohistochemistry results must be interpreted with caution and with regard to the overall morphologic pattern. Nonetheless, a positive B72.3 immunostain may be useful in identifying adenocarcinoma of the prostate, especially when an intense luminal reaction is found. A negative stain does not exclude the presence of adenocarcinoma, however.     

Identification of adenocarcinoma in cytospin preparations of effusions using monoclonal antibody B72.3

Metastatic adenocarcinoma in human effusions can be difficult to distinguish from reactive mesothelial cells, particularly if the malignant cells are rare, have bland cytologic features, or occur as single cells rather than clusters of cells. Monoclonal antibody (MAb) B72.3, generated against a membrane-enriched fraction of breast carcinoma, has been shown to be reactive with formalin-fixed, paraffin-embedded tissue sections of human breast and colon carcinomas but not with sarcomas, melanomas, hematopoietic neoplasms, or a variety of normal adult tissues. The authors have adapted the avidin-biotin-immunoperoxidase method using MAb B72.3 to evaluate cytospin preparations of pleural and peritoneal effusions for the presence of adenocarcinoma cells. The cytospin method was selected because it provides a rapid, efficient, and inexpensive means of evaluating effusions for cellular content. Reactive effusions from patients with no history of adenocarcinoma as well as obviously malignant effusions from patients with documented adenocarcinoma were studied. MAb B72.3 demonstrated no reactivity with any of the variety of cell types in the reactive specimens from patients without a history of adenocarcinoma. In contrast, in all of the malignant effusions studied, 10-90% of the nonhematopoietic cells demonstrated reactivity with MAb B72.3. In addition, MAb B72.3 highlighted occult adenocarcinoma cells in cytospin preparations of effusion fluids from patients with a primary diagnosis of adenocarcinoma in which no definite malignant cells could be identified using standard cytologic criteria.     

Diagnostic value of monoclonal antibody B72.3 in detecting adenocarcinoma cells in serous effusions

In serous effusions, the cytologic diagnosis of adenocarcinoma, and its distinction from mesothelial hyperplasia and malignant mesothelioma, is still difficult. This study compares the immunoreactivity of antibody B72.3 with that of anti-CEA, anti-LeuM1-antigen and anti-keratins in 41 serous effusions from patients with different malignant disorders. B72.3 selectively stained adenocarcinoma cells in 80% of the cases and was as good as anti-CEA and superior to anti-LeuM1-antigen and anti-keratins. It is concluded that B72.3 forms a useful supplement to conventional cytomorphology in the distinction between adenocarcinoma cells, reactive mesothelial cells and malignant mesothelial cells in serous effusions.     

Immunohistochemistry with monoclonal antibody B72.3 as an adjunct in the cytologic diagnosis of pancreatic carcinoma

Due to the morbidity of open tissue biopsy, the cytologic diagnosis of pancreatic carcinoma by fine needle aspiration or examination of biliary tree fluid is highly desirable. Immunohistochemistry with monoclonal antibody B72.3 has been advocated as an adjunct in the identification of tumor cells in body fluids. To assess its usefulness as an adjunct in the diagnosis of pancreatic carcinoma, we examined cytologic specimens of the pancreas from 35 patients [24 pancreatic carcinoma, 6 metastases (4 adenocarcinoma and 1 each of Hodgkin's disease and melanoma), 5 with benign conditions] with an immunohistochemical procedure using B72.3 directly over the Papanicolaou-stained slides. Of the pancreatic carcinomas, 21 of 24 (87%) were cytologically positive and 21 of 24 (87%) marked with B72.3. With both techniques, 23 of 24 cases (96%) could be identified. Three of four metastatic adenocarcinomas were positive by both cytology and B72.3. No staining occurred in the metastatic melanoma, Hodgkin's disease, or 3 of 5 benign conditions. In two benign duodenal aspirates, an unusual reticular B72.3 staining occurred in the mucin of acinar and goblet cells which could be misinterpreted as positive staining. In our experience, B72.3 enhances the sensitivity of the cytologic diagnosis of pancreatic cancer. Unrecognized single tumor cells, cytologically uninterpretable cells, and tumor cell clusters that could be misinterpreted as reactive epithelium mark with B72.3. Care should be taken to avoid misinterpretation of nonspecific mucin staining with this antibody.     

Frequent anti-V-region immune response to mouse B72.3 monoclonal antibody

The immune response of 56 colorectal cancer patients to a single infusion of 1 mg of radiolabeled (¹¹¹In) mouse B72.3-GYK-DTPA immunoconjugate was examined using a double-antigen radiometric assay system. The incidence of antibody response was 48% to polyclonal mouse IgG, 71% to mouse B72.3, and 62% to chimeric B72.3. Twelve patients (23%) had an antibody response to B72.3 V region in the absence of binding to polyclonal mouse IgG. An antiidiotype response was demonstrated in sera from 36% of 25 patients examined and correlated well with chimeric B72.3-GYK-DTPA immunoconjugate binding (r=0.72), moderately well with mouse B72.3 binding (r=0.56), and not at all with polyclonal mouse IgG binding (r=0.28). The peak antibody response occurred most frequently 2 weeks postinfusion, although a delayed peak response to chimeric B72.2 occurred in 29% of patients. This study suggests that mouse B72.3 causes an immune response in the majority of patients and that antibody response to the V region is common. Understanding the physiological significance of these antibody responses will require correlation with the kinetics and tumor localization of repeat infusions of such immunoconjugates.     

Chromosomal instability in pancreatic ductal cells from patients with chronic pancreatitis and pancreatic adenocarcinoma

Pancreatic adenocarcinoma is a disease with high mortality for which chronic pancreatitis confers a markedly increased risk. We hypothesize that chromosome instability and genomic damage occur in pre-neoplastic pancreatic ductal epithelium, and that this damage may be related to oxidative stress. We used dual-color fluorescence in situ hybridization with centromere probes and locus-specific arm probes for chromosome arms 11q, 17p, and 18q to identify genomic instability in cultures of normal-appearing human pancreatic ductal epithelium from normal organ donor controls compared to patients with chronic pancreatitis or pancreatic adenocarcinoma. To examine early pancreatic tumorigenesis, we studied only normal-appearing pancreatic ductal cells adjacent to pancreatitis or carcinoma. We found that, compared to the finding in normal controls, chromosomal abnormalities are present in normal-appearing human pancreatic ductal epithelia obtained from patients with chronic pancreatitis or pancreatic adenocarcinoma. Furthermore, these chromosomal abnormalities could be induced in cultured pancreatic ductal epithelium from normal organ donors by chronic exposure to dilute hydrogen peroxide, suggesting that oxidative stress may contribute to the development of chromosomal instability in the pancreas. These results elucidate a potential mechanism linking chronic pancreatitis to pancreatic cancer and suggest that chromosomal instability may be an early event in the pathogenesis of pancreatic cancer.     

Monoclonal antibody B72.3 in benign breast lesions.

It has been suggested that the monoclonal antibody B72.3 may be useful as a diagnostic tool in fine needle aspirates of breast masses because it recognises "tumour associated glycoprotein (TAG)-72". The antigen was sought in paraffin wax sections of 43 normal and benign breast biopsy specimens, using the avidin-biotin complex technique, to assess the extent of its presence in non-malignant tissue. Strong focal staining was seen in 21 (49%) cases. In 29 cases of fibrocystic change staining was present in 17 (59%). All areas of apocrine metaplasia were positive, as well as a few normal ducts and acini and occasional areas of adenosis. Focal positivity was present in five out of 12 foci of ductal epithelial hyperplasia and in three out of seven radial scars. Staining was absent in two areas of lobular hyperplasia, three areas of sclerosing adenosis, and in a focus of lactational change. Focal positivity was also seen in two out of five fibroadenomas and in two out of three intraduct papillomas. Five normal subareolar sections and a section of normal lactating breast were negative. It is concluded that B72.3 monoclonal antibody can show focal reactivity with a variety of normal and benign epithelial mammary structures, and it is doubtful that its use would be of any help in differentiating benign from malignant cells in fine needle aspirates.     

A tumor-associated antigen in carcinoma of the pancreas defined by monoclonal antibody B72.3

A retrospective analysis of 25 primary adenocarcinomas of the pancreas, 16 metastatic pancreatic tumors, 8 cases of chronic pancreatitis, and 3 adult normal pancreas was performed to ascertain the reactivity of monoclonal antibody (MAb) B72.3 to malignant and nonneoplastic pancreatic lesions. Formalin-fixed, paraffin-embedded sections of pancreas were evaluated by immunohistochemical techniques (avidin-biotin-peroxidase complex [ABC] method). Twenty-one of 25 malignant primary tumors were reactive, and all 16 metastatic sites expressed the B72.3 antigen. In contrast, all cases of pancreatitis and normal pancreas were either weakly reactive or nonreactive. Ten malignant and two benign pancreatic fine-needle aspirates provided results similar to those seen with fixed tissues. Because MAb B72.3 has selective reactivity for primary and metastatic pancreatic cancer, it may be of value as a diagnostic adjunct in cytologic examination or for radioimmunodetection of regional and/or distant metastases of adenocarcinoma of the pancreas.     

Differentiation of adenocarcinoma of the lung from mesothelioma. Periodic acid-Schiff, monoclonal antibodies B72.3, and Leu M1

The immunohistochemical reactivity of 38 mesotheliomas and 44 adeno-carcinomas or large cell carcinomas of the lung with monoclonal antibodies (MAb) B72.3 and Leu M1 was compared with their reactivity with the routine histochemic stains periodic acid-Schiff with diastase digestion (PAS-D) and alcian blue +/- hyaluronidase. Both MAbs reacted selectively with carcinomas when a positive test was set at greater than or equal to 10% reactive tumor cells. However, MAb B72.3 reacted with significantly more of the carcinomas (86%, chi-square test, P less than 0.01) and bound to a greater percentage of tumor cells (47 +/- 28%; mean +/- SD, t-test, P less than 0.001) than Leu M1 (57% and 25 +/- 28%, respectively). The similar reactivities of surgically resected tumor specimens and post mortem tissues with both antibodies confirmed antigen stability and suggested broad clinical utility. PAS-D stained 61% of the carcinomas. Using the markers for carcinomas (PAS-D, B72.3, and Leu M1), the tumors were classified into the correct group in 80 of 82 (98%) cases (95% confidence level: greater than 92% accuracy). The alcian blue stain was useful to confirm a diagnosis of dimorphic or epithelial mesothelioma (48% were positive).     

HFE genotypes in patients with chronic pancreatitis and pancreatic adenocarcinoma

PurposeThe homozygous p.C282Y variant of the HFE gene is a major risk factor for hereditary hemochromatosis, a disorder of iron metabolism resulting in progressive iron accumulation in a variety of organs including the pancreas. Heterozygosity of p.C282Y and p.H63D may increase susceptibility to chronic liver and pancreatic disease. This study determines the frequencies of p.C282Y and p.H63D alterations in patients with chronic pancreatitis and pancreatic adenocarcinoma.MethodsIn total, 958 patients (349 with alcoholic pancreatitis, 343 with idiopathic pancreatitis, 64 with familial chronic pancreatitis, 34 with acute pancreatitis, and 168 with pancreatic adenocarcinoma) were enrolled and compared with 681 healthy and 100 alcoholic controls. Furthermore, 45 parent–offspring trios were included for segregation analysis. Genotyping of p.C282Y and p.H63D was performed by restriction fragment length polymorphism or melting curve analyses.ResultsNo significant differences were found in heterozygosity for p.C282Y and p.H63D when patients with alcoholic (8.0/21.5%), idiopathic (7.3/24.5%), or familial (9.8/23.0%) pancreatitis, or pancreatic adenocarcinoma (5.4/28.6%) were compared with healthy (6.2/24.8%) and alcoholic (7.0/25.0%) controls. Neither genotype was associated with the presence of secondary diabetes mellitus in patients with chronic pancreatitis.ConclusionAlthough hemochromatosis is associated with pancreatic pathology, the p.C282Y and p.H63D variants do not play a significant role in the pathogenesis of chronic pancreatitis or pancreatic adenocarcinoma.     

Immunoperoxidase studies by monoclonal antibody B72.3 applied to breast aspirates: diagnostic considerations

The purpose of this study was to determine the reactivity of monoclonal antibody (MAb) B72.3 when applied directly to aspiration biopsy cytology (ABC) of the breast in the following conditions: (1) infiltration lobular carcinoma; (2) fibrocystic disease; (3) fibroadenoma; and (4) apocrine cysts. Nine of ten aspirates from infiltrating lobular carcinoma were positive in these assays, while 21 of 22 benign cases reacted negatively. The single false-positive benign aspirate manifested a staining pattern characteristic of apocrine cells. This study demonstrates that the MAb B72.3 can be employed as a potentially valuable diagnostic adjunct. It can be used on stained aspirates to assist in the interpretation of ABC from breast lesions.     

Use of monoclonal antibody B72.3 as a marker of metastatic carcinoma cells in neoplastic effusions

The value of monoclonal antibody B72.3 as a diagnostic discriminator between mesothelioma and carcinoma cells in malignant effusions was assessed using the ABC method in a series of cell blocks prepared from centrifuged fluids. These were obtained from either pleural or peritoneal neoplastic effusions in patients with histologically verified malignant mesothelioma (n:10) or carcinoma (n:20). Reactivity with MAb B72.3 in at least 10% or more of tumour cells was found in 16 (80%) out of 20 metastatic carcinoma, whereas 2 mesotheliomas displayed positive immunostaining in less than 5% and approximately 20% of the malignant cells respectively. Reactive mesothelial cells were consistently non-immunostained. These results suggest that B72.3 positivity in greater than 10% of tumour cells is certainly indicative, but not absolutely diagnostic, of a metastatic origin of malignant effusions.     

The novel monoclonal antibody HPC2 and N-cadherin distinguish pancreatic ductal adenocarcinoma from cholangiocarcinoma

Metastatic pancreatic ductal adenocarcinoma and primary cholangiocarcinoma are morphologically very similar and, therefore, challenging to distinguish in liver biopsies. The distinction is important because surgical management and prognosis differ significantly. Several immunohistochemical markers have been evaluated to aid this diagnosis, but aside from N-cadherin, which labels cholangiocarcinoma, few provide the combination of good sensitivity and specificity. Our laboratory recently developed the novel monoclonal antibody human pancreatic cancer fusion #2 (HPC2) that recognizes pancreatic cancer. We hypothesized that the combination of our new marker and N-cadherin can assist in distinguishing metastatic pancreatic cancer from cholangiocarcinoma. We immunostained resections of 60 pancreatic ductal adenocarcinomas and 31 cholangiocarcinomas for the HPC2 and N-cadherin antigens. We also stained 24 gallbladder adenocarcinomas, 11 ampullary adenocarcinomas, and 10 metastatic colonic adenocarcinomas to the liver. Sections were independently scored by 2 pathologists with good agreement using both markers (κ statistics, 0.62-0.64; P < .0001). HPC2 was observed in 80% of pancreatic cancers (48/60), 82% of ampullary (9/11), and 32% (10/31) of cholangiocarcinomas. N-cadherin stained 27% (16/60) of the pancreas cases and 58% (18/31) of the cholangiocarcinomas. Gallbladder and colon cancers were usually double negative (18/24 and 8/10, respectively). Each marker provided significant likelihood ratios to separate pancreatic cancer (HPC2, 2.48 [1.46-4.19]; P < .0001) from cholangiocarcinoma (N-cadherin, 2.17 [1.3-3.64]; P < .01). The combination of both markers provided even better specificity and positive likelihood ratios. We conclude that HPC2 and N-cadherin significantly improve accurate classification of pancreatic cancer and cholangiocarcinoma.     

The diagnostic distinction between malignant mesothelioma of the pleura and adenocarcinoma of the lung as defined by a monoclonal antibody (B72.3).

The correct distinction between malignant mesothelioma of the pleura and adenocarcinoma of the lung has become increasingly complex, with a variety of histochemical, immunohistochemical, and ultrastructural studies to be performed on biopsy material. The reliability of immunohistochemical studies has been hampered by the use of polyclonal antisera to "carcinoembryonic antigen (CEA)" and keratin. Hybridoma technology now offers monoclonal antibodies (MAbs) in unlimited quantity and standardized quality to selective ranges of specific antigenic determinants. MAb B72.3, generated against a membrane-enriched fraction of human metastatic breast carcinoma, was used to distinguish malignant mesothelioma of the pleura from adenocarcinoma of the lung in tissue sections and was compared in terms of diagnostic utility with polyclonal anti-keratin and anti-CEA to make the same distinction. Reactivity with MAb B72.3 in at least 10% of tumor cells or more was noted in 19 of 22 adenocarcinomas of the lung (P greater than 0.0001), whereas none of the 20 cases of malignant mesothelioma demonstrated comparable reactivity. Furthermore, MAb B72.3 showed no reactivity with benign mesothelial proliferations. MAb B72.3 thus appears to be an appropriate diagnostic adjunct capable of discriminating between these malignancies.     

Utility of serum IgG,IgG4 and carbonic anhydrase II antibodies in distinguishing autoimmune pancreatitis from pancreatic cancer and chronic pancreatitis

PurposeAutoimmune pancreatitis (AIP) can mimic pancreatic cancer in its clinical presentation, imaging features and laboratory parameters. The aim of our study was to compare IgG, IgG4 and anti-CAIIAb serum levels in patients with AIP, pancreatic adenocarcinoma (PA) and chronic pancreatitis (CP) and to assess their clinical significance and utility in differential diagnosis of pancreatic diseases.Patient/methodsThe study included 124 patients: 45 with PA, 24 with AIP and 55 with CP. Peripheral venous blood samples were obtained from all analyzed patients at the time of hospital admission and total IgG, IgG4 and anti-CAIIAB serum levels were measured using ELISA tests.ResultsSerum levels of IgG, IgG4 and anti-CAIIAb were significantly higher in patients with AIP compared to PA and CP patients (p < 0.001). In AIP patients the median IgG levels were 19.7 g/l, IgG4 levels – 301.9 mg/dl and anti-CAIIAb – 81.82 ng/ml, compared to 10.61 g/l, 123.2 mg/dl and 28.6 ng/ml, respectively, in PA patients. IgG4 for the cut-off 210 mg/dl showed the best sensitivity and specificity (83.8% and 89.5%) in AIP diagnosis compared to IgG (69.3% and 87.3%, respectively) and anti-CAIIAb (45.3% and 74.3%). However, 16 (35.5%) patients with PA and 14 (25.4%) patients with CP had IgG4 levels greater than 140 mg/dl. Moreover, in 3 (6.67%) patients with pancreatic cancer those values were greater than 280 mg/dl. No patients with CP had IgG4 more than 280 mg/dl.ConclusionsIgG4 at cut-off 210 mg/dl showed the best sensitivity and specificity in AIP diagnosis compared to IgG and anti-CAIIAb, however elevations of serum IgG4 may be seen in subjects without AIP, including pancreatic cancer.     

Maspin is useful in the distinction of pancreatic adenocarcinoma from chronic pancreatitis: a tissue microarray based study.

Maspin, a member of the serpin family of serine protease inhibitors, has been shown to limit invasion and metastases in breast and prostate carcinomas. Maspin gene expression is up-regulated in pancreatic cancer, but not in normal pancreatic tissue. Maspin expression has been documented using immunohistochemical studies in pancreatic adenocarcinoma and high-grade intraductal dysplasia. We studied pancreatic ductal adenocarcinomas and chronic pancreatitis utilizing tissue microarray technology to determine the utility of maspin in differentiating these lesions. Immunohistochemistry was performed on tissue microarrays made from 72 cases of pancreatic ductal adenocarcinoma and 24 cases of chronic pancreatitis. Carcinomas were graded as well, moderately, or poorly differentiated using the WHO criteria. The primary antibody used was monoclonal antimaspin antibody (clone G167-70, 1:800, PharMingen, San Diego, CA). Nuclear and/or cytoplasmic staining for maspin was qualitatively scored from 1 + to 3 + based on intensity. Cases were considered positive if one or more cores demonstrated staining. Cases of chronic pancreatitis showed focal, weak (1 + to 2 +) staining within occasional benign ductal epithelial cells in 29% of cases (7/24). Diffuse and intense (3 +) staining was present in ducts with squamous metaplasia (3 cases). The majority of ducts showed no staining. Ductal adenocarcinomas showed diffuse staining in 91% (66/72) of cases with generally more intense staining than cases of chronic pancreatitis. Maspin may be helpful in differentiating ductal adenocarcinoma from chronic pancreatitis, once squamous metaplasia is ruled out.     

Discrimination of pancreatic adenocarcinomas from chronic pancreatitis by morphometric analysis.

Chronic pancreatitis and pancreatic ductal adenocarcinoma show similar gross and microscopic anatomical features. Morphological examination alone is not always sufficient in diagnostic practice to make the clinically important discrimination between these two entities. Cases of pancreatic tumors were analysed in a morphometric study to evaluate the discriminatory value of nuclear and nucleolar features. Histologic sections of pancreas from 18 cases of chronic pancreatitis and 33 cases of ductal adenocarcinoma were included either into a learning or a test set. A multivariable discriminatory rule was derived from the learning set of 23 cases including nuclear polymorphism and nucleolar density. When applied to the test set, all 28 cases of adenocarcinomas and chronic pancreatitis were correctly classified. Distributional features describing nucleolar density and variation in nuclear size and shape were the most efficient discriminatory variables. Morphometry is shown to be a simple and fast cell analytical method which can support clinical judgement in distinguishing between chronic pancreatitis and pancreatic ductal adenocarcinoma.     

The value of monoclonal antibody B72.3 for the diagnosis of breast carcinoma: experience with the first commercially available source.

One hundred cases of invasive breast carcinoma were studied using the commercially available monoclonal antibody Anti-Human Tumor-Associated Glycoprotein-72 (MAb B72.3, Biomedical Technologies Inc, Stoughton, MA) prediluted at 8.5 micrograms/mL. Forty-three cases displayed positive reactivity with this antibody. Intensity and distribution of positive staining varied among the tumor cells. Twenty-two cases had 1% or less reactive cells, while eight cases contained 40% or more positive tumor cells. Apical cell membrane and diffuse cytoplasmic staining were present. In fifteen cases intracytoplasmic lumina and extra-cellular secretory material were highlighted by positive staining. Thirty-five cases had benign breast tissue adjacent to the tumor. Benign ductal and lobular epithelial cells were nonreactive except for two cases in which small foci of apocrine metaplasia were positive. Reactivity with MAb B72.3 was not dependent upon histologic grade, nuclear grade, nodal status, or patient age. Excluding the lower number of positively stained cases, our findings were similar to other MAb B72.3 investigations. The number of positively stained cases and the intensity of the positivity were increased by using MAb B72.3 at 5.0 micrograms/mL with overnight incubation, or by using MAb B72.3 at 40.0 micrograms/mL with 2 hours of incubation. Our findings confirm that MAb B72.3 shows reliable reactivity with breast carcinoma that is sensitive to antibody concentration and incubation time without loss of specificity for tumor cells. Our results are also consistent with the view that MAb B72.3 probably detects epithelial membrane-related antigens in breast carcinoma, as do several other antibodies.     

Determination of monoclonal antibody specificity by immunoadsorption and western blotting.

A rapid and simple method has been developed for identifying the specificity of monoclonal antibodies at an early stage in the production of hybridomas. The technique is a micro-method utilizing biotinylated crude antigen and the surface of microtiter plates as an immunoaffinity matrix. The monoclonal antibodies to be tested are adsorbed to the microtiter wells and incubated with the labeled antigen preparation. Non-specific binding can be reduced by blocking and repeated washing steps. The specific antigen is then eluted by SDS-containing buffers, subjected to SDS-PAGE, blotted onto nitrocellulose and detected by enzyme-labeled avidin in a Western blot assay. The amount of bound and removed antigen can be quantitated by developing eluted and non-eluted control wells by ELISA techniques. Since this ELISA can be performed rapidly, only samples which contain sufficient specific material can be selected for electrophoresis and blotting. The major advantages of the technique are (i) the use of a non-radioactive label resulting in an easy and time-saving procedure, (ii) the possibility of quantitating the amount of captured and detached antigen by ELISA, (iii) the need for only a minimal amount of antigen, (iv) the use of unpurified antibodies of all isotypes, (v) a high signal-to-noise ratio, and (vi) as with all immunoprecipitation techniques, the possibility of detecting SDS-sensitive epitopes and of using crude antigen preparations. Using this method we were able to characterize monoclonal antibodies against both soluble proteins (mouse and human C1q) and membrane determinants (human pan T cell CD5 and CD7).     

Comparison of methods for the generation of immunoreactive fragments of a monoclonal antibody (B72.3) reactive with human carcinomas

Immunoglobulin fragments, whether of polyclonal or monoclonal antibodies, offer a number of advantages over the intact immunoglobulin. The generation of immunoreactive fragments from monoclonal antibodies (MAb) is not always a straightforward task. Both pepsin and papain can be used to digest MAb to a bivalent molecule with a Mr of 100,000. However, pepsin pepsin digestions does not always result in immunoreactive fragments and a stable consistent product by papain digestion is often difficult to obtain. MAb B72.3 is an example of both situations. MAb B72.3 reacts with a glycoprotein (TAG-72) with a molecular weight greater than 10(6). MAb B72.3 has been shown to exhibit a high degree of selective reactivity with colon, breast and ovarian carcinomas and has been used for radioimmunodiagnosis in model systems and in clinical trials. A third enzyme, bromelain, in the same family of sulfhydryl proteases as papain, has been used to generate a fragment of MAb B72.3, with a Mr of approximately 100,000. The bromelain-generated fragment of MAb B72.3 retained 100% immunoreactivity as measured in competitive solid-phase radioimmunoassays and could be generated with consistent results from one preparation to another. Both the bromelain- and papain-generated fragments were radiolabelled with 125I without significant loss of the MAb's reactivity to tumor extracts. Differences were observed between the bromelain- and papain-generated fragment when compared in vivo. Fragmentation of MAb B72.3 with bromelain has yielded a superior bivalent fragment for radioimmunolocalization.